Current Advancements in Addressing Key Challenges of Therapeutic Antibody Design, Manufacture, and Formulation

Vicki Sifniotis; Esteban Cruz; Barbaros Eroglu; Veysel Kayser

doi:10.3390/antib8020036

Current Advancements in Addressing Key Challenges of Therapeutic Antibody Design, Manufacture, and Formulation

Sifniotis, Vicki;Cruz, Esteban;Eroglu, Barbaros;Kayser, Veysel 2019-06-03 00:00:00 antibodies Review Current Advancements in Addressing Key Challenges of Therapeutic Antibody Design, Manufacture, and Formulation Vicki Sifniotis , Esteban Cruz, Barbaros Eroglu and Veysel Kayser * School of Pharmacy, Faculty of Medicine and Health, The University of Sydney, Sydney 2006, Australia; vsif0221@uni.sydney.edu.au (V.S.); ecru7298@uni.sydney.edu.au (E.C.); barbaros.eroglu@sydney.edu.au (B.E.) * Correspondence: veysel.kayser@sydney.edu.au; Tel.: +61-2-9351-3391 Received: 16 April 2019; Accepted: 31 May 2019; Published: 3 June 2019 Abstract: Therapeutic antibody technology heavily dominates the biologics market and continues to present as a signiﬁcant industrial interest in developing novel and improved antibody treatment strategies. Many noteworthy advancements in the last decades have propelled the success of antibody development; however, there are still opportunities for improvement. In considering such interest to develop antibody therapies, this review summarizes the array of challenges and considerations faced in the design, manufacture, and formulation of therapeutic antibodies, such as stability, bioavailability and immunological engagement. We discuss the advancement of technologies that address these challenges, highlighting key antibody engineered formats that have been adapted. Furthermore, we examine the implication of novel formulation technologies such as nanocarrier delivery systems for the potential to formulate for pulmonary delivery. Finally, we comprehensively discuss developments in computational approaches for the strategic design of antibodies with modulated functions. Keywords: therapeutic antibody; stability; aggregation; manufacture challenges; formulation 1. Introduction Since the ﬁrst therapeutic monoclonal antibody (mAb) Orthoclone OKT3 (Janssen Biotech, Horsham, PA, USA) was approved by the USA Food and Drug Administration in 1986, whole antibody therapeutics have become and persistently remain the most dominant and signiﬁcant biologic therapeutic platform in the pharmaceutical industry [1,2]. To date, therapeutic antibodies treat a plethora of indications including cancers, infections, autoimmune disorders, and cardiovascular and neurological diseases [3]. The whole antibody therapeutics platform is regarded as the most promising class of pharmaceutical technology to date; it is continually being applied to newly identiﬁed biological targets and implemented in many formats to produce strategically engineered next generation antibody therapeutics, otherwise termed “biobetters” [4–9]. The international ImMunoGeneTics information ® ® system (IMGT , Montpellier, France) database reveals that as of December 2018, 65 whole antibodies and 18 next generation fragment or recombinant fusion antibody-based therapies are approved for clinical use, with hundreds more in clinical trials expected to reach market. Antibodies 2019, 8, 36; doi:10.3390/antib8020036 www.mdpi.com/journal/antibodies Antibodies 2019, 8, 36 2 of 23 Naked Whole mAbs Whole mAb formats 65 Naked whole mAbs 60 ADCs ADCs 4 Whole mAb bispecifics 1 Whole mAb Bispecifics Biosimilars Fragment mAb formats 18 Fabs Fabs 4 Fc fusions 10 Fc Fusions scFv fusions 3 scFv bispecifics (BiTE) 1 scFv Fusions Originator mAbs with scFv Bispecific (BiTE) approved biosimilars (a) (b) Figure 1. The proportions of therapeutic antibody formats approved for therapeutic use as of December , ® 2018 IMGT depicted through (a) a pie chart and (b) a table format. Whole therapeutic mAbs are presently the dominant antibody platform approved for clinical use (Figure 1), although antibody engineering technologies have advanced in recent years to produce highly optimized, strategically engineered biobetter therapies, along with biosimilar mAbs reaching market to compete against their originator. Further whole mAb formats include antibody–drug conjugates (ADCs), bispeciﬁcs, isotype-switched, and glycoengineered. These additional formats have been strategically designed to introduce exceptional potency, to engage dual biological targets, and to modulate Fc eector functions. Fragments of mAbs such as the crystallizable fragment (Fc), antigen binding fragment (Fab), and single-chain variable fragment (scFv) possess key functions such as speciﬁcity to a biological target or immunological activation. The isolation of these fragments for fusion with other mAb fragments, biologically functional proteins, cytotoxic drugs, or drug carriers has been the crux of ingenuity in developing the next generation of biobetter therapies [4–15]. Figure 2 depicts several examples of prominent biobetter formats, providing a general representation of current fragment mAbs, whole mAb bispeciﬁcs, fragment mAb multispeciﬁcs, and fragment mAb fusion therapeutics. Antibodies 2019, 8, 36 3 of 23 Figure 2. Schematic representation of a whole monoclonal antibody (mAb), a fragment mAb, and prominent fusion mAb formats that have been developed for strategic therapeutic uses. Proteins fused to mAb fragments are depicted as blue ovals for a general representation; however, fusion proteins may vary in size and structure. Fragment formats include the crystallizable (Fc), antigen binding (Fab and F(ab)2), and single-chain variable (scFv) fragments. Further whole mAb formats include the antibody–drug conjugate (ADC), triomab, dual variable domain immunoglobulin (DVD-Ig), and immunoglobulin–scFv fusion (IgG-scFv). Multispeciﬁc fragment formats include the F(ab)2 bispeciﬁc, bispeciﬁc T-cell engager (BiTE), dual anity re-targeting molecule (DART), and tandem diabody (tandAb). 2. Overview of mAb Production Challenges and Considerations Whole therapeutic mAbs require a mammalian expression system to produce the biologically functional product; however, a mAb fragment and recombinant fusion mAb products with simpliﬁed (or lacking) glycosylation are suitable for lower organism expression platforms [16]. Unlike oligopeptides, which can be chemically synthesized, whole therapeutic mAbs are considerably larger (with monomer ranging from 140–160 kDa) and comprise of four peptide chains (two heavy and two light chains) bound together by disulﬁde bonds and interchain non-covalent interactions. Further to this, antibodies contain glycosylation in a conserved region of the Fc (N297) that contributes Antibodies 2019, 8, 36 4 of 23 to its stability and immune eector functions [17,18]. Aside from peptide synthesis, cell machinery is required to glycosylate, fold, orient, and covalently bind the antibody peptide chains in order to produce the complete, biologically functional antibody product. Manufacturing biologically functional whole mAb product is therefore commercially unfeasible through chemical synthesis and insucient in lower organism expression platforms such as bacteria, yeast, insect, and plant cells that may not have the machinery to produce the equivalent tertiary structure and glycosylation proﬁles. In particular, many industrially relevant bacterial strains such as E. coli are completely deﬁcient in the machinery to add post-translational glycosylations; yeasts hyper-mannosylate glycans, which cause immunogenicity; and insect cells which are deﬁcient in sialylation machinery and produce immunogenic glycan structures [16]. A secretion of the mAb product for puriﬁcation is suboptimal for several lower organism expression platforms such as E. coli due to poor productivity and harsh culture conditions that promote product degradation. Protein is therefore produced intracellularly, as inclusion bodies and harvest involves further processing steps such as cell lysis, inclusion body recovery, protein solubilization, and renaturation prior to further downstream puriﬁcation steps [19,20]. Despite these pitfalls, the development of lower organism expression systems is of high commercial interest due to the simpliﬁed culture conditions, cheaper media requirements, rapid organism growth, and higher product yield as compared to mammalian expression systems [16,21]. In considering the requirements through the entire process of mAb discovery, manufacture, formulation, and disease treatment, several key challenges arise which have sparked overwhelming interest in pursuit of achieving better mAb manufacturing outcomes and treatment strategies. As with all biotherapeutics, mAbs and mAb-based therapeutics are limited to production in cell-based expression systems, which is considerably costly and inecient, can have varied yields depending on the product and expression system, and requires downstream processing to remove biological contaminants introduced from the expression system. Despite anity chromatography being a robust technology for the initial capture of a mAb for puriﬁcation, the capture process and further downstream processes such as viral inactivation applies the mAb product to harsh pH and salt conditions, which can chemically degrade the mAb, leading to product instability and loss [5,22,23]. Many factors through the manufacture process inﬂuence glycosylation and charge heterogeneity of mAbs, which aects their biophysical and pharmacological properties. Though not speciﬁcally discussed in this review, the improvement and control mAb production technologies address these variations to reduce formulation heterogeneity and o-target cytotoxicities. A common challenge, as seen with all biotherapeutics, is that mAbs and mAb-based therapeutics are currently restricted to lyophilised and liquid-based formulations for intravenous (IV) or subcutaneous (SC) delivery to achieve maximum bioavailability. Protein self-association and intrinsic stability drive this limitation, in that viscosity and propensity to aggregate are dependent on mAb concentration. Formulations are optimized to achieve the highest dosing concentration at the minimum achievable volume for injection, without compromising the quality of the mAb in formulation [24]. Viscosity remains a key limiting factor for formulating as a SC administration—certain mAb therapies are suitable and others not based on their solubility, self-association, and aggregation proﬁles. Alternative non-invasive administration strategies such as pulmonary delivery causes additional mechanical stress that further contribute to mAb instability and loss. Furthermore, oral delivery is unsuitable due to chemical and enzymatic degradation, as well as poor absorption in the gastric and intestinal environments [5,25–29]. A brief overview of considerations through the dierent concept stages of therapeutic mAb development is depicted in Figure 3. The main challenges and considerations in the manufacture and formulation of mAb therapeutics are brieﬂy summarized in Table 1. Antibodies 2019, 8, 36 5 of 23 Figure 3. Schematic representation of the concept stages of mAb drug development in which considerations follow on from one process to the next in the design and manufacture of mAb-based therapeutics. Antibodies 2019, 8, 36 6 of 23 Table 1. Summary of key technological advancements that address challenges and considerations in mAb design, manufacture, and formulation strategies. Challenges Advancements Manufacture Considerations Hybridoma technology produces immunogenic mAbs Humanization technologies [4–6] Yield from hybridoma technology is variable Commercial cell line development and recombinant technology [4–6]; lower organism systems for fragment mAbs, mammalian systems for whole mAbs, and Fc Signiﬁcance of post-translational modiﬁcations and higher-order structure in fusions [16,30,31] mAb product CHO expressed mAbs contain an immunogenic glycosylation proﬁle Human-based expression systems [16,30–32] HEK 293 expressed mAbs are prone to aggregation HKB-11 and PER.C6 cell lines [32] Undesirable byproducts produced in the manufacture process in vitro cell-free synthesis technology [33,34] Stability of mAb aects manufacture yield due to product loss through aggregation in Enhancing mAb stability through framework mutations and hyperglycosylation downstream processing steps [5,35–40] Treatment Considerations for Drug Design and Formulation Susceptibility of mAb to degradation limits delivery to intravenous and subcutaneous only Enhancing mAb stability through framework mutations, hyperglycosylation [5,35–40], and nanocarrier technologies [8,41] Stability of mAb limits concentration of formulation Concentration of mAb aects viscosity and injection pressure for Excipients, fragment mAbs, PEGylation, and hyperglycosylation [25–27,41] subcutaneous delivery Poor tissue penetration and biodistribution Fragment mAbs and nanocarrier technologies [8,41,42]; high anity ADCs [43–45] PEGylation, hyper-glycosylation and Fc fusion proteins [8,41,46]; modulation of FcRn Reduced half-life in low MW species recycling through Fc mutation [11,13] Modulation of immunological engagement Isotype switching, glycoengineering, and Fc mutations [11,13,15,43,46] Antibodies 2019, 8, 36 7 of 23 3. mAb Discovery and Manufacture Technologies Traditional technology for whole therapeutic antibody discovery required the immunization of animals—primarily mice with a target antigen for the generation of a mixed population of B-lymphocytes-producing antibody against the target. The B-lymphocytes would be isolated for immortalization to produce monoclonal hybridoma cell lines that secrete antibody candidates of interest for further panning through display technologies to isolate potential leads [5]. This method has led to the generation of highly speciﬁc mAb libraries of non-human origin that were potentially immunogenic and less ecacious at eliciting an immune response as compared to wholly human mAbs. Technologies arose to address immunogenicity by producing chimeric and humanized antibodies through the grafting of the variable domains (chimeric) or complementarity-determining region (CDR) residues (humanized) isolated from lead non-human antibodies to a human antibody framework. To further improve the humanization strategy, transgenic mice were developed with their murine antibody heavy and light chain genes replaced with equivalent human genes; they consequently expressed wholly human antibodies for discovery [4–6]. Phage display technology remains the most prominent selection technology for panning antibody gene pools for speciﬁcity to a target antigen, as it eectively couples the expression of mAb proteins to the genes that encode them for the panning of high anity leads. Antibody variable genes from B-lymphocyte pools are isolated through polymerase chain reaction (PCR), and cloned into a phage expression vector that presents the expressed mAb on the surface of the phage, the library of vectors is rescued in phage, and the phage library is screened against a speciﬁc target antigen. Phage pools undergo several rounds of selection to isolate high anity candidates, the variable genes of the lead mAb candidates can then be isolated and sequenced for the design of a mAb drug format and the development of a suitable expression platform [5,47]. Error-prone PCR-based mutagenesis has propelled display technology for the generation of enormous libraries for target anity screening. Yeast surface display further improves these screening technologies, as glycosylation sites introduced in CDRs are expressed through this platform [5,48]. The expression, yield, and quality of a mAb can vary quite substantially in hybridoma cell lines. High yielding mammalian expression systems have been developed to meet the commercial need for high expression eciency, scalability, quality, and reproducibility. Antibody genes of interest are introduced into suitable expression vectors and transfected into highly ecient mammalian cell lines for antibody expression and secretion; a mAb can then be directly captured from the culture supernatant through anity chromatography. Currently, the majority of mammalian expression systems for commercial whole therapeutic antibody expression are based on stable chinese hamster ovary (CHO), mouse myeloma (NS0), and mouse hybridoma (Sp2/0) cell lines [4,5]. However, in the context of research and development, the transient expression in human cell lines such as embryonic kidney (HEK 293), amniotic (CAP), a hybrid of HEK 293 and lymphoma (HKB-11), and embryonic retina (PER.C6) are favored over stable CHO expression due to the ease and speed of production of workable quantities of antibody for preliminary studies [16,30–32,49,50]. The generated mAb library undergoes relevant in vitro testing along with formulation stability screening to exclude candidates that have poor manufacturability attributes [51]. Lead mAb candidates that show potential for further investigation would then be developed for high yielding stable expression and more rigorous characterization leading up to therapeutic development. However, a drawback from changing from human to hamster expression systems is the dierence in glycosylation proﬁle. CHO expressed mAbs produce immunogenic non-human glycoform Neu5Gc and have a higher composition of sialylation, which may result in reduced antibody-dependent cellular cytotoxicity (ADCC). Therefore in the early stages of development, leads need to be identiﬁed and thoroughly characterized in the expression system to be developed for commercial manufacture before committing to industrial scale up [16,30,32]. Amongst other biotherapeutics, approved mAb Fc fusion therapeutics dulglutide (Trulicity , Eli Lilly, ® ® Indianapolis, IN USA), efmoroctocog alpha and eftrenonacog alpha (Eloctate and Alprolix , Biogen, Cambridge, MA, USA), are manufactured in a HEK 293 expression system, which is giving rise to Antibodies 2019, 8, 36 8 of 23 the acceptance of human-based expression systems for the production of mAb-based therapeutics [4]. However, HEK 293 expression systems are prone to inducing mAb aggregation in cultures, which is detrimental to cell viability and creates a loss of product during manufacture. Hence, HKB-11 and PER.C6 are the preferred commercial human cell lines for mAb expression in human cell lines, speciﬁcally [16,31,32]. Established lower organism expression systems for fragment or recombinant fusion mAb therapeutics include bacteria such as E. coli, yeasts such as S. cerevisiae and P. pastoris, plants such as tobacco, algae, and insects such as silkworm [5,16,52–54]. Expression platforms that use E. coli in particular are considered high risk due to the potential endotoxin contamination in the mAb product, of which complete endotoxin removal requires further puriﬁcation steps [19]. However, several approved mAb fragment- and recombinant-based therapies are produced in an E. coli-based expression system, including pegol conjugated Fab’ certolizumab pegol (Cimzia , Celltech UCB, Brussels, Belgium), Fab ranibizumab (Lucentis , Genentech, San Francisco, CA, USA), and recombinant Fc fusion romiplostim (Nplate , Amgen, Thousand Oaks, CA, USA). Lower organism expression platforms such as E. coli and P. pastoris continue to be the preferred option for the manufacture of fragment mAb formats due to the relative ease, high yield, and reduced cost for manufacture as compared to mammalian expression platforms. An emerging in vitro cell-free synthesis technology is being developed with bacterial and CHO cell lysates which have the potential to alleviate formation of undesirable biological byproducts, as the machinery from the cell lysates purely express protein from the mAb genes that are introduced to the system. Though this technology is not currently applicable for industrial scale manufacture, it holds much promise as an alternative to live culture. For instance, culture maintenance and highly deﬁned media are no longer necessary, reactions can run continuously, lysates can be recycled with reproducible results, unmodiﬁed linear DNA is suitable for the system (thus alleviating a need for multiple cloning steps), and additional enzymes can be introduced to the system for the engineering of speciﬁc post-translational modiﬁcations [33,34,55–58]. Despite the apparent advantages of this technology for mAb manufacture, cell lysates encounter the same challenges as their host cell expression system. That is, post-translational modiﬁcations of mAb product from lysates from lower level organisms are limited by the endogenous machinery of that host organism. Furthermore, preparation of mammalian cell lysates is challenging, and yield produced from these lysates is considerably low [59,60]. Current bioprocess engineering and cell line development strategies have been crucial in enhancing the manufacturability of mAbs. Many current mAb therapeutic expression platforms make use of CHO cell lines that have been commercially developed with dihydrofolate reductase or glutamine synthetase deﬁciency for enhanced stability selection through resistance to methotrexate and methionine sulfoximine inhibition, respectively [50,61]. Mammalian cell lines have been adapted to suspension culture, as they support higher cell densities and mAb titers in the absence of serum from the culture media, for large scale fed-batches, perfusion systems, or continuous culture systems. Further to this, cell lines are continually being engineered for enhanced metabolic functions, introducing glycosylation pathways, superior secretion, and resistance to apoptosis for prolonged survival, in eorts to generate super-producer cell lines that can sustain a continuous culture [62–68]. Gene and expression vector design and development are speciﬁcally tailored to the expression system to maximize mAb expression and cell line stabilization through host cell codon optimization and the addition of highly ecient transcription, secretion, selection, and integration elements [69–73]. Vectors can contain a single site for gene insertion for the subsequent co-transfection of vectors that carry the antibody heavy and light chains, or both chains can be cloned into a dual expression vector. In more elaborately designed recombinant mAb drug formats that require the expression of several genes, vector technologies have implemented multicistronic expression to enhance the eciency of the vector system. The stable or transient transfection of vectors is performed on highly viable cell cultures with high cell density. Antibody heavy and light chain ratios may require adjustment in transfection for optimizing expression and secretion. A reporter vector that expresses green ﬂuorescent protein is typically included Antibodies 2019, 8, 36 9 of 23 to observe transfection eciency during optimization [74,75]. Liposome-mediated transfection is preferential over all other mammalian transfection strategies and is induced chemically through the complexing of DNA to a cationic lipid prior to or during addition to a culture. Polyethylenimine is the most prevalently used transfection reagent despite the development of superior reagents such as Lipofectamine— 2000 and Freestyle— MAX (Thermo Fisher Scientiﬁc, Waltham, MA, USA), LyoVec— (InvivoGen, San Diego, CA, USA), FuGENE 6— (Promega, Madison, WI, USA), and TransIT-PRO (Mirus, Madison, WI, USA), owing to its lower cost and relative eciency [73,75–78]. The commercial manufacture of mAbs has transitioned to serum-free, chemically deﬁned, and animal-free media which has removed a source of expression variability and has been driven particularly from the advent of mad cow disease [50,79]. Several media supplements such as plant and yeast digests (peptones/hydrolysates), surfactants (e.g., Pluronic F-68), DNA methyltransferase (azacytidine), and histone deacetylase (sodium butyrate, valproic acid) inhibitors have been found to support cell viability and enhance mAb expression [79–85]. Mild hypothermia of the culture has also demonstrated to reduce cell expansion, support prolonged cell viability, and enhance mAb expression [73,86–90]. Further to this, continuous supplementation in expression cultures of uridine, manganese chloride, and galactose demonstrated successful the glycan engineering of expressed mAb, where mAb hyper-galactosylation was promoted to enhance complement-dependent cytotoxicity (CDC) activity [91]. In the manufacturing process of fed-batch, perfusion, and continuous culture systems, media is continuously fed in the culture system, whilst parameters such as cell viability, cell density, and metabolite levels are monitored in real time until the parameters indicate that it is the optimal time to harvest the expressed mAb from the culture supernatant. In perfusion and continuous culture systems, a feed is removed from the culture simultaneously to the media addition, the dierence being that the cell dilution rate in continuous culture is optimized to remain equal or higher than the cell growth rate, which allows the perpetuation of mAb expression and requires the continuous harvest of the supernatant [92–94]. The harvest of the supernatant requires clariﬁcation technologies such as the use of precipitants/ﬂocculants (e.g., polyethylene glycol, diethylaminoethyl dextran, caprylic acid, and polyethylenimine), high throughput centrifugation, and ﬁltration methods to remove cells and biological debris, for the lowering the loading burden in the lead up to mAb capture through anity chromatography [19,95–100]. The mechanical stresses from centrifugation and ﬁltration are unavoidable, although they can induce mild mAb product loss through fragmentation and aggregation. Protein A-ligand-based anity chromatography is the most robust, ecient, and prevalently used mAb capture technology, owing to its selectivity and high anity to various human Fc, as well as its ecient dissociation of captured mAbs at a low pH for reuse. Fragment mAbs and recombinant formats based on Fabs and single chain variable fragments (scFv) are unable to take advantage of protein A anity capture, and alternatives have been developed, such as capturing proteins G, M, and L, which bind at dierent mAb epitopes; ion exchange chromatography; and polyhistidine tagged capture through immobilized metal chromatography [100–102]. A further advantage of anity chromatography in manufacture is the integration of a viral inactivation step by holding the low pH-eluted mAb product prior to further puriﬁcation steps; however, this applies the mAb product to low pH at high concentrations, which can induce mild mAb instability, aggregation, and the formation of acidic variants [5,22,100,103,104]. Post mAb capture, further polishing steps are required to remove further contaminants such as host cell proteins and DNA, as well as leached anity chromatography ligand- and mAb-degradation products such as fragments, aggregates, and ionic variants. The polishing steps are designed based on the speciﬁc properties of the mAb product, such as the isoelectric point and molecular weight (MW), to implement appropriate chromatographic technologies such as anion and cation exchanges, hydrophobic interaction, and multimodal and size exclusion that will separate the mAb product from the manufacture-introduced impurities [22,100,103]. The additional chromatographic steps unavoidably apply the mAb product to buers of varying ionic strength and high concentrations, which can again induce mild mAb instability and aggregation. Post polishing steps, the puriﬁed Antibodies 2019, 8, 36 10 of 23 mAb product undergoes a further viral removal step, such as ﬁltration, and then it undergoes buer exchange and concentration through ultraﬁltration or diaﬁltration methods to prepare the bulk mAb product for formulation [100,105]. 4. Formulation Strategies and Considerations Strategies to improve the formulation of mAbs and mAb-based therapies continues to be an ongoing challenge, as is faced with all biotherapeutics. Firstly, whole therapeutic mAbs are unsuitable for non-invasive oral, nasal, or pulmonary routes of administration as they are susceptible to chemical and enzymatic degradation in the gastrointestinal tract. The bioavailability of mAbs through these routes is poor, owing to mAbs’ polar surface charge and relatively large MW, limiting transport through mucosal membranes. Fragment mAb platforms, together with excipient and PEGylation technologies, have been developed to help circumvent transportation limitations for pulmonary delivery, speciﬁcally. However, physical stresses applied to the mAb (e.g., shear stresses from the aerosolization of liquid formulations for a pressurized meter dose and a nebulization delivery, or from the production of dry powder for inhalation) pose further challenges of mAb instability, leading to degradation and reduced ecacy. [8,26,28]. Few biologics have been successfully approved for pulmonary delivery, most notably insulin formulation Afrezza (MannKind, Westlake Village, CA, USA), and, currently, an erythropoietin-Fc fusion and a nanobody-targeting respiratory syncytial virus are in clinical trials, showing promise for the further development of this administration strategy for both the systemic and localized delivery of mAb-based therapeutics [5,28]. Several drug carrier technologies such as microencapsulation, liposome, and nanoparticle formulations inherently enhance the stability and control the release of mAbs, which can prolong their half-life. These nanocarrier formulation strategies are of intense interest, as they hold promise for developing less invasive inhaled formulations of mAb-based therapeutics with superior attributes to currently established formulations [41,106–109]. The majority of currently approved mAb therapeutics are formulated for IV, although commercial interest has directed technologies to develop injectable mAb formulations which has seen success in many approved mAb therapies thus far, primarily SC for systemic delivery, along with a few intravitreal and intramuscular formulations for tissue-speciﬁc indications. Amongst others, anti-HER2 antibody trastuzumab was originally developed as an IV formulation and was successfully repurposed as a SC formulation [110–112], whereas next generation therapies have directly moved towards SC formulation, such as with anti-TNF- antibody adalimumab [113]. Injectable administrations oer several advantages over IV administration, especially in the treatment of chronic diseases in regards to a reduced burden to allied health services, patient tolerance, and adherence to treatment; however, the intrinsic physicochemical properties of mAbs may be undesirable for injectable formulation. In considering the low volume, injection pressure, and the typically high (>100 mg) eective dose of mAb for injectable delivery, the viscosity and aggregation propensity of mAbs become key formulation challenges to address, as they are dependent on mAb concentration. Certain mAb therapies are suitable, and others are not based on their solubility, viscosity, self-association, intrinsic stability, aggregation, and precipitation proﬁles. Injectable mAb formulations can be further improved with the use of excipients to increase solubility, reduce viscosity, and enhance the stability of mAbs. Excipients are considered for a formulation based on their physicochemical properties, pharmacokinetics, and safety. For example, polysorbates are commonly used in biologics as a stabilizing agent; however, their addition in high concentrations can denature proteins and cause adverse side eects such as injection site reactions [114–116]. Injectable mAb formulations are co-formulated with recombinant human hyaluronidase, speciﬁcally as a permeation enhancer for more ecient absorption into tissue, although the inclusion of this additional biologic adds further burden to the formulation’s viscosity and propensity to aggregate [5,8,25–28,117–123]. Antibody therapies for IV administration are prepared as Antibodies 2019, 8, 36 11 of 23 lyophilised powder for reconstitution and further dilution, and injectable administrations are prepared as liquid-based formulations in pre-ﬁlled syringes. Liquid formulations of mAbs are more susceptible to physiochemical degradation, are less stable, and have a reduced shelf-life as compared to lyophilised formulations. However, drying technologies to produce lyophilised formulations apply the mAb to physical stresses that induce instability and degradation, leading to reduced ecacy [124–126]. Long-term stability predictions are elucidated from formulation screening in accelerated aggregation studies, as part of the preliminary screening process of generated mAb libraries [127]. The stability proﬁles of mAbs can be greatly aected by the amino acid composition, structure, and potential glycosylation in their variable region CDRs that are isolated through the screening and maturation process. Elucidating the causes for reduced solubility or increased aggregation propensity has to be considered together with the molecular interactions between the mAb and the biological target as to not compromise anity. Computational tools have been developed to elucidate amino acids within the mAb CDR structure that have a high propensity to aggregate, and strategies for improving solubility and aggregation proﬁles have been developed through direct amino acid substitutions and the strategic addition of glycosylation sites [128–132]. The characterization of mAb stability and target interaction for optimization is considered a fundamental step as part of preliminary drug discovery, as the applicability for development, manufacture, and formulation of the mAb therapeutic is governed by the mAbs stability proﬁle. 5. Improving mAb Tissue Penetration for Cancer Treatment Antibody therapies are currently restricted to invasive parenteral routes of administration for maximum bioavailability and systemic distribution; however, delivery to the speciﬁc target tissue, such as tumors for cancer treatments, continues to be a challenge. Penetration to tissue from blood vessels is again poor, owing to mAbs’ polar surface charge and relatively large MW, limiting transport through physiological barriers. The eciency of tissue penetration is further inﬂuenced by systemic and local mAb clearance rates. Enhancement to mAbs’ anity to their biological target through maturation and strategic mutation technologies have led to faster diusion rates of mAb to target for increased ecacy. However, for tumourous tissue speciﬁcally, penetration through tumors is restricted by the tumor ’s binding site barrier, in which antigens expressed on the tumors periphery capture the majority of the mAb released to surrounding tissue; this eect is exaggerated with overexpressed antigen. The enhancement of whole mAbs’ anity beyond 1 nM has speciﬁcally shown that there is no further improvement of tumor diusion rates, tissue penetration, and accumulation [42,43,133]. An increased anity of mAbs to cellular targets also leads to the increased uptake, internalization, and catabolism of mAbs, which reduces ADCC and increases the mAb clearance rate. This mechanism is exploited through the development of high anity ADCs to target the delivery and release of potent drugs, inducing targeted cell death [43–45]. Fragment mAb platforms have shown better tissue penetration and biodistribution than whole mAb therapeutics; however, a pitfall of smaller peptides lacking an Fc region is a highly reduced in vivo half-life and poor retention times. Technologies to improve the half-life of mAb fragments have been primarily through PEGylation, along with strategic Fc mutation and glycosylation engineering to enhance neonatal Fc receptor (FcRn) recycling. Furthermore, hyper-glycosylation technology has been successfully applied in other biotherapeutics, such as with glycosylated erythropoietin Aranesp (Amgen, Thousand Oaks, CA, USA). Conversely, nanocarrier platforms that are relatively larger as compared to whole mAbs have demonstrated superior pharmacokinetics and tumor retention, as well as previously mentioned enhanced stability and the sustained, controlled release of mAbs [41]. These enhanced properties have generated considerable interest in developing the systemic delivery of mAb-nanoparticle platforms for superior tumor penetration, as well as targeted and sustained therapeutic drug delivery. Further to nanocarriers, additional formulation strategies developed for the controlled release of mAbs include hydrogels and crystalline antibodies, which have shown success as stable, injectable formulations for development [5,8,15,41–43,134–136]. Antibodies 2019, 8, 36 12 of 23 In addition, to further enhance the stability and half-life of other biotherapeutics, recombinant technologies allowed their fusion to mAb Fc as to introduce FcRn recycling as a protection mechanism, which has innovatively expanded the applicability of mAb-based therapeutic platforms. Notable examples include the TNF Receptor–Fc fusion protein Enbrel (Amgen, Thousand Oaks, CA, USA), which acts as an inhibitor to overexpressed TNF- in autoimmune diseases, and the Factor IX–Fc fusion protein Alprolix (Biogen, Cambridge, MA, USA), which is a blood factor supplement for hemophiliacs [11,46,133]. 6. Strategic Modulation of mAb Immune Eector Functions The modulation of mAbs eector functions through isotype switching, glycoengineering, and strategic mutations have proved advantageous for the development of more eective mAb treatment strategies. Antibody binding to Cq1 promotes the complement cascade, Fc R1A/B, Fc R2A, and Fc R3A/B receptors to activate immune eector functions; Fc R2B counter-balances the eector response, and FcRn prolongs mAb half-life. Binding to these receptors is primarily done through sites in the C , C 2, and the conserved glycosylation region in the Fc (N297). hinge H IgG3 does not eciently bind to FcRn, which reduces its half-life to approximately seven days, as opposed to a 21 day half-life for the other IgG isotypes. In most instances of mAb design, an extended half-life is preferred as to prolong the eective dose of a mAb in serum [15]. IgG2 and IgG4 mAbs have a reduced eector function as compared to IgG1 and are thus used in instances where minimal engagement to the immune system is warranted to increase safety of the mAb therapy—with ADCs reducing o-target cytotoxicity, for instance. Eculizumab (Soliris , Alexion Pharmaceuticals, New Haven, CT, USA) is the ﬁrst (and thus far only) approved recombinant IgG 2(C 1/C )–4(C 2/C 3) H hinge H H hybrid whole mAb. Further cross-sub-class variant mAb therapies are in development, with key amino acid substitutions from the IgG2 and IgG4 sub-classes introduced for intentionally suppressed eector functions [4,13]. The removal of the conserved glycosylation site through amino acid substitution of N297 or T299 (aglycosylation) has also demonstrated reduced eector function; however, this happens at the expense of mAb stability and is therefore not a suitable strategy for whole mAb therapeutics [11,14,15,137–140]. On the other hand, enhancing ADCC and CDC is useful for engaging the immune system to tumor tissues in the absence of a conjugated cytotoxic drug. Certain glycoengineered modiﬁcations to the conserved glycosylation region in the Fc, such as deﬁciency in core fucose (afucosylation) and hyper-galactosylation, have demonstrated enhanced mAb binding to Fc R3A and Cq1, speciﬁcally for an enhanced ADCC and CDC eect [13,43]. Afucosylated mAbs benralizumab (Fasenra—, AstraZeneca, London, UK) and mogamulizumab (Poteligeo , Kyowa Hakko Kirin, Tokyo, Japan), as well as low fucose content mAb obinutuzumab (Gazyva , Genentech, San Francisco, CA, USA), are currently approved therapies, with several further in development (along with clinical trials), which demonstrates the commercial interest and applicability of this technology in improving mAb therapeutics through enhancing ADCC. However, the signiﬁcance of manipulating sialylation in mAb therapeutics is a controversial topic, with several reports demonstrating a reduction in ADCC and CDC and others reporting no observable dierence. This highlights a clear need to characterize and deﬁne the in vivo ecacy of such manipulation [11,13,14,43]. No currently approved mAb therapies contain amino acid substitutions for an improved half-life through modulated binding properties to FcRn, improved CDC through binding to complement factor Cq1, or improved ADCC through enhanced binding anity to Fc R1A and 3A. However, several mutations have been reported, patented, and are in clinical development, demonstrating the commercial interest in this technology for improving mAb therapeutics [6,11,13,15,133,137,140–143]. For a more comprehensive understanding of mAb engineering strategies for modulated immune eector functions, we direct the reader to the following reviews [13–15,140]. Antibodies 2019, 8, 36 13 of 23 7. Computational Approaches for Aggregation Prediction and Rational Design of mAbs The advancement of in silico analysis of mAb peptide sequences, structures, conformation, and their associated biological interaction has been integral to the development of various computational tools for characterizing, designing, and optimizing mAb-based therapeutics. The generation and continued pursuit of mAb structural data for in silico analysis has led to the development of several integral databases, notably IMGT , serving as a key resource for data mining [144–146]. Molecular dynamic (MD) simulation analysis has driven the computational analysis of the discovery and characterization of relevant molecular interactions within the mAb molecule, mAb binding to biological targets and mAb surface association with the surrounding environment. These interactions can infer intrinsic stability, target binding associations, solubility and aggregation propensity of the mAb. MD simulations and free energy calculations from crystal structures remain key for the highly speciﬁc elucidation of mAb-target intermolecular interactions that correlate to binding anity, as signiﬁcant interactions such as hydrogen-bond formation can be predicted and determine the strength of the molecular associations [147]. However, elucidation of mAb self-association, solubility, and aggregation propensity have been driven by the development of computational modelling and simulation tools that simultaneously analyses mAb topography and surface polarity. Notably, AGGRESCAN3D, TANGO, and PASTA are the most prominent tools for predicting the site speciﬁc aggregation propensity of mAbs [35,148]. The preliminary elucidation of mAb structural data—that being amino acid sequences and higher order structure (HOS)—is experimentally derived to produce crystal structures that model the solid-state 3D structure of the mAb. Mass spectrometry technologies have come of age to produce high throughput and orthogonal analysis to elucidate mAb peptide sequences, oxidation, deamidation, and glycosylation heterogeneity, as well as, more recently, for native, destabilized, and aggregated HOS elucidation [149,150]. X-ray crystallography technologies have been the underlying workhorse for elucidating the crystal structure of mAbs. Producing crystalline mAbs is challenging, owing to the complexity of mAb HOS and the degree of mAb conformational heterogeneity. However, several complementing technologies have evolved in recent years to ascertain structure and interactions, including circular dichroism (CD), infrared (IR) and raman spectroscopy, cryogenic-electron microscopy, and nuclear magnetic resonance (NMR) spectroscopy [148,150–157]. Of the technologies available for HOS elucidation, 2D-NMR and X-Ray crystallography (followed by MS) provide the highest sensitivity and local speciﬁcity. In comparison, CD, IR, and raman are high-throughput methods, although they have much lower sensitivity [150,157]. Thus far, only four whole IgG antibodies have a successfully determined crystal structure (PDB ID: 1HZH, 1IGT, 1IGY, and 5DK3), notably 1HZH as a wholly human IgG1 and 5DK3 as a humanized IgG4/, both of which have been used as model structures for MD simulations [158]. However, fragment mAbs yield much higher success with producing crystal structures, which has led to much pursuit and contribution in generating mAb fragments and targetting complexing crystal structure libraries for data mining and in silico analysis [159,160]. Upon obtaining relevant crystal structures for a candidate mAb, detailed crystal structure and MD simulation analysis has been extensively used to elucidate the mAbs intramolecular interactions that confer intrinsic stability and intermolecular interactions to confer interactions to biological targets and self-association. The particular binding interface of interest is analyzed, and amino acids in the mAb structure are identiﬁed for substitution that can disrupt molecular interactions in the interface in the instance of diminishing target binding and identify potentially unutilized bonding sites and polarity mismatches in the interface that can be improved in the instance of enhancing target binding. MD simulations are then carried out for the native and substituted mAb structures to elucidate any relevant bond formations, interactions, and more favorable binding free energy produced by the substituted structure, of which promising candidates can be synthesized and validated through in vitro analysis. This predictive approach has been successfully applied to identify mutations in mAbs for modulating eector functions, target binding, optimizing anity capture for manufacturing, and, more Antibodies 2019, 8, 36 14 of 23 recently, for designing antibodies de novo from targets of interest [11,161–165]. Mutations have been speciﬁcally identiﬁed for enhanced binding to Fc R2A, Fc R3A, and Cq1, as well as a reduced binding to Fc R2B for a more pronounced ADCC and CDC eect, an enhanced binding to FcRn for an extended half-life, and a reduced binding to Fc Rs and Cq1 for a diminished ADCC and CDC eect, all of which are transferrable to mAbs of the same isotype sub-class [137,141,142]. Further to this, the molecular interactions of glycoengineered mAb variants to FcRs have been characterized through MD simulations to corroborate predictions with the observed modulated eector functions [13,15,137,141,142]. Self-association, solubility, and aggregation propensity are further evaluated by computational modelling tools that speciﬁcally characterize the topography and surface polarity of mAbs, concurrently analyzing the local spatial arrangement of amino acids in the mAb structure, solvent accessibility, and local surface charge [5]. In particular, surface exposed hydrophobicity is sought as the lead mechanism for protein self-association driving aggregation propensity, in which aggregation-prone regions (APRs) are identiﬁed in the mAb structure. The substitution of identiﬁed APRs to gatekeeper (i.e., polar) amino acids has experimentally demonstrated resistance to aggregation and improved solubility, which validates this strategy for improving mAb stability [36–40,166–170]. The majority of APRs identiﬁed are located in biologically relevant mAb regions—those being the CDRs and the Fc. Speciﬁcally targeting these APRs through mutation may disrupt important biological functions and mAb structure. Analyses of the mAbs intermolecular interactions are necessary to validate the intrinsic stability of substituted mAb variants; if conformational ﬂuctuations are elucidated, then the biologically active interface is likely disrupted. Interestingly, mutations for enhancing biological functions have demonstrated a negative impact on mAb solubility and stability, further suggesting that the self-association proﬁle of mAbs is linked to its biological activity [36,128,171]. Aside from direct residue substitution in mAb structures, other strategies reported to profoundly interfere with the eects of APRs include isotype switching and the strategic addition of N-linked glycans [39,128,172,173]. Additionally, N-linked glycosylation in the CDR regions of mAbs, although uncommon, has demonstrated improved solubility and stability proﬁles of mAbs without impacting their target anity, as compared to the same mAbs with removed CDR glycans [128,174]. The rationale behind strategic glycan addition is based on the election of N-linked glycosylation sites that have apparent spacial proximity to APRs, with the introduced glycan therefore sterically hindering the APR from self-association interactions. The beneﬁts of extending mAb half-life with strategic glycan addition, as seen with hyper-glycosylated biotherapeutics (as well as improving mAb solubility and stability for improved formulation strategies) has yet to be realized and suggests a very intriguing and highly relevant technology for perusal. 8. Concluding Remarks Therapeutic antibodies have come of age as continuing to be a key, dominant technology in the biopharmaceutical industry. The repurpose of antibodies to many formats have made them versatile to design and tailor highly specialized treatments, including ADCs as a targeted drug delivery system, bispeciﬁc and fragment mAb platforms for tailored engagement and increased bioavailability, and recombinant Fc-fusion proteins for an increased half-life and introduced immunological engagement. Further advancements include the modulation of Fc eector functions through manipulations of the Fc, either through isotype switching, glycoengineering, or strategic mutations in the Fc region, along with the PEGylation of fragment mAbs for enhanced half-life. Advancements in discovery, manufacture, and formulation technologies have further propelled the success of therapeutic antibodies, notably through expression system development and the transition from IV to SC formulations. Human-based expression systems have been extensively used in mAb development and are becoming an accepted manufacturing platform for mAb therapeutics. Furthermore, cell-free synthesis technology is giving rise to the potential for higher eciency in the manufacture process. Though developments for further generation antibody therapies have led to great strides in producing improved therapeutic outcomes, many facets of the manufacture process and formulation Antibodies 2019, 8, 36 15 of 23 development strategy pose as challenges to be considered. Antibody-based therapies are susceptible to chemical and enzymatic degradation through oral, nasal, or pulmonary routes of administration and are therefore currently restricted IV or SC delivery. Despite achieving maximum bioavailability through IV/SC administration, tissue penetration of mAb-based therapies is poor, which limits their local bioavailability, requiring high concentrations to achieve an eective dose. The stability of mAbs-based therapeutics is a highly pronounced and recurring challenge to be considered, as it aects manufacture yield and formulation considerations. Several strategies are in development to improve the stability of mAbs in order to potentially produce formulations for pulmonary or oral administration. Notably, computational tools have come of age, complementing experimental techniques to derive antibody structure and aggregation prediction. Through these methods, the stability and aggregation propensity of mAb-based therapies have demonstrated improvement through rational mutation and glycosylation within the framework region, which is potentially translatable to all mAbs within the same isotype. Furthermore, nanocarrier technologies have been shown to enhance the stability and potentially control the release of mAbs. The reﬁnement of rational mAb design coupled with nanocarrier technologies has the potential to overcome these challenges, to develop superior treatment strategies, and ultimately to formulate for non-invasive administration routes such as pulmonary delivery. Author Contributions: V.S. revised the ﬁgure and prepared the scope, literature review, and original manuscript draft. E.C prepared the original ﬁgure and reviewed the manuscript. B.E. and V.K. equally reviewed and edited the manuscript. Funding: This research received no external funding. Conﬂicts of Interest: The authors declare no conﬂict of interest. References 1. Urquhart, L. Top drugs and companies by sales in 2017. Nat. Rev. Drug Discov. 2018, 17, 232. [CrossRef] [PubMed] 2. Ecker, D.; Dana Jones, S.; Levine, H.L. The therapeutic monoclonal antibody market. mAbs 2015, 7, 9–14. [CrossRef] [PubMed] 3. An, Z. “Magic bullets” at the center stage of immune therapy: A special issue on therapeutic antibodies. Protein Cell 2018, 9, 1–2. [CrossRef] [PubMed] 4. Santos, M.L.d.; Quintilio, W.; Manieri, T.M.; Tsuruta, L.R.; Moro, A.M. Advances and challenges in therapeutic monoclonal antibodies drug development. Bras. J. Pharm. Sci. 2018, 54. [CrossRef] 5. Elgundi, Z.; Reslan, M.; Cruz, E.; Sifniotis, V.; Kayser, V. The state-of-play and future of antibody therapeutics. Adv. Drug Del. Rev. 2017, 122, 2–19. [CrossRef] 6. Almagro, J.C.; Daniels-Wells, T.R.; Perez-Tapia, S.M.; Penichet, M.L. Progress and challenges in the design and clinical development of antibodies for cancer therapy. Front. Immunol. 2018, 8, 1751. [CrossRef] [PubMed] 7. Liu, B.N.; Luo, J.H. Research and development of innovative antibody-based drugs. Yaoxue Xuebao 2017, 52, 1811–1819. 8. Awwad, S.; Angkawinitwong, U. Overview of antibody drug delivery. Pharmaceutics 2018, 10, 83. [CrossRef] [PubMed] 9. Klein, C. Special issue: Monoclonal antibodies. Antibodies 2018, 7, 17. [CrossRef] 10. Wang, C.; Xu, P.; Zhang, L.; Huang, J.; Zhu, K.; Luo, C. Current strategies and applications for precision drug design. Front. Pharmacol. 2018, 9, 787. [CrossRef] [PubMed] 11. Strohl, W.R. Current progress in innovative engineered antibodies. Protein Cell 2018, 9, 86–120. [CrossRef] [PubMed] 12. Homann, R.M.; Coumbe, B.G.T.; Josephs, D.H.; Mele, S.; Ilieva, K.M.; Cheung, A.; Tutt, A.N.; Spicer, J.F.; Thurston, D.E.; Crescioli, S.; et al. Antibody structure and engineering considerations for the design and function of antibody drug conjugates (adcs). OncoImmunology 2018, 7, e1395127. [CrossRef] [PubMed] 13. Wang, X.; Mathieu, M.; Brezski, R.J. Igg fc engineering to modulate antibody eector functions. Protein Cell 2018, 9, 63–73. [CrossRef] [PubMed] Antibodies 2019, 8, 36 16 of 23 14. Pawlowski, J.W.; Bajardi-Taccioli, A.; Houde, D.; Feschenko, M.; Carlage, T.; Kaltashov, I.A. Inﬂuence of glycan modiﬁcation on igg1 biochemical and biophysical properties. J. Pharm. Biomed. Anal. 2018, 151, 133–144. [CrossRef] [PubMed] 15. Fonseca, M.H.G.; Furtado, G.P.; Bezerra, M.R.L.; Pontes, L.Q.; Fernandes, C.F.C. Boosting half-life and eector functions of therapeutic antibodies by fc-engineering: An interaction-function review. Int. J. Biol. Macromol. 2018, 119, 306–311. [CrossRef] [PubMed] 16. Mizukami, A.; Caron, A.L.; Picanço-Castro, V.; Swiech, K. Platforms for recombinant therapeutic glycoprotein production. In Recombinant Glycoprotein Production; Humana Press Inc.: New York, NY, USA, 2018; Volume 1674, pp. 1–14. 17. Thomson, C.A. Igg structure and function. In Encyclopedia of Immunobiology; Elsevier Inc.: Amsterdam, The Netherlands, 2016; Volume 2, pp. 15–22. 18. Kayser, V.; Chennamsetty, N.; Voynov, V.; Forrer, K.; Helk, B.; Trout, B.L. Glycosylation inﬂuences on the aggregation propensity of therapeutic monoclonal antibodies. Biotechnol. J. 2011, 6, 38–44. [CrossRef] 19. Pieracci, J.P.; Armando, J.W.; Westoby, M.; Thommes, J. Chapter 9—Industry review of cell separation and product harvesting methods. In Biopharmaceutical Processing; Jagschies, G., Lindskog, E., Łacki, ˛ K., Galliher, P., Eds.; Elsevier: Amsterdam, The Netherlands, 2018; pp. 165–206. 20. Huang, C.-J.; Lin, H.; Yang, X. Industrial production of recombinant therapeutics in escherichia coli and its recent advancements. J. Ind. Microbiol. Biotechnol. 2012, 39, 383–399. [CrossRef] 21. Behme, S. Manufacturing of Pharmaceutical Proteins: From Technology to Economy: Second, Revised and Expanded Edition; Wiley: Hoboken, NJ, USA, 2015; pp. 1–427. 22. Ulmer, N.; Vogg, S.; Müller-Späth, T.; Morbidelli, M. Chapter 7—Puriﬁcation of human monoclonal antibodies and their fragments. In Human monoclonal Antibodies: Methods and Protocols; Steinitz, M., Ed.; Springer: New York, NY, USA, 2019; pp. 163–188. 23. Arosio, P.; Barolo, G.; Müller-Späth, T.; Wu, H.; Morbidelli, M. Aggregation stability of a monoclonal antibody during downstream processing. Pharm. Res. 2011, 28, 1884–1894. [CrossRef] 24. Kayser, V.; Chennamsetty, N.; Voynov, V.; Helk, B.; Trout, B.L. Conformational stability and aggregation of therapeutic monoclonal antibodies studied with ans and thioﬂavin t binding. mAbs 2011, 3, 408–411. [CrossRef] 25. Bittner, B.; Richter, W.; Schmidt, J. Subcutaneous administration of biotherapeutics: An overview of current challenges and opportunities. Biodrugs 2018, 32, 425–440. [CrossRef] 26. Viola, M.; Sequeira, J.; Seiça, R.; Veiga, F.; Serra, J.; Santos, A.C.; Ribeiro, A.J. Subcutaneous delivery of monoclonal antibodies: How do we get there? J. Control. Release 2018, 286, 301–314. [CrossRef] [PubMed] 27. Otvos, L. 6.05—Basic principles of formulation for biotherapeutics: Approaches to alternative drug delivery. In Comprehensive Medicinal Chemistry III; Chackalamannil, S., Rotella, D., Ward, S.E., Eds.; Elsevier: Oxford, UK, 2017; pp. 131–156. 28. Morales, J.O.; Fathe, K.R.; Brunaugh, A.; Ferrati, S.; Li, S.; Montenegro-Nicolini, M.; Mousavikhamene, Z.; McConville, J.T.; Prausnitz, M.R.; Smyth, H.D.C. Challenges and future prospects for the delivery of biologics: Oral mucosal, pulmonary, and transdermal routes. AAPS J. 2017, 19, 652–668. [CrossRef] [PubMed] 29. Muheem, A.; Shakeel, F.; Jahangir, M.A.; Anwar, M.; Mallick, N.; Jain, G.K.; Warsi, M.H.; Ahmad, F.J. A review on the strategies for oral delivery of proteins and peptides and their clinical perspectives. Saudi Pharm. J. 2016, 24, 413–428. [CrossRef] [PubMed] 30. Hunter, M.; Yuan, P.; Vavilala, D.; Fox, M. Optimization of protein expression in mammalian cells. Curr. Protoc. Protein Sci. 2019, 95, e77. [CrossRef] [PubMed] 31. Lindskog, E.K.; Fischer, S.; Wenger, T.; Schulz, P. Chapter 6—Host cells. In Biopharmaceutical Processing; Jagschies, G., Lindskog, E., Łacki, ˛ K., Galliher, P., Eds.; Elsevier: Amsterdam, The Netherlands, 2018; pp. 111–130. 32. Fliedl, L.; Grillari, J.; Grillari-Voglauer, R. Human cell lines for the production of recombinant proteins: On the horizon. New Biotechnol. 2015, 32, 673–679. [CrossRef] 33. Matsuda, T.; Ito, T.; Takemoto, C.; Katsura, K.; Ikeda, M.; Wakiyama, M.; Kukimoto-Niino, M.; Yokoyama, S.; Kurosawa, Y.; Shirouzu, M. Cell-free synthesis of functional antibody fragments to provide a structural basis for antibody–antigen interaction. PLoS ONE 2018, 13, e0193158. [CrossRef] 34. Stech, M.; Kubick, S. Cell-free synthesis meets antibody production: A review. Antibodies 2015, 4, 12. [CrossRef] Antibodies 2019, 8, 36 17 of 23 35. Pujols, J.; Peña-Díaz, S.; Ventura, S. Aggrescan3d: Toward the prediction of the aggregation propensities of protein structures. In Methods Mol. Biol.; Humana Press Inc.: New York, NY, USA, 2018; Volume 1762, pp. 427–443. 36. Van der Kant, R.; Karow-Zwick, A.R.; Van Durme, J.; Blech, M.; Gallardo, R.; Seeliger, D.; Aßfalg, K.; Baatsen, P.; Compernolle, G.; Gils, A.; et al. Prediction and reduction of the aggregation of monoclonal antibodies. J. Mol. Biol. 2017, 429, 1244–1261. [CrossRef] 37. Lerch, T.F.; Sharpe, P.; Mayclin, S.J.; Edwards, T.E.; Lee, E.; Conlon, H.D.; Polleck, S.; Rouse, J.C.; Luo, Y.; Zou, Q. Inﬂiximab crystal structures reveal insights into self-association. mAbs 2017, 9, 874–883. [CrossRef] 38. Dobson, C.L.; Devine, P.W.A.; Phillips, J.J.; Higazi, D.R.; Lloyd, C.; Popovic, B.; Arnold, J.; Buchanan, A.; Lewis, A.; Goodman, J.; et al. Engineering the surface properties of a human monoclonal antibody prevents self-association and rapid clearance in vivo. Sci. Rep. 2016, 6, 38644. [CrossRef] 39. Courtois, F.; Agrawal, N.J.; Lauer, T.M.; Trout, B.L. Rational design of therapeutic mabs against aggregation through protein engineering and incorporation of glycosylation motifs applied to bevacizumab. mAbs 2016, 8, 99–112. [CrossRef] 40. Courtois, F.; Schneider, C.P.; Agrawal, N.J.; Trout, B.L. Rational design of biobetters with enhanced stability. J. Pharm. Sci. 2015, 104, 2433–2440. [CrossRef] 41. Yu, M.; Wu, J.; Shi, J.; Farokhzad, O.C. Nanotechnology for protein delivery: Overview and perspectives. J. Control. Release 2016, 240, 24–37. [CrossRef] 42. Shi, J.; Kanto, P.W.; Wooster, R.; Farokhzad, O.C. Cancer nanomedicine: Progress, challenges and opportunities. Nat. Rev. Cancer 2016, 17, 20. [CrossRef] 43. Dalziel, M.; Beers, S.A.; Cragg, M.S.; Crispin, M. Through the barricades: Overcoming the barriers to eective antibody-based cancer therapeutics. Glycobiology 2018, 28, 697–712. [CrossRef] 44. Lambert, J.M.; Berkenblit, A. Antibody-drug conjugates for cancer treatment. Annu. Rev. Med. 2018, 69, 191–207. [CrossRef] 45. Lambert, J.M.; Morris, C.Q. Antibody–drug conjugates (adcs) for personalized treatment of solid tumors: A review. Adv. Ther. 2017, 34, 1015–1035. [CrossRef] 46. Levin, D.; Golding, B.; Strome, S.E.; Sauna, Z.E. Fc fusion as a platform technology: Potential for modulating immunogenicity. Trends Biotechnol. 2015, 33, 27–34. [CrossRef] 47. Henry, K.A. Next-generation DNA sequencing of vh/vl repertoires: A primer and guide to applications in single-domain antibody discovery. In Phage Display: Methods and Protocols; Hust, M., Lim, T.S., Eds.; Springer: New York, NY, USA, 2018; pp. 425–446. 48. Frenzel, A.; Roskos, L.; Klakamp, S.; Liang, M.; Arends, R.; Green, L. Antibody anity. In Handbook of Therapeutic Antibodies, 2nd ed.; Wiley Blackwell: Hoboken, NJ, USA, 2014; Volume 1–4, pp. 115–140. 49. Hu, J.; Han, J.; Li, H.; Zhang, X.; Liu, L.; Chen, F.; Zeng, B. Human embryonic kidney 293 cells: A vehicle for biopharmaceutical manufacturing, structural biology, and electrophysiology. Cells Tissues Organs 2018, 205, 1–8. [CrossRef] 50. Jacobi, A.; Enenkel, B.; Garidel, P.; Eckermann, C.; Knappenberger, M.; Presser, I.; Kaufmann, H. Process development and manufacturing of therapeutic antibodies. In Handbook of Therapeutic Antibodies, 2nd ed.; Wiley Blackwell: Hoboken, NJ, USA, 2014; Volume 2–4, pp. 601–664. 51. Kayser, V.; Chennamsetty, N.; Voynov, V.; Helk, B.; Forrer, K.; Trout, B.L. A screening tool for therapeutic monoclonal antibodies: Identifying the most stable protein and its best formulation based on thioﬂavin t binding. Biotechnol. J. 2012, 7, 127–132. [CrossRef] 52. Tada, M.; Tatematsu, K.-I.; Ishii-Watabe, A.; Harazono, A.; Takakura, D.; Hashii, N.; Sezutsu, H.; Kawasaki, N. Characterization of anti-cd20 monoclonal antibody produced by transgenic silkworms (bombyx mori). mAbs 2015, 7, 1138–1150. [CrossRef] 53. Maccani, A.; Landes, N.; Stadlmayr, G.; Maresch, D.; Leitner, C.; Maurer, M.; Gasser, B.; Ernst, W.; Kunert, R.; Mattanovich, D. Pichia pastoris secretes recombinant proteins less eciently than chinese hamster ovary cells but allows higher space-time yields for less complex proteins. Biotechnol. J. 2014, 9, 526–537. [CrossRef] 54. Frenzel, A.; Hust, M.; Schirrmann, T. Expression of recombinant antibodies. Front. Immunol. 2013, 4, 217. [CrossRef] 55. Thoring, L.; Kubick, S. Versatile cell-free protein synthesis systems based on chinese hamster ovary cells. In Recombinant Protein Expression in Mammalian Cells: Methods and Protocols; Hacker, D.L., Ed.; Springer: New York, NY, USA, 2018; pp. 289–308. Antibodies 2019, 8, 36 18 of 23 56. Tran, K.; Gurramkonda, C.; Cooper, M.A.; Pilli, M.; Taris, J.E.; Selock, N.; Han, T.-C.; Tolosa, M.; Zuber, A.; Peñalber-Johnstone, C.; et al. Cell-free production of a therapeutic protein: Expression, puriﬁcation, and characterization of recombinant streptokinase using a cho lysate. Biotechnol. Bioeng. 2018, 115, 92–102. [CrossRef] 57. Stech, M.; Nikolaeva, O.; Thoring, L.; Stöcklein, W.F.M.; Wüstenhagen, D.A.; Hust, M.; Dübel, S.; Kubick, S. Cell-free synthesis of functional antibodies using a coupled in vitro transcription-translation system based on cho cell lysates. Sci. Rep. 2017, 7, 12030. [CrossRef] 58. Li, J.; Wang, H.; Kwon, Y.-C.; Jewett, M.C. Establishing a high yielding streptomyces-based cell-free protein synthesis system. Biotechnol. Bioeng. 2017, 114, 1343–1353. [CrossRef] 59. Jaroentomeechai, T.; Stark, J.C.; Natarajan, A.; Glasscock, C.J.; Yates, L.E.; Hsu, K.J.; Mrksich, M.; Jewett, M.C.; DeLisa, M.P. Single-pot glycoprotein biosynthesis using a cell-free transcription-translation system enriched with glycosylation machinery. Nat. Commun. 2018, 9, 2686. [CrossRef] 60. Gurramkonda, C.; Rao, A.; Borhani, S.; Pilli, M.; Deldari, S.; Ge, X.; Pezeshk, N.; Han, T.-C.; Tolosa, M.; Kostov, Y.; et al. Improving the recombinant human erythropoietin glycosylation using microsome supplementation in cho cell-free system. Biotechnol. Bioeng. 2018, 115, 1253–1264. [CrossRef] 61. Nakamura, T.; Omasa, T. Optimization of cell line development in the gs-cho expression system using a high-throughput, single cell-based clone selection system. J. Biosci. Bioeng. 2015, 120, 323–329. [CrossRef] 62. Zhou, Y.; Raju, R.; Alves, C.; Gilbert, A. Debottlenecking protein secretion and reducing protein aggregation in the cellular host. Curr. Opin. Biotechnol. 2018, 53, 151–157. [CrossRef] 63. Inwood, S.; Betenbaugh, M.J.; Lal, M.; Shiloach, J. Genome-wide high-throughput rnai screening for identiﬁcation of genes involved in protein production. In Recombinant Protein Expression in Mammalian Cells: Methods and Protocols; Hacker, D.L., Ed.; Springer: New York, NY, USA, 2018; pp. 209–219. 64. Dangi, A.K.; Sinha, R.; Dwivedi, S.; Gupta, S.K.; Shukla, P. Cell line techniques and gene editing tools for antibody production: A review. Front. Pharmacol. 2018, 9, 630. [CrossRef] 65. Delic, M.; Göngrich, R.; Mattanovich, D.; Gasser, B. Engineering of protein folding and secretion—Strategies to overcome bottlenecks for ecient production of recombinant proteins. Antioxid. Redox Signal. 2014, 21, 414–437. [CrossRef] 66. Zhong, X.; Wright, J.F. Biological insights into therapeutic protein modiﬁcations throughout tracking and their biopharmaceutical applications. Int. J. Cell Biol. 2013, 2013, 19. [CrossRef] 67. Altamirano, C.; Berrios, J.; Vergara, M.; Becerra, S. Advances in improving mammalian cells metabolism for recombinant protein production. Electron. J. Biotechnol. 2013, 16. [CrossRef] 68. Parola, C.; Mason, D.M.; Zingg, A.; Neumeier, D.; Reddy, S.T. Genome engineering of hybridomas to generate stable cell lines for antibody expression. In Recombinant Protein Expression in Mammalian Cells: Methods and Protocols; Hacker, D.L., Ed.; Springer: New York, NY, USA, 2018; pp. 79–111. 69. You, M.; Yang, Y.; Zhong, C.; Chen, F.; Wang, X.; Jia, T.; Chen, Y.; Zhou, B.; Mi, Q.; Zhao, Q.; et al. Ecient mab production in cho cells with optimized signal peptide, codon, and utr. Appl. Microbiol. Biotechnol. 2018, 102, 5953–5964. [CrossRef] 70. Mauro, V.P.; Chappell, S.A. Considerations in the use of codon optimization for recombinant protein expression. In Recombinant Protein Expression in Mammalian Cells: Methods and Protocols; Hacker, D.L., Ed.; Springer: New York, NY, USA, 2018; pp. 275–288. 71. Michael, I.P.; Nagy, A. Inducible protein production in 293 cells using the piggybac transposon system. In Recombinant Protein Expression in Mammalian Cells: Methods and Protocols; Hacker, D.L., Ed.; Springer: New York, NY, USA, 2018; pp. 57–68. 72. Balasubramanian, S. Recombinant cho cell pool generation using piggybac transposon system. In Recombinant Protein Expression in Mammalian Cells: Methods and Protocols; Hacker, D.L., Ed.; Springer: New York, NY, USA, 2018; pp. 69–78. 73. Jäger, V.; Büssow, K.; Schirrmann, T. Transient recombinant protein expression in mammalian cells. In Animal Cell Culture; Al-Rubeai, M., Ed.; Springer International Publishing: Cham, Switzerland, 2015; pp. 27–64. 74. Wijesuriya, S.D.; Pongo, E.; Tomic, M.; Zhang, F.; Garcia-Rodriquez, C.; Conrad, F.; Farr-Jones, S.; Marks, J.D.; Horwitz, A.H. Antibody engineering to improve manufacturability. Protein Expression Purif. 2018, 149, 75–83. [CrossRef] Antibodies 2019, 8, 36 19 of 23 75. L’Abbé, D.; Bisson, L.; Gervais, C.; Grazzini, E.; Durocher, Y. Transient gene expression in suspension hek293-ebna1 cells. In Recombinant Protein Expression in Mammalian Cells: Methods and Protocols; Hacker, D.L., Ed.; Springer: New York, NY, USA, 2018; pp. 1–16. 76. Arena, T.A.; Harms, P.D.; Wong, A.W. High throughput transfection of hek293 cells for transient protein production. In Recombinant Protein Expression in Mammalian Cells: Methods and Protocols; Hacker, D.L., Ed.; Springer: New York, NY, USA, 2018; pp. 179–187. 77. Agrawal, V.; Yu, B.; Pagila, R.; Yang, B.; Simonsen, C.; Beske, O. A high-yielding, cho-k1-based transient transfection system. BioProcess Int. 2013, 11, 28–35. 78. Hacker, D.L.; Kiseljak, D.; Rajendra, Y.; Thurnheer, S.; Baldi, L.; Wurm, F.M. Polyethyleneimine-based transient gene expression processes for suspension-adapted hek-293e and cho-dg44 cells. Protein Expression Purif. 2013, 92, 67–76. [CrossRef] 79. Ritacco, F.V.; Wu, Y.; Khetan, A. Cell culture media for recombinant protein expression in chinese hamster ovary (cho) cells: History, key components, and optimization strategies. Biotechnol. Prog. 2018, 34, 1407–1426. [CrossRef] 80. Whitford, W.G.; Lundgren, M.; Fairbank, A. Chapter 8—Cell culture media in bioprocessing. In Biopharmaceutical Processing; Jagschies, G., Lindskog, E., Łacki, ˛ K., Galliher, P., Eds.; Elsevier: Amsterdam, The Netherlands, 2018; pp. 147–162. 81. Davami, F.; Eghbalpour, F.; Barkhordari, F.; Mahboudi, F. Eect of peptone feeding on transient gene expression process in cho dg44. Avicenna J. Med. Biotechnol. 2014, 6, 147–155. 82. Mahboudi, F.; Abolhassan, M.R.; Azarpanah, A.; Aghajani-Lazarjani, H.; Sadeghi-Haskoo, M.A.; Maleknia, S.; Vaziri, B. The role of dierent supplements in expression level of monoclonal antibody against human cd20. Avicenna J. Med. Biotechnol. 2013, 5, 140–147. 83. You, M.; Liu, Y.; Chen, Y.; Guo, J.; Wu, J.; Fu, Y.; Shen, R.; Qi, R.; Luo, W.; Xia, N. Maximizing antibody production in suspension-cultured mammalian cells by the customized transient gene expression method. Biosci. Biotechnol. Biochem. 2013, 77, 1207–1213. [CrossRef] 84. Backliwal, G.; Hildinger, M.; Kuettel, I.; Delegrange, F.; Hacker, D.L.; Wurm, F.M. Valproic acid: A viable alternative to sodium butyrate for enhancing protein expression in mammalian cell cultures. Biotechnol. Bioeng. 2008, 101, 182–189. [CrossRef] 85. Elgundi, Z.; Sifniotis, V.; Reslan, M.; Cruz, E.; Kayser, V. Laboratory scale production and puriﬁcation of a therapeutic antibody. JoVE 2017. [CrossRef] 86. Kim, S.J.; Ha, G.S.; Lee, G.; Lim, S.I.; Lee, C.M.; Yang, Y.H.; Lee, J.; Kim, J.E.; Lee, J.H.; Shin, Y.; et al. Enhanced expression of soluble antibody fragments by low-temperature and overdosing with a nitrogen source. Enzyme Microb. Technol. 2018, 115, 9–15. [CrossRef] 87. Lalonde, M.-E.; Durocher, Y. Therapeutic glycoprotein production in mammalian cells. J. Biotechnol. 2017, 251, 128–140. [CrossRef] 88. Rajendra, Y.; Kiseljak, D.; Baldi, L.; Hacker, D.L.; Wurm, F.M. A simple high-yielding process for transient gene expression in cho cells. J. Biotechnol. 2011, 153, 22–26. [CrossRef] 89. Codamo, J.; Munro, T.P.; Hughes, B.S.; Song, M.; Gray, P.P. Enhanced cho cell-based transient gene expression with the epi-cho expression system. Mol. Biotechnol. 2011, 48, 109–115. [CrossRef] 90. Codamo, J.; Hou, J.J.C.; Hughes, B.S.; Gray, P.P.; Munro, T.P. Ecient mab production in cho cells incorporating pei-mediated transfection, mild hypothermia and the co-expression of xbp-1. J. Chem. Technol. Biotechnol. 2011, 86, 923–934. [CrossRef] 91. Castan, A.; Schulz, P.; Wenger, T.; Fischer, S. Chapter 7—Cell line development. In Biopharmaceutical Processing; Jagschies, G., Lindskog, E., Łacki, ˛ K., Galliher, P., Eds.; Elsevier: Amsterdam, The Netherlands, 2018; pp. 131–146. 92. Lindskog, E.K. Chapter 31—The upstream process: Principal modes of operation. In Biopharmaceutical Processing; Jagschies, G., Lindskog, E., Łacki, ˛ K., Galliher, P., Eds.; Elsevier: Amsterdam, The Netherlands, 2018; pp. 625–635. 93. Fisher, A.C.; Kamga, M.-H.; Agarabi, C.; Brorson, K.; Lee, S.L.; Yoon, S. The current scientiﬁc and regulatory landscape in advancing integrated continuous biopharmaceutical manufacturing. Trends Biotechnol. 2018, 37, 253–267. [CrossRef] 94. Rathore, A.S.; Agarwal, H.; Sharma, A.K.; Pathak, M.; Muthukumar, S. Continuous processing for production of biopharmaceuticals. Prep. Biochem. Biotechnol. 2015, 45, 836–849. [CrossRef] Antibodies 2019, 8, 36 20 of 23 95. Shukla, A.A.; Suda, E. Chapter 3—Harvest and recovery of monoclonal antibodies: Cell removal and clariﬁcation. In Process Scale Puriﬁcation of Antibodies; Gottschalk, U., Ed.; John Wiley & Sons, Inc.: Hoboken, NJ, USA, 2017; pp. 55–79. 96. Thömmes, J.; Twyman, R.M.; Gottschalk, U. Chapter 10—Alternatives to packed-bed chromatography for antibody extraction and puriﬁcation. In Process Scale Puriﬁcation of Antibodies; Gottschalk, U., Ed.; John Wiley & Sons: Hoboken, NJ, USA, 2017; pp. 215–231. 97. Glynn, J. Chapter 11—Process-scale precipitation of impurities in mammalian cell culture broth. In Process Scale Puriﬁcation of Antibodies; Gottschalk, U., Ed.; John Wiley & Sons, Inc.: Hoboken, NJ, USA, 2017; pp. 233–246. 98. Singh, N.; Arunkumar, A.; Chollangi, S.; Tan, Z.G.; Borys, M.; Li, Z.J. Clariﬁcation technologies for monoclonal antibody manufacturing processes: Current state and future perspectives. Biotechnol. Bioeng. 2016, 113, 698–716. [CrossRef] 99. Singh, N.; Chollangi, S. Chapter 4—Next-generation clariﬁcation technologies for the downstream processing of antibodies. In Process scale Puriﬁcation of Antibodies; Gottschalk, U., Ed.; John Wiley & Sons, Inc.: Hoboken, NJ, USA, 2017; pp. 81–112. 100. Kelley, B. Chapter 1—Downstream processing of monoclonal antibodies: Current practices and future opportunities. In Process Scale Puriﬁcation of Antibodies; Gottschalk, U., Ed.; John Wiley & Sons, Inc.: Hoboken, NJ, USA, 2017; pp. 1–21. 101. Danielsson, Å. Chapter 17—Anity chromatography. In Biopharmaceutical Processing; Jagschies, G., Lindskog, E., Łacki, ˛ K., Galliher, P., Eds.; Elsevier: Amsterdam, The Netherlands, 2018; pp. 367–378. 102. Kinna, A.; Tolner, B.; Rota, E.M.; Titchener-Hooker, N.; Nesbeth, D.; Chester, K. Imac capture of recombinant protein from unclariﬁed mammalian cell feed streams. Biotechnol. Bioeng. 2016, 113, 130–140. [CrossRef] 103. Ghose, S.; Jin, M.; Liu, J.; Hickey, J.; Lee, S. Chapter 14—Integrated polishing steps for monoclonal antibody puriﬁcation. In Process Scale Puriﬁcation of Antibodies; Gottschalk, U., Ed.; John Wiley & Sons, Inc.: Hoboken, NJ, USA, 2017; pp. 303–323. 104. Joshi, V.; Shivach, T.; Kumar, V.; Yadav, N.; Rathore, A. Avoiding antibody aggregation during processing: Establishing hold times. Biotechnol. J. 2014, 9, 1195–1205. [CrossRef] 105. Liderfelt, J.; Royce, J. Chapter 23—Filtration methods for use in puriﬁcation processes (concentration and buer exchange). In Biopharmaceutical Processing; Jagschies, G., Lindskog, E., Łacki, ˛ K., Galliher, P., Eds.; Elsevier: Amsterdam, The Netherlands, 2018; pp. 441–453. 106. Anselmo, A.C.; Gokarn, Y.; Mitragotri, S. Non-invasive delivery strategies for biologics. Nat. Rev. Drug Discov. 2018, 18, 19. [CrossRef] 107. Sousa, D.; Ferreira, D.; Rodrigues, J.L.; Rodrigues, L.R. Chapter 14—Nanotechnology in targeted drug delivery and therapeutics. In Applications of Targeted Nano Drugs and Delivery Systems; Mohapatra, S.S., Ranjan, S., Dasgupta, N., Mishra, R.K., Thomas, S., Eds.; Elsevier: Amsterdam, The Netherlands, 2019; pp. 357–409. 108. Jani, R.; Krupa, G.; Rupal, J. Active targeting of nanoparticles: An innovative technology for drug delivery in cancer therapeutics. J. Drug Deliv. Ther. 2019. [CrossRef] 109. Abdelaziz, H.M.; Gaber, M.; Abd-Elwakil, M.M.; Mabrouk, M.T.; Elgohary, M.M.; Kamel, N.M.; Kabary, D.M.; Freag, M.S.; Samaha, M.W.; Mortada, S.M.; et al. Inhalable particulate drug delivery systems for lung cancer therapy: Nanoparticles, microparticles, nanocomposites and nanoaggregates. J. Control. Release 2018, 269, 374–392. [CrossRef] 110. Jackisch, C.; Kim, S.B.; Semiglazov, V.; Melichar, B.; Pivot, X.; Hillenbach, C.; Stroyakovskiy, D.; Lum, B.L.; Elliott, R.; Weber, H.A.; et al. Subcutaneous versus intravenous formulation of trastuzumab for her2-positive early breast cancer: Updated results from the phase iii hannah study. Ann. Oncol. 2015, 26, 320–325. [CrossRef] 111. Lambertini, M.; Pondé, N.F.; Solinas, C.; de Azambuja, E. Adjuvant trastuzumab: A 10-year overview of its beneﬁt. Expert Rev. Anticancer Ther. 2017, 17, 61–74. [CrossRef] 112. Sanford, M. Subcutaneous trastuzumab: A review of its use in her2-positive breast cancer. Target. Oncol. 2014, 9, 85–94. [CrossRef] 113. Reichert, J.M. Adalimumab (humira ). In Handbook of Therapeutic Antibodies, 2nd ed.; Wiley Blackwell: Hoboken, NJ, USA, 2014; Volume 3–4, pp. 1309–1322. Antibodies 2019, 8, 36 21 of 23 114. Crommelin, D.J.A.; Hawe, A.; Jiskoot, W. Formulation of biologics including biopharmaceutical considerations. In Pharmaceutical Biotechnology: Fundamentals and Applications; Crommelin, D.J.A., Sindelar, R.D., Meibohm, B., Eds.; Springer International Publishing: Cham, Switzerland, 2019; pp. 83–103. 115. Singh, S.K.; Mahler, H.-C.; Hartman, C.; Stark, C.A. Are injection site reactions in monoclonal antibody therapies caused by polysorbate excipient degradants? J. Pharm. Sci. 2018, 107, 2735–2741. [CrossRef] 116. Rayaprolu, B.M.; Strawser, J.J.; Anyarambhatla, G. Excipients in parenteral formulations: Selection considerations and eective utilization with small molecules and biologics. Drug Dev. Ind. Pharm. 2018, 44, 1565–1571. [CrossRef] 117. Pimentel, F.F.; Morgan, G.; Tiezzi, D.G.; de Andrade, J.M. Development of new formulations of biologics: Expectations, immunogenicity, and safety for subcutaneous trastuzumab. Pharmaceut. Med. 2018, 32, 319–325. [CrossRef] 118. Garidel, P.; Kuhn, A.B.; Schäfer, L.V.; Karow-Zwick, A.R.; Blech, M. High-concentration protein formulations: How high is high? Eur. J. Pharm. Biopharm. 2017, 119, 353–360. [CrossRef] 119. Kemter, K.; Altrichter, J.; Derwand, R.; Kriehuber, T.; Reinauer, E.; Scholz, M. Amino acid-based advanced liquid formulation development for highly concentrated therapeutic antibodies balances physical and chemical stability and low viscosity. Biotechnol. J. 2018, 13, e1700523. [CrossRef] 120. Mandal, A.; Pal, D.; Agrahari, V.; Trinh, H.M.; Joseph, M.; Mitra, A.K. Ocular delivery of proteins and peptides: Challenges and novel formulation approaches. Adv. Drug Del. Rev. 2018, 126, 67–95. [CrossRef] 121. Reslan, M.; Kayser, V. The eect of deuterium oxide on the conformational stability and aggregation of bovine serum albumin. Pharm. Dev. Technol. 2016, 1–7. [CrossRef] 122. Reslan, M.; Ranganathan, V.; Macfarlane, D.R.; Kayser, V. Choline ionic liquid enhances the stability of herceptin(r) (trastuzumab). Chem. Commun. (Camb.) 2018, 54, 10622–10625. [CrossRef] 123. Reslan, M.; Kayser, V. Ionic liquids as biocompatible stabilizers of proteins. Biophys. Rev. 2018, 10, 781–793. [CrossRef] 124. Emami, F.; Vatanara, A.; Park, E.J.; Na, D.H. Drying technologies for the stability and bioavailability of biopharmaceuticals. Pharmaceutics 2018, 10, 131. [CrossRef] 125. Izutsu, K.I. Applications of freezing and freeze-drying in pharmaceutical formulations. In Adv. Exp. Med. Biol.; Springer: New York, NY, USA, 2018; Volume 1081, pp. 371–383. 126. Schermeyer, M.T.; Wöll, A.K.; Kokke, B.; Eppink, M.; Hubbuch, J. Characterization of highly concentrated antibody solution—A toolbox for the description of protein long-term solution stability. mAbs 2017, 9, 1169–1185. [CrossRef] 127. Kayser, V.; Chennamsetty, N.; Voynov, V.; Helk, B.; Forrer, K.; Trout, B.L. Evaluation of a non-arrhenius model for therapeutic monoclonal antibody aggregation. J. Pharm. Sci. 2011, 100, 2526–2542. [CrossRef] 128. Rabia, L.A.; Desai, A.A.; Jhajj, H.S.; Tessier, P.M. Understanding and overcoming trade-os between antibody anity, speciﬁcity, stability and solubility. Biochem. Eng. J. 2018, 137, 365–374. [CrossRef] 129. Van de Bovenkamp, F.S.; Derksen, N.I.L.; van Breemen, M.J.; de Taeye, S.W.; Ooijevaar-de Heer, P.; Sanders, R.W.; Rispens, T. Variable domain n-linked glycans acquired during antigen-speciﬁc immune responses can contribute to immunoglobulin g antibody stability. Front. Immunol. 2018, 9, 740. [CrossRef] 130. Kuhn, A.B.; Kube, S.; Karow-Zwick, A.R.; Seeliger, D.; Garidel, P.; Blech, M.; Schäfer, L.V. Improved solution-state properties of monoclonal antibodies by targeted mutations. J. Phys. Chem. B 2017, 121, 10818–10827. [CrossRef] 131. Singh, S.N.; Yadav, S.; Shire, S.J.; Kalonia, D.S. Dipole-dipole interaction in antibody solutions: Correlation with viscosity behavior at high concentration. Pharm. Res. 2014, 31, 2549–2558. [CrossRef] 132. Ionescu, R.M.; Vlasak, J.; Price, C.; Kirchmeier, M. Contribution of variable domains to the stability of humanized igg1 monoclonal antibodies. J. Pharm. Sci. 2008, 97, 1414–1426. [CrossRef] 133. Liu, L. Pharmacokinetics of monoclonal antibodies and fc-fusion proteins. Protein Cell 2018, 9, 15–32. [CrossRef] 134. Schweizer, D.; Serno, T.; Goepferich, A. Controlled release of therapeutic antibody formats. Eur. J. Pharm. Biopharm. 2014, 88, 291–309. [CrossRef] 135. Rudnick, S.I.; Adams, G.P. Anity and avidity in antibody-based tumor targeting. Cancer Biother. Radiopharm. 2009, 24, 155–161. [CrossRef] 136. Ickenstein, L.M.; Garidel, P. Hydrogel formulations for biologicals: Current spotlight from a commercial perspective. Ther. Deliv. 2018, 9, 221–230. [CrossRef] Antibodies 2019, 8, 36 22 of 23 137. Saxena, A.; Wu, D. Advances in therapeutic fc engineering—Modulation of igg-associated eector functions and serum half-life. Front. Immunol. 2016, 7, 580. [CrossRef] 138. Jeeris, R. Glycosylation of antibody molecules. In Handbook of Therapeutic Antibodies, 2nd ed.; Wiley Blackwell: Hoboken, NJ, USA, 2014; Volume 1–4, pp. 171–200. 139. Zheng, K.; Bantog, C.; Bayer, R. The impact of glycosylation on monoclonal antibody conformation and stability. mAbs 2011, 3, 568–576. [CrossRef] 140. Lei, C.; Gong, R.; Ying, T. Editorial: Antibody fc engineering: Towards better therapeutics. Front. Immunol. 2018, 9, 2450. [CrossRef] 141. Booth, B.J.; Ramakrishnan, B.; Narayan, K.; Wollacott, A.M.; Babcock, G.J.; Shriver, Z.; Viswanathan, K. Extending human igg half-life using structure-guided design. mAbs 2018, 10, 1098–1110. [CrossRef] 142. Kellner, C.; Otte, A.; Cappuzzello, E.; Klausz, K.; Peipp, M. Modulating cytotoxic eector functions by fc engineering to improve cancer therapy. Transfus. Med. Hemoth. 2017, 44, 327–336. [CrossRef] 143. Wang, Q.; Chen, Y.; Pelletier, M.; Cvitkovic, R.; Bonnell, J.; Chang, C.Y.; Koksal, A.C.; O’Connor, E.; Gao, X.; Yu, X.Q.; et al. Enhancement of antibody functions through fc multiplications. mAbs 2017, 9, 393–403. [CrossRef] 144. Lefranc, M.P.; Ehrenmann, F.; Kossida, S.; Giudicelli, V.; Duroux, P. Use of imgt databases and tools for antibody engineering and humanization. In Methods Mol. Biol.; Humana Press Inc.: New York, NY, USA, 2018; Volume 1827, pp. 35–69. 145. Lefranc, M.-P.; Giudicelli, V.; Duroux, P.; Jabado-Michaloud, J.; Folch, G.; Aouinti, S.; Carillon, E.; Duvergey, H.; ® ® Houles, A.; Paysan-Lafosse, T.; et al. Imgt( ), the international immunogenetics information system( ) 25 years on. Nucleic Acids Res. 2015, 43, D413–D422. [CrossRef] 146. Martin, A.C.R.; Allen, J. Bioinformatics tools for analysis of antibodies. In Handbook of Therapeutic Antibodies, 2nd ed.; Wiley Blackwell: Hoboken, NJ, USA, 2014; Volume 1–4, pp. 201–228. 147. Sledz, ´ P.; Caﬂisch, A. Protein structure-based drug design: From docking to molecular dynamics. Curr. Opin. Struct. Biol. 2018, 48, 93–102. [CrossRef] 148. Pandya, A.; Howard, M.J.; Zloh, M.; Dalby, P.A. An evaluation of the potential of nmr spectroscopy and computational modelling methods to inform biopharmaceutical formulations. Pharmaceutics 2018, 10, 1–24. [CrossRef] 149. Rathore, D.; Faustino, A.; Schiel, J.; Pang, E.; Boyne, M.; Rogstad, S. The role of mass spectrometry in the characterization of biologic protein products. Expert Rev. Proteomic. 2018, 15, 431–449. [CrossRef] 150. Wang, X.; An, Z.; Luo, W.; Xia, N.; Zhao, Q. Molecular and functional analysis of monoclonal antibodies in support of biologics development. Protein Cell 2018, 9, 74–85. [CrossRef] 151. Baker Edward, N. Crystallography and the development of therapeutic medicines. IUCrJ 2018, 5, 118–119. [CrossRef] 152. Vénien-Bryan, C.; Li, Z.; Vuillard, L.; Boutin, J.A. Cryo-electron microscopy and X-ray crystallography: Complementary approaches to structural biology and drug discovery. Acta Crystallogr. Sect. F Struct. Biol. Commun. 2017, 73, 174–183. [CrossRef] 153. Brader, M.L.; Baker, E.N.; Dunn, M.F.; Laue, T.M.; Carpenter, J.F. Using x-ray crystallography to simplify and accelerate biologics drug development. J. Pharm. Sci. 2017, 106, 477–494. [CrossRef] 154. Nero, T.L.; Parker, M.W.; Morton, C.J. Protein structure and computational drug discovery. Biochem. Soc. Trans. 2018, 46, 1367–1379. [CrossRef] 155. Blaert, J.; Haeri, H.H.; Blech, M.; Hinderberger, D.; Garidel, P. Spectroscopic methods for assessing the molecular origins of macroscopic solution properties of highly concentrated liquid protein solutions. Anal. Biochem. 2018, 561–562, 70–88. [CrossRef] 156. Brinson, R.G.; Marino, J.P.; Delaglio, F.; Arbogast, L.W.; Evans, R.M.; Kearsley, A.; Gingras, G.; Ghasriani, H.; Aubin, Y.; Pierens, G.K.; et al. Enabling adoption of 2d-nmr for the higher order structure assessment of monoclonal antibody therapeutics. mAbs 2019, 11, 94–105. [CrossRef] 157. Young, J.A.; Gabrielson, J.P. Higher order structure methods for similarity assessment. In Biosimilars: Regulatory, Clinical, and Biopharmaceutical Development; Gutka, H.J., Yang, H., Kakar, S., Eds.; Springer International Publishing: Cham, Switzerland, 2018; pp. 321–337. 158. Kumar, S.; Plotnikov, N.V.; Rouse, J.C.; Singh, S.K. Biopharmaceutical informatics: Supporting biologic drug development via molecular modelling and informatics. J. Pharm. Pharmacol. 2018, 70, 595–608. [CrossRef] Antibodies 2019, 8, 36 23 of 23 159. Westbrook, J.D.; Burley, S.K. How structural biologists and the protein data bank contributed to recent fda new drug approvals. Structure 2018, 27, 211–217. [CrossRef] 160. Burley, S.K.; Berman, H.M.; Christie, C.; Duarte, J.M.; Feng, Z.; Westbrook, J.; Young, J.; Zardecki, C. Rcsb protein data bank: Sustaining a living digital data resource that enables breakthroughs in scientiﬁc research and biomedical education. Protein Sci. 2018, 27, 316–330. [CrossRef] 161. Kuroda, D.; Tsumoto, K. Antibody anity maturation by computational design. In Methods Mol. Biol.; Humana Press Inc.: New York, NY, USA, 2018; Volume 1827, pp. 15–34. 162. Fischman, S.; Ofran, Y. Computational design of antibodies. Curr. Opin. Struct. Biol. 2018, 51, 156–162. [CrossRef] 163. Adolf-Bryfogle, J.; Kalyuzhniy, O.; Kubitz, M.; Weitzner, B.D.; Hu, X.; Adachi, Y.; Schief, W.R.; Dunbrack, R.L., Jr. Rosettaantibodydesign (rabd): A general framework for computational antibody design. PLoS Comp. Biol. 2018, 14, e1006112. [CrossRef] 164. Branco, R.J.; Dias, A.M.; Roque, A.C. Understanding the molecular recognition between antibody fragments and protein a biomimetic ligand. J. Chromatogr. A 2012, 1244, 106–115. [CrossRef] 165. Huang, B.; Liu, F.F.; Dong, X.Y.; Sun, Y. Molecular mechanism of the anity interactions between protein a and human immunoglobulin g1 revealed by molecular simulations. J. Phys. Chem. B 2011, 115, 4168–4176. [CrossRef] 166. Van der Kant, R.; van Durme, J.; Rousseau, F.; Schymkowitz, J. Solubis: Optimizing protein solubility by minimal point mutations. In Methods Mol. Biol.; Humana Press Inc.: New York, NY, USA, 2019; Volume 1873, pp. 317–333. 167. Gil-Garcia, M.; Banó-Polo, M.; Varejao, N.; Jamroz, M.; Kuriata, A.; Díaz-Caballero, M.; Lascorz, J.; Morel, B.; Navarro, S.; Reverter, D.; et al. Combining structural aggregation propensity and stability predictions to redesign protein solubility. Mol. Pharm. 2018, 15, 3846–3859. [CrossRef] 168. Seeliger, D.; Schulz, P.; Litzenburger, T.; Spitz, J.; Hoerer, S.; Blech, M.; Enenkel, B.; Studts, J.M.; Garidel, P.; Karow, A.R. Boosting antibody developability through rational sequence optimization. mAbs 2015, 7, 505–515. [CrossRef] 169. Chennamsetty, N.; Voynov, V.; Kayser, V.; Helk, B.; Trout, B.L. Prediction of aggregation prone regions of therapeutic proteins. J. Phys. Chem. B 2010, 114, 6614–6624. [CrossRef] 170. Chennamsetty, N.; Voynov, V.; Kayser, V.; Helk, B.; Trout, B.L. Design of therapeutic proteins with enhanced stability. Proc. Natl. Acad. Sci. USA 2009, 106, 11937–11942. [CrossRef] 171. Majumder, S.; Jones, M.T.; Kimmel, M.; Alphonse Ignatius, A. Probing conformational diversity of fc domains in aggregation-prone monoclonal antibodies. Pharm. Res. 2018, 35, 220. [CrossRef] 172. Nakamura, H.; Oda-Ueda, N.; Ueda, T.; Ohkuri, T. Introduction of a glycosylation site in the constant region decreases the aggregation of adalimumab fab. Biochem. Biophys. Res. Commun. 2018, 503, 752–756. [CrossRef] 173. Pepinsky, R.B.; Silvian, L.; Berkowitz, S.A.; Farrington, G.; Lugovskoy, A.; Walus, L.; Eldredge, J.; Capili, A.; Mi, S.; Gra, C.; et al. Improving the solubility of anti-lingo-1 monoclonal antibody li33 by isotype switching and targeted mutagenesis. Protein Sci. 2010, 19, 954–966. [CrossRef] 174. Wu, S.-J.; Luo, J.; O’Neil, K.T.; Kang, J.; Lacy, E.R.; Canziani, G.; Baker, A.; Huang, M.; Tang, Q.M.; Raju, T.S.; et al. Structure-based engineering of a monoclonal antibody for improved solubility. Protein Eng. Des. Sel. 2010, 23, 643–651. [CrossRef] © 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/). http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Antibodies Multidisciplinary Digital Publishing Institute http://www.deepdyve.com/lp/multidisciplinary-digital-publishing-institute/current-advancements-in-addressing-key-challenges-of-therapeutic-K2mvDxOXaN

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References (198)

Stefan Behme (2009)
Manufacturing of Pharmaceutical Proteins: From Technology to Economy
Alexander Kinna, B. Tolner, E. Rota, N. Titchener-Hooker, D. Nesbeth, K. Chester (2015)
IMAC capture of recombinant protein from unclarified mammalian cell feed streams
Biotechnology and Bioengineering, 113
Naresh Chennamsetty, V. Voynov, Veysel Kayser, B. Helk, B. Trout (2009)
Design of therapeutic proteins with enhanced stability
Proceedings of the National Academy of Sciences, 106
(2019)
Chapter 7—Purification of human monoclonal antibodies and their fragments
Human monoclonal Antibodies: Methods and Protocols
C. Altamirano, J. Berrios, M. Vergara, S. Becerra (2013)
Advances in improving mammalian cells metabolism for recombinant protein production
Electronic Journal of Biotechnology, 16
Xiaotian Zhong, Jill Wright (2013)
Biological Insights into Therapeutic Protein Modifications throughout Trafficking and Their Biopharmaceutical Applications
International Journal of Cell Biology, 2013
V. Mauro, S. Chappell (2018)
Considerations in the Use of Codon Optimization for Recombinant Protein Expression.
Methods in molecular biology, 1850
Lena Thoring, S. Kubick (2018)
Versatile Cell-Free Protein Synthesis Systems Based on Chinese Hamster Ovary Cells.
Methods in molecular biology, 1850
E. Baker (2018)
Crystallography and the development of therapeutic medicines
IUCrJ, 5
M. Lalonde, Y. Durocher (2017)
Therapeutic glycoprotein production in mammalian cells.
Journal of biotechnology, 251
(2018)
Chapter 7—Cell line development
C. Dobson, P. Devine, J. Phillips, D. Higazi, C. Lloyd, B. Popovic, J. Arnold, A. Buchanan, A. Lewis, Joanne Goodman, C. Walle, P. Thornton, Lisa Vinall, D. Lowne, A. Aagaard, Lise-Lotte Olsson, A. Wollberg, Fraser Welsh, T. Karamanos, C. Pashley, M. Iadanza, N. Ranson, A. Ashcroft, A. Kippen, T. Vaughan, S. Radford, D. Lowe (2016)
Engineering the surface properties of a human monoclonal antibody prevents self-association and rapid clearance in vivo
Scientific Reports, 6
Veysel Kayser, Naresh Chennamsetty, V. Voynov, B. Helk, Kurt Forrer, B. Trout (2011)
Evaluation of a non-Arrhenius model for therapeutic monoclonal antibody aggregation.
Journal of pharmaceutical sciences, 100 7
A. Shukla, Jagannadha Kandula (2008)
Harvest and Recovery of Monoclonal Antibodies: Cell Removal and Clarification
Nicole Ulmer, Sebastian Vogg, T. Müller-Späth, M. Morbidelli (2018)
Purification of Human Monoclonal Antibodies and Their Fragments.
Methods in molecular biology, 1904
Tsuyoshi Nakamura, T. Omasa (2015)
Optimization of cell line development in the GS-CHO expression system using a high-throughput, single cell-based clone selection system.
Journal of bioscience and bioengineering, 120 3
J. Thömmes, U. Gottschalk (2008)
Alternatives to Packed‐Bed Chromatography for Antibody Extraction and Purification
M. Fonseca, G. Furtado, M. Bezerra, Larissa Pontes, C. Fernandes (2018)
Boosting half-life and effector functions of therapeutic antibodies by Fc-engineering: An interaction-function review.
International journal of biological macromolecules, 119
B. Bittner, W. Richter, Johannes Schmidt (2018)
Subcutaneous Administration of Biotherapeutics: An Overview of Current Challenges and Opportunities
Biodrugs, 32
Xin Wang, Z. An, Wen-xin Luo, N. Xia, Qinjian Zhao (2017)
Molecular and functional analysis of monoclonal antibodies in support of biologics development
Protein & Cell, 9
M. Lambertini, N. Pondé, C. Solinas, E. Azambuja (2017)
Adjuvant trastuzumab: a 10-year overview of its benefit
Expert Review of Anticancer Therapy, 17
Nripen Singh, A. Arunkumar, S. Chollangi, Zhijun Tan, Michael Borys, Z. Li (2016)
Clarification technologies for monoclonal antibody manufacturing processes: Current state and future perspectives
Biotechnology and Bioengineering, 113
S. Rudnick, G. Adams (2009)
Affinity and avidity in antibody-based tumor targeting.
Cancer biotherapy & radiopharmaceuticals, 24 2
D. Levin, B. Golding, S. Strome, Z. Sauna (2015)
Fc fusion as a platform technology: potential for modulating immunogenicity.
Trends in biotechnology, 33 1
Subhabrata Majumder, Michael Jones, M. Kimmel, Arun Ignatius (2018)
Probing Conformational Diversity of Fc Domains in Aggregation-Prone Monoclonal Antibodies
Pharmaceutical Research, 35
P. Garidel, Alexander Kuhn, Lars Schäfer, Anne Karow-Zwick, M. Blech (2017)
High‐concentration protein formulations: How high is high?
European Journal of Pharmaceutics and Biopharmaceutics, 119
Veysel Kayser, Naresh Chennamsetty, V. Voynov, Kurt Forrer, B. Helk, B. Trout (2011)
Glycosylation influences on the aggregation propensity of therapeutic monoclonal antibodies.
Biotechnology journal, 6 1
Hadeer Abdelaziz, M. Gaber, Mahmoud Abd-Elwakil, Moustafa Mabrouk, Mayada Elgohary, N. Kamel, Dalia Kabary, May Freag, M. Samaha, S. Mortada, Kadria Elkhodairy, Jia-You Fang, Ahmed Elzoghby (2018)
Inhalable particulate drug delivery systems for lung cancer therapy: Nanoparticles, microparticles, nanocomposites and nanoaggregates.
Journal of controlled release : official journal of the Controlled Release Society, 269
D. Crommelin, A. Hawe, W. Jiskoot (2019)
Formulation of Biologics Including Biopharmaceutical Considerations
Pharmaceutical Biotechnology
T. Matsuda, Takuhiro Ito, C. Takemoto, Kazushige Katsura, Mariko Ikeda, M. Wakiyama, M. Kukimoto-Niino, S. Yokoyama, Y. Kurosawa, M. Shirouzu (2018)
Cell-free synthesis of functional antibody fragments to provide a structural basis for antibody–antigen interaction
PLoS ONE, 13
Abhirup Mandal, D. Pal, Vibhuti Agrahari, H. Trinh, M. Joseph, A. Mitra (2018)
Ocular delivery of proteins and peptides: Challenges and novel formulation approaches☆
Advanced Drug Delivery Reviews, 126
(2014)
Antibody affinity
Handbook of Therapeutic Antibodies, Volume 1–4
(2018)
Versatile cell-free protein synthesis systems based on chinese hamster ovary cells
Recombinant Protein Expression in Mammalian Cells: Methods and Protocols
E. Lindskog (2018)
The Upstream Process: Principal Modes of Operation
(2017)
Chapter 11—Process-scale precipitation of impurities in mammalian cell culture broth
Fabienne Courtois, Neeraj Agrawal, Timothy Lauer, B. Trout (2016)
Rational design of therapeutic mAbs against aggregation through protein engineering and incorporation of glycosylation motifs applied to bevacizumab
mAbs, 8
R. Ionescu, J. Vlasák, Colleen Price, M. Kirchmeier (2008)
Contribution of variable domains to the stability of humanized IgG1 monoclonal antibodies.
Journal of pharmaceutical sciences, 97 4
(2018)
Chapter 9—Industry review of cell separation and product harvesting methods
Biopharmaceutical Processing
Marie-Therese Schermeyer, A. Wöll, B. Kokke, M. Eppink, J. Hubbuch (2017)
Characterization of highly concentrated antibody solution - A toolbox for the description of protein long-term solution stability
mAbs, 9
Robert Brinson, J. Marino, F. Delaglio, Luke Arbogast, Ryan Evans, Anthony Kearsley, G. Gingras, H. Ghasriani, Y. Aubin, G. Pierens, Xinying Jia, M. Mobli, H. Grant, D. Keizer, K. Schweimer, Jonas Ståhle, G. Widmalm, E. Zartler, Chad Lawrence, P. Reardon, J. Cort, P. Xu, F. Ni, S. Yanaka, Koichi Kato, S. Parnham, D. Tsao, A. Blomgren, T. Rundlöf, Nils Trieloff, P. Schmieder, A. Ross, K. Skidmore, Kang Chen, D. Keire, D. Freedberg, Thea Suter-Stahel, G. Wider, Gregor Ilc, J. Plavec, S. Bradley, D. Baldisseri, M. Sforça, A. Zeri, J. Wei, C. Szabo, Carlos Amezcua, J. Jordan, M. Wikström (2018)
Enabling adoption of 2D-NMR for the higher order structure assessment of monoclonal antibody therapeutics
mAbs, 11
(2018)
High throughput transfection of hek293 cells for transient protein production
Recombinant Protein Expression in Mammalian Cells: Methods and Protocols
S. Wijesuriya, E. Pongo, M. Tomic, Fangjiu Zhang, Consuelo Garcia-Rodriquez, Fraser Conrad, S. Farr-Jones, J. Marks, A. Horwitz (2018)
Antibody engineering to improve manufacturability.
Protein expression and purification, 149
(2017)
Research and development of innovative antibody-based drugs
M. Brader, E. Baker, M. Dunn, T. Laue, J. Carpenter (2017)
Using X-Ray Crystallography to Simplify and Accelerate Biologics Drug Development.
Journal of pharmaceutical sciences, 106 2
(2013)
A high-yielding, cho-k1-based transient transfection system
Veysel Kayser, Naresh Chennamsetty, V. Voynov, B. Helk, B. Trout (2011)
Conformational stability and aggregation of therapeutic monoclonal antibodies studied with ANS and Thioflavin T binding
mAbs, 3
Amanda Mizukami, A. Caron, V. Picanço-Castro, K. Swiech (2018)
Platforms for Recombinant Therapeutic Glycoprotein Production.
Methods in molecular biology, 1674
L. Otvos (2017)
Basic Principles of Formulation for Biotherapeutics: Approaches to Alternative Drug Delivery
, 6
V. Joshi, Tarun Shivach, Vijesh Kumar, N. Yadav, A. Rathore (2014)
Avoiding antibody aggregation during processing: Establishing hold times
Biotechnology Journal, 9
Andreas Maccani, Nils Landes, G. Stadlmayr, D. Maresch, C. Leitner, Michael Maurer, Brigitte Gasser, W. Ernst, R. Kunert, D. Mattanovich (2014)
Pichia pastoris secretes recombinant proteins less efficiently than Chinese hamster ovary cells but allows higher space-time yields for less complex proteins
Biotechnology Journal, 9
Joe Codamo, T. Munro, Benjamin Hughes, Michael Song, P. Gray (2011)
Enhanced CHO Cell-Based Transient Gene Expression with the Epi-CHO Expression System
Molecular Biotechnology, 48
Jinjun Shi, P. Kantoff, R. Wooster, O. Farokhzad (2016)
Cancer nanomedicine: progress, challenges and opportunities
Nature Reviews Cancer, 17
G. Backliwal, M. Hildinger, Ivan Kuettel, Fanny Delegrange, D. Hacker, F. Wurm (2008)
Valproic acid: a viable alternative to sodium butyrate for enhancing protein expression in mammalian cell cultures.
Biotechnology and bioengineering, 101 1
(2014)
Controlled release of therapeutic antibody
S. Burley, H. Berman, Cole Christie, Jose Duarte, Zukang Feng, J. Westbrook, Jasmine Young, C. Zardecki (2017)
RCSB Protein Data Bank: Sustaining a living digital data resource that enables breakthroughs in scientific research and biomedical education
Protein Science : A Publication of the Protein Society, 27
(2018)
Advances and challenges in therapeutic monoclonal antibodies drug development. Bras
Jared Adolf-Bryfogle, O. Kalyuzhniy, M. Kubitz, Brian Weitzner, Xiaozhen Hu, Yumiko Adachi, W. Schief, Roland Dunbrack (2017)
RosettaAntibodyDesign (RAbD): A general framework for computational antibody design
PLoS Computational Biology, 14
F. Pimentel, G. Morgan, D. Tiezzi, J. Andrade (2018)
Development of New Formulations of Biologics: Expectations, Immunogenicity, and Safety for Subcutaneous Trastuzumab
Pharmaceutical Medicine, 32
J. Morales, Kristin Fathe, A. Brunaugh, S. Ferrati, Song Li, Miguel Montenegro‐Nicolini, Zeynab Mousavikhamene, J. McConville, M. Prausnitz, H. Smyth (2017)
Challenges and Future Prospects for the Delivery of Biologics: Oral Mucosal, Pulmonary, and Transdermal Routes
The AAPS Journal, 19
(2017)
Chapter 1—Downstream processing of monoclonal antibodies: Current practices and future opportunities
Abdul Muheem, F. Shakeel, M. Jahangir, Mohammed Anwar, N. Mallick, G. Jain, M. Warsi, F. Ahmad (2014)
A review on the strategies for oral delivery of proteins and peptides and their clinical perspectives
Saudi Pharmaceutical Journal : SPJ, 24
Bo Huang, Fu-Feng Liu, Xiaoyan Dong, Y. Sun (2011)
Molecular mechanism of the affinity interactions between protein A and human immunoglobulin G1 revealed by molecular simulations.
The journal of physical chemistry. B, 115 14
R. Kant, J. Durme, F. Rousseau, J. Schymkowitz (2018)
SolubiS: Optimizing Protein Solubility by Minimal Point Mutations.
Methods in molecular biology, 1873
Kai Zheng, Christopher Bantog, R. Bayer (2011)
The impact of glycosylation on monoclonal antibody conformation and stability
mAbs, 3
Naresh Chennamsetty, V. Voynov, Veysel Kayser, B. Helk, B. Trout (2010)
Prediction of aggregation prone regions of therapeutic proteins.
The journal of physical chemistry. B, 114 19
Cheng Lei, R. Gong, T. Ying (2018)
Editorial: Antibody Fc Engineering: Towards Better Therapeutics
Frontiers in Immunology, 9
F. Ritacco, Yongqi Wu, Anurag Khetan (2018)
Cell culture media for recombinant protein expression in Chinese hamster ovary (CHO) cells: History, key components, and optimization strategies
Biotechnology Progress, 34
R. Jefferis, M. Goodall, V. Tishchenko, P. Nash, J. Lund (1995)
Glycosylation of Antibody Molecules
Jakob Liderfelt, Jonathan Royce (2018)
Filtration Methods for Use in Purification Processes (Concentration and Buffer Exchange)
Z. Elgundi, Vicki Sifniotis, Mouhamad Reslan, Esteban Cruz, Veysel Kayser (2017)
Laboratory Scale Production and Purification of a Therapeutic Antibody.
Journal of visualized experiments : JoVE, 119
Lilia Rabia, Alec Desai, Harkamal Jhajj, P. Tessier (2018)
Understanding and overcoming trade-offs between antibody affinity, specificity, stability and solubility.
Biochemical engineering journal, 137
Shubhadra Singh, S. Yadav, S. Shire, D. Kalonia (2014)
Dipole-Dipole Interaction in Antibody Solutions: Correlation with Viscosity Behavior at High Concentration
Pharmaceutical Research, 31
(2018)
Chapter 31—The upstream process: Principal modes of operation
Biopharmaceutical Processing
Y. Zhou, Ravali Raju, Christina Alves, Alan Gilbert (2018)
Debottlenecking protein secretion and reducing protein aggregation in the cellular host.
Current opinion in biotechnology, 53
Jared Young, John Gabrielson (2018)
Higher Order Structure Methods for Similarity Assessment
R. Kant, Anne Karow-Zwick, J. Durme, M. Blech, R. Gallardo, Daniel Seeliger, Kerstin Assfalg, P. Baatsen, G. Compernolle, A. Gils, J. Studts, P. Schulz, P. Garidel, J. Schymkowitz, F. Rousseau (2017)
Prediction and Reduction of the Aggregation of Monoclonal Antibodies
Journal of Molecular Biology, 429
A. Torosian (1980)
[Antibody affinity].
Uspekhi sovremennoi biologii, 89 3
S. Singh, Hanns-Christian Mahler, C. Hartman, C. Stark (2018)
Are Injection Site Reactions in Monoclonal Antibody Therapies Caused by Polysorbate Excipient Degradants?
Journal of pharmaceutical sciences, 107 11
P. Śledź, A. Caflisch (2018)
Protein structure-based drug design: from docking to molecular dynamics.
Current opinion in structural biology, 48
(2019)
Chapter 14—Nanotechnology in targeted drug delivery and therapeutics
Applications of Targeted Nano Drugs and Delivery Systems
(2018)
Transient gene expression in suspension hek293-ebna1 cells
Recombinant Protein Expression in Mammalian Cells: Methods and Protocols
Fabienne Courtois, C. Schneider, Neeraj Agrawal, B. Trout (2015)
Rational Design of Biobetters with Enhanced Stability.
Journal of pharmaceutical sciences, 104 8
D. Sousa, D. Ferreira, J. Rodrigues, L. Rodrigues (2019)
Nanotechnology in Targeted Drug Delivery and Therapeutics
Applications of Targeted Nano Drugs and Delivery Systems
Marizela Delic, Rebecca Göngrich, D. Mattanovich, Brigitte Gasser (2014)
Engineering of protein folding and secretion-strategies to overcome bottlenecks for efficient production of recombinant proteins.
Antioxidants & redox signaling, 21 3
(2018)
Chapter 17—Affinity chromatography
(2014)
Adalimumab (humira®)
P. Arosio, Giuliano Barolo, T. Müller-Späth, Hua Wu, M. Morbidelli (2011)
Aggregation Stability of a Monoclonal Antibody During Downstream Processing
Pharmaceutical Research, 28
Akash Pandya, M. Howard, M. Zloh, P. Dalby (2018)
An Evaluation of the Potential of NMR Spectroscopy and Computational Modelling Methods to Inform Biopharmaceutical Formulations
Pharmaceutics, 10
J. Westbrook, S. Burley (2019)
How Structural Biologists and the Protein Data Bank Contributed to Recent FDA New Drug Approvals.
Structure, 27 2
Lukas Fliedl, J. Grillari, Regina Grillari-Voglauer (2015)
Human cell lines for the production of recombinant proteins: on the horizon.
New biotechnology, 32 6
Mouhamad Reslan, Veysel Kayser (2018)
Ionic liquids as biocompatible stabilizers of proteins
Biophysical Reviews, 10
B. Rayaprolu, Jonathan Strawser, G. Anyarambhatla (2018)
Excipients in parenteral formulations: selection considerations and effective utilization with small molecules and biologics
Drug Development and Industrial Pharmacy, 44
A. Rathore, H. Agarwal, Abhishek Sharma, Mili Pathak, S. Muthukumar (2015)
Continuous Processing for Production of Biopharmaceuticals
Preparative Biochemistry and Biotechnology, 45
R. Pepinsky, L. Silvian, S. Berkowitz, G. Farrington, A. Lugovskoy, L. Walus, J. Eldredge, A. Capili, S. Mi, Christilyn Graff, E. Garber (2010)
Improving the solubility of anti‐LINGO‐1 monoclonal antibody Li33 by isotype switching and targeted mutagenesis
Protein Science, 19
Liming Liu (2017)
Pharmacokinetics of monoclonal antibodies and Fc-fusion proteins
Protein & Cell, 9
Se Kim, Gyong-Sik Ha, Gyunghwa Lee, S. Lim, C. Lee, Yoo Yang, Jaemin Lee, Ju Kim, Jae Lee, Y. Shin, Chan-wha Kim, Dong Lee (2018)
Enhanced expression of soluble antibody fragments by low-temperature and overdosing with a nitrogen source.
Enzyme and microbial technology, 115
A. Frenzel, M. Hust, T. Schirrmann (2013)
Expression of Recombinant Antibodies
Frontiers in Immunology, 4
Veysel Kayser, Naresh Chennamsetty, V. Voynov, B. Helk, Kurt Forrer, B. Trout (2012)
A screening tool for therapeutic monoclonal antibodies: Identifying the most stable protein and its best formulation based on thioflavin T binding
Biotechnology Journal, 7
Kevin Henry (2018)
Next-Generation DNA Sequencing of VH/VL Repertoires: A Primer and Guide to Applications in Single-Domain Antibody Discovery.
Methods in molecular biology, 1701
M. Lefranc, F. Ehrenmann, C. Ginestoux, V. Giudicelli, P. Duroux (2012)
Use of IMGT(®) databases and tools for antibody engineering and humanization.
Methods in molecular biology, 907
Cristina Parola, Derek Mason, A. Zingg, D. Neumeier, S. Reddy (2018)
Genome Engineering of Hybridomas to Generate Stable Cell Lines for Antibody Expression.
Methods in molecular biology, 1850
(2018)
Genome-wide high-throughput rnai screening for identification of genes involved in protein production
Recombinant Protein Expression in Mammalian Cells: Methods and Protocols
Sheng‐Jiun Wu, Jinquan Luo, K. O'Neil, James Kang, E. Lacy, G. Canziani, Audrey Baker, Maggie Huang, Q. Tang, T. Raju, S. Jacobs, A. Teplyakov, G. Gilliland, Yiqing Feng (2010)
Structure-based engineering of a monoclonal antibody for improved solubility.
Protein engineering, design & selection : PEDS, 23 8
(2018)
Next-generation DNA sequencing of vh/vl repertoires: A primer and guide to applications in single-domain antibody discovery
Phage Display: Methods and Protocols
W. Strohl (2017)
Current progress in innovative engineered antibodies
Protein & Cell, 9
I. Michael, A. Nagy (2018)
Inducible Protein Production in 293 Cells Using the piggyBac Transposon System.
Methods in molecular biology, 1850
(2018)
Considerations in the use of codon optimization for recombinant protein expression
Recombinant Protein Expression in Mammalian Cells: Methods and Protocols
(2018)
Genome engineering of hybridomas to generate stable cell lines for antibody expression
Recombinant Protein Expression in Mammalian Cells: Methods and Protocols
Joe Codamo, J. Hou, Benjamin Hughes, P. Gray, T. Munro (2011)
Efficient mAb production in CHO cells incorporating PEI-mediated transfection, mild hypothermia and the co-expression of XBP-1
Journal of Chemical Technology & Biotechnology, 86
Ricarda Hoffmann, B. Coumbe, D. Josephs, S. Mele, Kristina Ilieva, A. Cheung, A. Tutt, J. Spicer, D. Thurston, S. Crescioli, S. Karagiannis (2017)
Antibody structure and engineering considerations for the design and function of Antibody Drug Conjugates (ADCs)
Oncoimmunology, 7
Sanchayita Ghose, Mi Jin, Jia Liu, J. Hickey (2008)
INTEGRATED POLISHING STEPS FOR MONOCLONAL ANTIBODY PURIFICATION
C. Kellner, A. Otte, E. Cappuzzello, K. Klausz, M. Peipp (2017)
Modulating Cytotoxic Effector Functions by Fc Engineering to Improve Cancer Therapy
Transfusion Medicine and Hemotherapy, 44
Lisa Urquhart (2018)
Market watch: Top drugs and companies by sales in 2017
Nature Reviews Drug Discovery, 17
Sarah Inwood, M. Betenbaugh, Madhu Lal, J. Shiloach (2018)
Genome-Wide High-Throughput RNAi Screening for Identification of Genes Involved in Protein Production.
Methods in molecular biology, 1850
D. L'abbe, Louis Bisson, C. Gervais, E. Grazzini, Y. Durocher (2018)
Transient Gene Expression in Suspension HEK293-EBNA1 Cells.
Methods in molecular biology, 1850
John Pieracci, J. Armando, M. Westoby, J. Thömmes (2018)
Industry Review of Cell Separation and Product Harvesting Methods
V. Jäger, K. Büssow, T. Schirrmann (2015)
Transient Recombinant Protein Expression in Mammalian Cells
(2018)
Chapter 6—Host cells
Daniel Seeliger, P. Schulz, T. Litzenburger, J. Spitz, S. Hoerer, M. Blech, B. Enenkel, J. Studts, P. Garidel, Anne Karow (2015)
Boosting antibody developability through rational sequence optimization
mAbs, 7
(2017)
Chapter 14—Integrated polishing steps for monoclonal antibody purification
Process Scale Purification of Antibodies
C. Vénien-Bryan, Zhuolun Li, L. Vuillard, J. Boutin (2017)
Cryo-electron microscopy and X-ray crystallography: complementary approaches to structural biology and drug discovery.
Acta crystallographica. Section F, Structural biology communications, 73 Pt 4
D. Hacker, Divor Kiseljak, Yashas Rajendra, S. Thurnheer, L. Baldi, F. Wurm (2013)
Polyethyleneimine-based transient gene expression processes for suspension-adapted HEK-293E and CHO-DG44 cells
Protein Expression and Purification, 92
(2018)
Chapter 23—Filtration methods for use in purification processes (concentration and buffer exchange)
Biopharmaceutical Processing
Margarida Viola, Joana Sequeira, Raquel Seiça, F. Veiga, J. Serra, A. Santos, A. Ribeiro (2018)
Subcutaneous delivery of monoclonal antibodies: How do we get there?
Journal of controlled release : official journal of the Controlled Release Society, 286
Z. An (2017)
“Magic Bullets” at the center stage of immune therapy: a special issue on therapeutic antibodies
Protein & Cell, 9
K. Izutsu (2018)
Applications of Freezing and Freeze-Drying in Pharmaceutical Formulations.
Advances in experimental medicine and biology, 1081
L. Ickenstein, P. Garidel (2018)
Hydrogel formulations for biologicals: current spotlight from a commercial perspective.
Therapeutic delivery, 9 3
Molly Hunter, P. Yuan, D. Vavilala, M. Fox (2018)
Optimization of Protein Expression in Mammalian Cells
Current Protocols in Protein Science, 95
A. Saxena, Donghui Wu (2016)
Advances in Therapeutic Fc Engineering – Modulation of IgG-Associated Effector Functions and Serum Half-life
Frontiers in Immunology, 7
J. Lambert, Charles Morris (2017)
Antibody–Drug Conjugates (ADCs) for Personalized Treatment of Solid Tumors: A Review
Advances in Therapy, 34
J. Lambert, A. Berkenblit (2018)
Antibody-Drug Conjugates for Cancer Treatment.
Annual review of medicine, 69
(2017)
Chapter 10—Alternatives to packed-bed chromatography for antibody extraction and purification
Process Scale Purification of Antibodies
Deepali Rathore, Anneliese Faustino, John Schiel, Eric Pang, M. Boyne, S. Rogstad (2018)
The role of mass spectrometry in the characterization of biologic protein products
Expert Review of Proteomics, 15
R. Jani, Gohil Krupa (2019)
Active targeting of nanoparticles: An innovative technology for drug delivery in cancer therapeutics
Journal of Drug Delivery and Therapeutics
K. Tran, Chandrasekhar Gurramkonda, Merideth Cooper, Manohar Pilli, Joseph Taris, Nicholas Selock, Tzu-Chiang Han, Michael Tolosa, Adil Zuber, Chariz Peñalber-Johnstone, Christina Dinkins, Niloufar Pezeshk, Y. Kostov, D. Frey, L. Tolosa, D. Wood, G. Rao (2018)
Cell‐free production of a therapeutic protein: Expression, purification, and characterization of recombinant streptokinase using a CHO lysate
Biotechnology and Bioengineering, 115
Chen Wang, Pan Xu, Luyu Zhang, Jing Huang, K. Zhu, C. Luo (2018)
Current Strategies and Applications for Precision Drug Design
Frontiers in Pharmacology, 9
Z. Elgundi, Mouhamad Reslan, Esteban Cruz, Vicki Sifniotis, Veysel Kayser (2017)
The state‐of‐play and future of antibody therapeutics☆
Advanced Drug Delivery Reviews, 122
Qun Wang, Yan Chen, M. Pelletier, R. Cvitkovic, J. Bonnell, Chien-ying Chang, Adem Koksal, Ellen O'Connor, Xizhe Gao, Xiang-Qing Yu, Herren Wu, C. Stover, W. Dall'acqua, Xiaodong Xiao (2017)
Enhancement of antibody functions through Fc multiplications
mAbs, 9
Daisuke Kuroda, K. Tsumoto (2018)
Antibody Affinity Maturation by Computational Design.
Methods in molecular biology, 1827
Andrew Martin, James Allen (2014)
Bioinformatics Tools for Analysis of Antibodies
(2018)
Inducible protein production in 293 cells using the piggybac transposon system
Recombinant Protein Expression in Mammalian Cells: Methods and Protocols
M. Lefranc, V. Giudicelli, P. Duroux, J. Jabado-Michaloud, G. Folch, Safa Aouinti, Emilie Carillon, Hugo Duvergey, Amélie Houles, T. Paysan-Lafosse, Saida Hadi-Saljoqi, S. Sasorith, G. Lefranc, S. Kossida (2014)
IMGT®, the international ImMunoGeneTics information system® 25 years on
Nucleic Acids Research, 43
(2018)
Rosettaantibodydesign (rabd): A general framework for computational antibody design
PLoS Comp. Biol., 14
Jian Li, He Wang, Yong-Chan Kwon, M. Jewett (2017)
Establishing a high yielding streptomyces‐based cell‐free protein synthesis system
Biotechnology and Bioengineering, 114
Chandrasekhar Gurramkonda, Aniruddha Rao, Shayan Borhani, Manohar Pilli, Sevda Deldari, X. Ge, Niloufar Pezeshk, Tzu-Chiang Han, Michael Tolosa, Y. Kostov, L. Tolosa, D. Wood, K. Vattem, D. Frey, G. Rao (2018)
Improving the recombinant human erythropoietin glycosylation using microsome supplementation in CHO cell‐free system
Biotechnology and Bioengineering, 115
Jordi Pujols, Samuel Peña-Díaz, S. Ventura (2018)
AGGRESCAN3D: Toward the Prediction of the Aggregation Propensities of Protein Structures.
Methods in molecular biology, 1762
Fleur Bovenkamp, N. Derksen, M. Breemen, S. Taeye, P. Heer, R. Sanders, T. Rispens (2018)
Variable Domain N-Linked Glycans Acquired During Antigen-Specific Immune Responses Can Contribute to Immunoglobulin G Antibody Stability
Frontiers in Immunology, 9
A. Jacobi, B. Enenkel, P. Garidel, C. Eckermann, M. Knappenberger, Ingo Presser, H. Kaufmann (2014)
Process Development and Manufacturing of Therapeutic Antibodies
F. Emami, A. Vatanara, Eun Park, D. Na (2018)
Drying Technologies for the Stability and Bioavailability of Biopharmaceuticals
Pharmaceutics, 10
A. Dangi, R. Sinha, Shailja Dwivedi, Sanjeev Gupta, Pratyoosh Shukla (2018)
Cell Line Techniques and Gene Editing Tools for Antibody Production: A Review
Frontiers in Pharmacology, 9
S. Balasubramanian (2018)
Recombinant CHO Cell Pool Generation Using piggyBac Transposon System.
Methods in molecular biology, 1850
(2018)
Chapter 8—Cell culture media in bioprocessing
Chung‐Jr Huang, Henry Lin, Xiaoming Yang (2012)
Industrial production of recombinant therapeutics in Escherichia coli and its recent advancements
Journal of Industrial Microbiology & Biotechnology, 39
Marcos Gil-Garcia, Manuel Bañó-Polo, N. Varejão, Michal Jamróz, Aleksander Kuriata, Marta Díaz-Caballero, Jara Lascorz, Bertrand Morel, Susanna Navarro, D. Reverter, Sebastian Kmiecik, S. Ventura (2018)
Combining Structural Aggregation Propensity and Stability Predictions To Redesign Protein Solubility.
Molecular pharmaceutics, 15 9
Mariana Santos, W. Quintilio, T. Manieri, L. Tsuruta, A. Moro (2018)
Advances and challenges in therapeutic monoclonal antibodies drug development
Brazilian Journal of Pharmaceutical Sciences, 54
Fatemeh Davami, F. Eghbalpour, F. Barkhordari, F. Mahboudi (2014)
Effect of Peptone Feeding on Transient Gene Expression Process in CHO DG44
Avicenna Journal of Medical Biotechnology, 6
K. Kemter, J. Altrichter, R. Derwand, Thomas Kriehuber, E. Reinauer, M. Scholz (2018)
Amino Acid-Based Advanced Liquid Formulation Development for Highly Concentrated Therapeutic Antibodies Balances Physical and Chemical Stability and Low Viscosity.
Biotechnology journal, 13 7
T. Lerch, P. Sharpe, S. Mayclin, T. Edwards, Eunhee Lee, H. Conlon, S. Polleck, J. Rouse, Yin Luo, Qin Zou (2017)
Infliximab crystal structures reveal insights into self-association
mAbs, 9
(2018)
Recombinant cho cell pool generation using piggybac transposon system
Recombinant Protein Expression in Mammalian Cells: Methods and Protocols
Alexander Kuhn, Sebastian Kube, Anne Karow-Zwick, Daniel Seeliger, P. Garidel, M. Blech, Lars Schäfer (2017)
Improved Solution-State Properties of Monoclonal Antibodies by Targeted Mutations.
The journal of physical chemistry. B, 121 48
J. Almagro, T. Daniels-Wells, S. Pérez-Tapia, M. Penichet (2018)
Progress and Challenges in the Design and Clinical Development of Antibodies for Cancer Therapy
Frontiers in Immunology, 8
Mouhamad Reslan, Vijayaraghavan Ranganathan, D. Macfarlane, Veysel Kayser (2018)
Choline ionic liquid enhances the stability of Herceptin® (trastuzumab).
Chemical communications, 54 75
Min You, Yanning Liu, Yingwei Chen, Jiyuan Guo, Jun Wu, Yajuan Fu, Rong Shen, Rui Qi, Wen-xin Luo, N. Xia (2013)
Maximizing Antibody Production in Suspension-Cultured Mammalian Cells by the Customized Transient Gene Expression Method
Bioscience, Biotechnology, and Biochemistry, 77
M. Dalziel, S. Beers, M. Cragg, M. Crispin (2018)
Through the barricades: overcoming the barriers to effective antibody-based cancer therapeutics.
Glycobiology, 28 9
M. Tada, K. Tatematsu, A. Ishii-Watabe, A. Harazono, Daisuke Takakura, N. Hashii, H. Sezutsu, N. Kawasaki (2015)
Characterization of anti-CD20 monoclonal antibody produced by transgenic silkworms (Bombyx mori)
mAbs, 7
R. Branco, A. Dias, A. Roque (2012)
Understanding the molecular recognition between antibody fragments and protein A biomimetic ligand.
Journal of chromatography. A, 1244
C. Klein (2018)
Special Issue: Monoclonal Antibodies
Antibodies, 7
Marlitt Stech, O. Nikolaeva, O. Nikolaeva, Lena Thoring, Lena Thoring, W. Stöcklein, D. Wüstenhagen, M. Hust, S. Dübel, S. Kubick (2017)
Cell-free synthesis of functional antibodies using a coupled in vitro transcription-translation system based on CHO cell lysates
Scientific Reports, 7
C. Thomson (2016)
IgG Structure and Function
D. Ecker, S. Jones, H. Levine (2015)
The therapeutic monoclonal antibody market
mAbs, 7
Xinhua Wang, M. Mathieu, Randall Brezski (2017)
IgG Fc engineering to modulate antibody effector functions
Protein & Cell, 9
Min You, Yi Yang, Chuanqi Zhong, Fentian Chen, Xin Wang, Tianrong Jia, Yuanzhi Chen, Bing-xi Zhou, Qingyu Mi, Qinjian Zhao, Z. An, Wen-xin Luo, N. Xia (2018)
Efficient mAb production in CHO cells with optimized signal peptide, codon, and UTR
Applied Microbiology and Biotechnology, 102
Hitomi Nakamura, N. Oda-Ueda, T. Ueda, T. Ohkuri (2018)
Introduction of a glycosylation site in the constant region decreases the aggregation of adalimumab Fab.
Biochemical and biophysical research communications, 503 2
S. Awwad, Ukrit Angkawinitwong (2018)
Overview of Antibody Drug Delivery
Pharmaceutics, 10
Jacob Blaffert, H. Haeri, M. Blech, D. Hinderberger, P. Garidel (2018)
Spectroscopic methods for assessing the molecular origins of macroscopic solution properties of highly concentrated liquid protein solutions.
Analytical biochemistry, 561-562
Tia Arena, Peter Harms, Athena Wong (2018)
High Throughput Transfection of HEK293 Cells for Transient Protein Production.
Methods in molecular biology, 1850
Jianwen Hu, Jizhong Han, Haoran Li, Xian Zhang, Lanlan Liu, Fei Chen, B. Zeng (2018)
Human Embryonic Kidney 293 Cells: A Vehicle for Biopharmaceutical Manufacturing, Structural Biology, and Electrophysiology
Cells Tissues Organs, 205
B. Booth, B. Ramakrishnan, Kristin Narayan, A. Wollacott, G. Babcock, Z. Shriver, Karthik Viswanathan (2018)
Extending human IgG half-life using structure-guided design
mAbs, 10
M. Yu, Jun Wu, Jinjun Shi, O. Farokhzad (2016)
Nanotechnology for protein delivery: Overview and perspectives.
Journal of controlled release : official journal of the Controlled Release Society, 240
Marlitt Stech, S. Kubick, S. Ohtake (2015)
Cell-Free Synthesis Meets Antibody Production: A Review
Antibodies, 4
Yashas Rajendra, Divor Kiseljak, L. Baldi, D. Hacker, F. Wurm (2011)
A simple high-yielding process for transient gene expression in CHO cells.
Journal of biotechnology, 153 1-2
D. Schweizer, T. Serno, A. Goepferich (2014)
Controlled release of therapeutic antibody formats.
European journal of pharmaceutics and biopharmaceutics : official journal of Arbeitsgemeinschaft fur Pharmazeutische Verfahrenstechnik e.V, 88 2
(2019)
Chapter 7—Purification of human monoclonal antibodies and their fragments. In Human monoclonal Antibodies: Methods and Protocols
Thapakorn Jaroentomeechai, Jessica Stark, A. Natarajan, Cameron Glasscock, Laura Yates, Karen Hsu, M. Mrksich, M. Jewett, M. DeLisa (2018)
Single-pot glycoprotein biosynthesis using a cell-free transcription-translation system enriched with glycosylation machinery
Nature Communications, 9
Sandeep Kumar, N. Plotnikov, J. Rouse, Satishwer Singh (2018)
Biopharmaceutical Informatics: supporting biologic drug development via molecular modelling and informatics
Journal of Pharmacy and Pharmacology, 70
A. Anselmo, Y. Gokarn, S. Mitragotri (2018)
Non-invasive delivery strategies for biologics
Nature Reviews Drug Discovery, 18
(2017)
Chapter 3—Harvest and recovery of monoclonal antibodies: Cell removal and clarification
Process Scale Purification of Antibodies
(2018)
Platforms for recombinant therapeutic glycoprotein production
Recombinant Glycoprotein Production, Volume 1674
Nripen Singh, S. Chollangi (2017)
NEXT‐GENERATION CLARIFICATION TECHNOLOGIES FOR THE DOWNSTREAM PROCESSING OF ANTIBODIES
Mouhamad Reslan, Veysel Kayser (2018)
The effect of deuterium oxide on the conformational stability and aggregation of bovine serum albumin
Pharmaceutical Development and Technology, 23
Sharon Fischman, Yanay Ofran (2018)
Computational design of antibodies.
Current opinion in structural biology, 51
C. Jackisch, So-Woon Kim, V. Semiglazov, B. Melichar, X. Pivot, C. Hillenbach, D. Stroyakovskiy, Bert Lum, R. Elliott, H. Weber, Gustavo Ismael (2015)
Subcutaneous versus intravenous formulation of trastuzumab for HER2-positive early breast cancer: updated results from the phase III HannaH study.
Annals of oncology : official journal of the European Society for Medical Oncology, 26 2
T. Nero, M. Parker, C. Morton (2018)
Protein structure and computational drug discovery.
Biochemical Society transactions, 46 5
Jake Pawlowski, Adriana Bajardi-Taccioli, Damian Houde, Marina Feschenko, Tyler Carlage, I. Kaltashov (2018)
Influence of glycan modification on IgG1 biochemical and biophysical properties
Journal of Pharmaceutical and Biomedical Analysis, 151
M. Sanford (2014)
Subcutaneous trastuzumab: a review of its use in HER2-positive breast cancer
Targeted Oncology, 9
F. Mahboudi, M. Abolhassan, A. Azarpanah, Hamideh Aghajani-Lazarjani, Mohammad Sadeghi-Haskoo, S. Maleknia, B. Vaziri (2013)
The Role of Different Supplements in Expression Level of Monoclonal Antibody against Human CD20
Avicenna Journal of Medical Biotechnology, 5
This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license
Adam Fisher, M. Kamga, C. Agarabi, K. Brorson, Sau Lee, Seongkyu Yoon (2019)
The Current Scientific and Regulatory Landscape in Advancing Integrated Continuous Biopharmaceutical Manufacturing.
Trends in biotechnology, 37 3

Publisher: Multidisciplinary Digital Publishing Institute
Copyright: © 1996-2019 MDPI (Basel, Switzerland) unless otherwise stated
ISSN: 2073-4468
DOI: 10.3390/antib8020036
Publisher site: See Article on Publisher Site

Abstract

antibodies Review Current Advancements in Addressing Key Challenges of Therapeutic Antibody Design, Manufacture, and Formulation Vicki Sifniotis , Esteban Cruz, Barbaros Eroglu and Veysel Kayser * School of Pharmacy, Faculty of Medicine and Health, The University of Sydney, Sydney 2006, Australia; vsif0221@uni.sydney.edu.au (V.S.); ecru7298@uni.sydney.edu.au (E.C.); barbaros.eroglu@sydney.edu.au (B.E.) * Correspondence: veysel.kayser@sydney.edu.au; Tel.: +61-2-9351-3391 Received: 16 April 2019; Accepted: 31 May 2019; Published: 3 June 2019 Abstract: Therapeutic antibody technology heavily dominates the biologics market and continues to present as a signiﬁcant industrial interest in developing novel and improved antibody treatment strategies. Many noteworthy advancements in the last decades have propelled the success of antibody development; however, there are still opportunities for improvement. In considering such interest to develop antibody therapies, this review summarizes the array of challenges and considerations faced in the design, manufacture, and formulation of therapeutic antibodies, such as stability, bioavailability and immunological engagement. We discuss the advancement of technologies that address these challenges, highlighting key antibody engineered formats that have been adapted. Furthermore, we examine the implication of novel formulation technologies such as nanocarrier delivery systems for the potential to formulate for pulmonary delivery. Finally, we comprehensively discuss developments in computational approaches for the strategic design of antibodies with modulated functions. Keywords: therapeutic antibody; stability; aggregation; manufacture challenges; formulation 1. Introduction Since the ﬁrst therapeutic monoclonal antibody (mAb) Orthoclone OKT3 (Janssen Biotech, Horsham, PA, USA) was approved by the USA Food and Drug Administration in 1986, whole antibody therapeutics have become and persistently remain the most dominant and signiﬁcant biologic therapeutic platform in the pharmaceutical industry [1,2]. To date, therapeutic antibodies treat a plethora of indications including cancers, infections, autoimmune disorders, and cardiovascular and neurological diseases [3]. The whole antibody therapeutics platform is regarded as the most promising class of pharmaceutical technology to date; it is continually being applied to newly identiﬁed biological targets and implemented in many formats to produce strategically engineered next generation antibody therapeutics, otherwise termed “biobetters” [4–9]. The international ImMunoGeneTics information ® ® system (IMGT , Montpellier, France) database reveals that as of December 2018, 65 whole antibodies and 18 next generation fragment or recombinant fusion antibody-based therapies are approved for clinical use, with hundreds more in clinical trials expected to reach market. Antibodies 2019, 8, 36; doi:10.3390/antib8020036 www.mdpi.com/journal/antibodies Antibodies 2019, 8, 36 2 of 23 Naked Whole mAbs Whole mAb formats 65 Naked whole mAbs 60 ADCs ADCs 4 Whole mAb bispecifics 1 Whole mAb Bispecifics Biosimilars Fragment mAb formats 18 Fabs Fabs 4 Fc fusions 10 Fc Fusions scFv fusions 3 scFv bispecifics (BiTE) 1 scFv Fusions Originator mAbs with scFv Bispecific (BiTE) approved biosimilars (a) (b) Figure 1. The proportions of therapeutic antibody formats approved for therapeutic use as of December , ® 2018 IMGT depicted through (a) a pie chart and (b) a table format. Whole therapeutic mAbs are presently the dominant antibody platform approved for clinical use (Figure 1), although antibody engineering technologies have advanced in recent years to produce highly optimized, strategically engineered biobetter therapies, along with biosimilar mAbs reaching market to compete against their originator. Further whole mAb formats include antibody–drug conjugates (ADCs), bispeciﬁcs, isotype-switched, and glycoengineered. These additional formats have been strategically designed to introduce exceptional potency, to engage dual biological targets, and to modulate Fc eector functions. Fragments of mAbs such as the crystallizable fragment (Fc), antigen binding fragment (Fab), and single-chain variable fragment (scFv) possess key functions such as speciﬁcity to a biological target or immunological activation. The isolation of these fragments for fusion with other mAb fragments, biologically functional proteins, cytotoxic drugs, or drug carriers has been the crux of ingenuity in developing the next generation of biobetter therapies [4–15]. Figure 2 depicts several examples of prominent biobetter formats, providing a general representation of current fragment mAbs, whole mAb bispeciﬁcs, fragment mAb multispeciﬁcs, and fragment mAb fusion therapeutics. Antibodies 2019, 8, 36 3 of 23 Figure 2. Schematic representation of a whole monoclonal antibody (mAb), a fragment mAb, and prominent fusion mAb formats that have been developed for strategic therapeutic uses. Proteins fused to mAb fragments are depicted as blue ovals for a general representation; however, fusion proteins may vary in size and structure. Fragment formats include the crystallizable (Fc), antigen binding (Fab and F(ab)2), and single-chain variable (scFv) fragments. Further whole mAb formats include the antibody–drug conjugate (ADC), triomab, dual variable domain immunoglobulin (DVD-Ig), and immunoglobulin–scFv fusion (IgG-scFv). Multispeciﬁc fragment formats include the F(ab)2 bispeciﬁc, bispeciﬁc T-cell engager (BiTE), dual anity re-targeting molecule (DART), and tandem diabody (tandAb). 2. Overview of mAb Production Challenges and Considerations Whole therapeutic mAbs require a mammalian expression system to produce the biologically functional product; however, a mAb fragment and recombinant fusion mAb products with simpliﬁed (or lacking) glycosylation are suitable for lower organism expression platforms [16]. Unlike oligopeptides, which can be chemically synthesized, whole therapeutic mAbs are considerably larger (with monomer ranging from 140–160 kDa) and comprise of four peptide chains (two heavy and two light chains) bound together by disulﬁde bonds and interchain non-covalent interactions. Further to this, antibodies contain glycosylation in a conserved region of the Fc (N297) that contributes Antibodies 2019, 8, 36 4 of 23 to its stability and immune eector functions [17,18]. Aside from peptide synthesis, cell machinery is required to glycosylate, fold, orient, and covalently bind the antibody peptide chains in order to produce the complete, biologically functional antibody product. Manufacturing biologically functional whole mAb product is therefore commercially unfeasible through chemical synthesis and insucient in lower organism expression platforms such as bacteria, yeast, insect, and plant cells that may not have the machinery to produce the equivalent tertiary structure and glycosylation proﬁles. In particular, many industrially relevant bacterial strains such as E. coli are completely deﬁcient in the machinery to add post-translational glycosylations; yeasts hyper-mannosylate glycans, which cause immunogenicity; and insect cells which are deﬁcient in sialylation machinery and produce immunogenic glycan structures [16]. A secretion of the mAb product for puriﬁcation is suboptimal for several lower organism expression platforms such as E. coli due to poor productivity and harsh culture conditions that promote product degradation. Protein is therefore produced intracellularly, as inclusion bodies and harvest involves further processing steps such as cell lysis, inclusion body recovery, protein solubilization, and renaturation prior to further downstream puriﬁcation steps [19,20]. Despite these pitfalls, the development of lower organism expression systems is of high commercial interest due to the simpliﬁed culture conditions, cheaper media requirements, rapid organism growth, and higher product yield as compared to mammalian expression systems [16,21]. In considering the requirements through the entire process of mAb discovery, manufacture, formulation, and disease treatment, several key challenges arise which have sparked overwhelming interest in pursuit of achieving better mAb manufacturing outcomes and treatment strategies. As with all biotherapeutics, mAbs and mAb-based therapeutics are limited to production in cell-based expression systems, which is considerably costly and inecient, can have varied yields depending on the product and expression system, and requires downstream processing to remove biological contaminants introduced from the expression system. Despite anity chromatography being a robust technology for the initial capture of a mAb for puriﬁcation, the capture process and further downstream processes such as viral inactivation applies the mAb product to harsh pH and salt conditions, which can chemically degrade the mAb, leading to product instability and loss [5,22,23]. Many factors through the manufacture process inﬂuence glycosylation and charge heterogeneity of mAbs, which aects their biophysical and pharmacological properties. Though not speciﬁcally discussed in this review, the improvement and control mAb production technologies address these variations to reduce formulation heterogeneity and o-target cytotoxicities. A common challenge, as seen with all biotherapeutics, is that mAbs and mAb-based therapeutics are currently restricted to lyophilised and liquid-based formulations for intravenous (IV) or subcutaneous (SC) delivery to achieve maximum bioavailability. Protein self-association and intrinsic stability drive this limitation, in that viscosity and propensity to aggregate are dependent on mAb concentration. Formulations are optimized to achieve the highest dosing concentration at the minimum achievable volume for injection, without compromising the quality of the mAb in formulation [24]. Viscosity remains a key limiting factor for formulating as a SC administration—certain mAb therapies are suitable and others not based on their solubility, self-association, and aggregation proﬁles. Alternative non-invasive administration strategies such as pulmonary delivery causes additional mechanical stress that further contribute to mAb instability and loss. Furthermore, oral delivery is unsuitable due to chemical and enzymatic degradation, as well as poor absorption in the gastric and intestinal environments [5,25–29]. A brief overview of considerations through the dierent concept stages of therapeutic mAb development is depicted in Figure 3. The main challenges and considerations in the manufacture and formulation of mAb therapeutics are brieﬂy summarized in Table 1. Antibodies 2019, 8, 36 5 of 23 Figure 3. Schematic representation of the concept stages of mAb drug development in which considerations follow on from one process to the next in the design and manufacture of mAb-based therapeutics. Antibodies 2019, 8, 36 6 of 23 Table 1. Summary of key technological advancements that address challenges and considerations in mAb design, manufacture, and formulation strategies. Challenges Advancements Manufacture Considerations Hybridoma technology produces immunogenic mAbs Humanization technologies [4–6] Yield from hybridoma technology is variable Commercial cell line development and recombinant technology [4–6]; lower organism systems for fragment mAbs, mammalian systems for whole mAbs, and Fc Signiﬁcance of post-translational modiﬁcations and higher-order structure in fusions [16,30,31] mAb product CHO expressed mAbs contain an immunogenic glycosylation proﬁle Human-based expression systems [16,30–32] HEK 293 expressed mAbs are prone to aggregation HKB-11 and PER.C6 cell lines [32] Undesirable byproducts produced in the manufacture process in vitro cell-free synthesis technology [33,34] Stability of mAb aects manufacture yield due to product loss through aggregation in Enhancing mAb stability through framework mutations and hyperglycosylation downstream processing steps [5,35–40] Treatment Considerations for Drug Design and Formulation Susceptibility of mAb to degradation limits delivery to intravenous and subcutaneous only Enhancing mAb stability through framework mutations, hyperglycosylation [5,35–40], and nanocarrier technologies [8,41] Stability of mAb limits concentration of formulation Concentration of mAb aects viscosity and injection pressure for Excipients, fragment mAbs, PEGylation, and hyperglycosylation [25–27,41] subcutaneous delivery Poor tissue penetration and biodistribution Fragment mAbs and nanocarrier technologies [8,41,42]; high anity ADCs [43–45] PEGylation, hyper-glycosylation and Fc fusion proteins [8,41,46]; modulation of FcRn Reduced half-life in low MW species recycling through Fc mutation [11,13] Modulation of immunological engagement Isotype switching, glycoengineering, and Fc mutations [11,13,15,43,46] Antibodies 2019, 8, 36 7 of 23 3. mAb Discovery and Manufacture Technologies Traditional technology for whole therapeutic antibody discovery required the immunization of animals—primarily mice with a target antigen for the generation of a mixed population of B-lymphocytes-producing antibody against the target. The B-lymphocytes would be isolated for immortalization to produce monoclonal hybridoma cell lines that secrete antibody candidates of interest for further panning through display technologies to isolate potential leads [5]. This method has led to the generation of highly speciﬁc mAb libraries of non-human origin that were potentially immunogenic and less ecacious at eliciting an immune response as compared to wholly human mAbs. Technologies arose to address immunogenicity by producing chimeric and humanized antibodies through the grafting of the variable domains (chimeric) or complementarity-determining region (CDR) residues (humanized) isolated from lead non-human antibodies to a human antibody framework. To further improve the humanization strategy, transgenic mice were developed with their murine antibody heavy and light chain genes replaced with equivalent human genes; they consequently expressed wholly human antibodies for discovery [4–6]. Phage display technology remains the most prominent selection technology for panning antibody gene pools for speciﬁcity to a target antigen, as it eectively couples the expression of mAb proteins to the genes that encode them for the panning of high anity leads. Antibody variable genes from B-lymphocyte pools are isolated through polymerase chain reaction (PCR), and cloned into a phage expression vector that presents the expressed mAb on the surface of the phage, the library of vectors is rescued in phage, and the phage library is screened against a speciﬁc target antigen. Phage pools undergo several rounds of selection to isolate high anity candidates, the variable genes of the lead mAb candidates can then be isolated and sequenced for the design of a mAb drug format and the development of a suitable expression platform [5,47]. Error-prone PCR-based mutagenesis has propelled display technology for the generation of enormous libraries for target anity screening. Yeast surface display further improves these screening technologies, as glycosylation sites introduced in CDRs are expressed through this platform [5,48]. The expression, yield, and quality of a mAb can vary quite substantially in hybridoma cell lines. High yielding mammalian expression systems have been developed to meet the commercial need for high expression eciency, scalability, quality, and reproducibility. Antibody genes of interest are introduced into suitable expression vectors and transfected into highly ecient mammalian cell lines for antibody expression and secretion; a mAb can then be directly captured from the culture supernatant through anity chromatography. Currently, the majority of mammalian expression systems for commercial whole therapeutic antibody expression are based on stable chinese hamster ovary (CHO), mouse myeloma (NS0), and mouse hybridoma (Sp2/0) cell lines [4,5]. However, in the context of research and development, the transient expression in human cell lines such as embryonic kidney (HEK 293), amniotic (CAP), a hybrid of HEK 293 and lymphoma (HKB-11), and embryonic retina (PER.C6) are favored over stable CHO expression due to the ease and speed of production of workable quantities of antibody for preliminary studies [16,30–32,49,50]. The generated mAb library undergoes relevant in vitro testing along with formulation stability screening to exclude candidates that have poor manufacturability attributes [51]. Lead mAb candidates that show potential for further investigation would then be developed for high yielding stable expression and more rigorous characterization leading up to therapeutic development. However, a drawback from changing from human to hamster expression systems is the dierence in glycosylation proﬁle. CHO expressed mAbs produce immunogenic non-human glycoform Neu5Gc and have a higher composition of sialylation, which may result in reduced antibody-dependent cellular cytotoxicity (ADCC). Therefore in the early stages of development, leads need to be identiﬁed and thoroughly characterized in the expression system to be developed for commercial manufacture before committing to industrial scale up [16,30,32]. Amongst other biotherapeutics, approved mAb Fc fusion therapeutics dulglutide (Trulicity , Eli Lilly, ® ® Indianapolis, IN USA), efmoroctocog alpha and eftrenonacog alpha (Eloctate and Alprolix , Biogen, Cambridge, MA, USA), are manufactured in a HEK 293 expression system, which is giving rise to Antibodies 2019, 8, 36 8 of 23 the acceptance of human-based expression systems for the production of mAb-based therapeutics [4]. However, HEK 293 expression systems are prone to inducing mAb aggregation in cultures, which is detrimental to cell viability and creates a loss of product during manufacture. Hence, HKB-11 and PER.C6 are the preferred commercial human cell lines for mAb expression in human cell lines, speciﬁcally [16,31,32]. Established lower organism expression systems for fragment or recombinant fusion mAb therapeutics include bacteria such as E. coli, yeasts such as S. cerevisiae and P. pastoris, plants such as tobacco, algae, and insects such as silkworm [5,16,52–54]. Expression platforms that use E. coli in particular are considered high risk due to the potential endotoxin contamination in the mAb product, of which complete endotoxin removal requires further puriﬁcation steps [19]. However, several approved mAb fragment- and recombinant-based therapies are produced in an E. coli-based expression system, including pegol conjugated Fab’ certolizumab pegol (Cimzia , Celltech UCB, Brussels, Belgium), Fab ranibizumab (Lucentis , Genentech, San Francisco, CA, USA), and recombinant Fc fusion romiplostim (Nplate , Amgen, Thousand Oaks, CA, USA). Lower organism expression platforms such as E. coli and P. pastoris continue to be the preferred option for the manufacture of fragment mAb formats due to the relative ease, high yield, and reduced cost for manufacture as compared to mammalian expression platforms. An emerging in vitro cell-free synthesis technology is being developed with bacterial and CHO cell lysates which have the potential to alleviate formation of undesirable biological byproducts, as the machinery from the cell lysates purely express protein from the mAb genes that are introduced to the system. Though this technology is not currently applicable for industrial scale manufacture, it holds much promise as an alternative to live culture. For instance, culture maintenance and highly deﬁned media are no longer necessary, reactions can run continuously, lysates can be recycled with reproducible results, unmodiﬁed linear DNA is suitable for the system (thus alleviating a need for multiple cloning steps), and additional enzymes can be introduced to the system for the engineering of speciﬁc post-translational modiﬁcations [33,34,55–58]. Despite the apparent advantages of this technology for mAb manufacture, cell lysates encounter the same challenges as their host cell expression system. That is, post-translational modiﬁcations of mAb product from lysates from lower level organisms are limited by the endogenous machinery of that host organism. Furthermore, preparation of mammalian cell lysates is challenging, and yield produced from these lysates is considerably low [59,60]. Current bioprocess engineering and cell line development strategies have been crucial in enhancing the manufacturability of mAbs. Many current mAb therapeutic expression platforms make use of CHO cell lines that have been commercially developed with dihydrofolate reductase or glutamine synthetase deﬁciency for enhanced stability selection through resistance to methotrexate and methionine sulfoximine inhibition, respectively [50,61]. Mammalian cell lines have been adapted to suspension culture, as they support higher cell densities and mAb titers in the absence of serum from the culture media, for large scale fed-batches, perfusion systems, or continuous culture systems. Further to this, cell lines are continually being engineered for enhanced metabolic functions, introducing glycosylation pathways, superior secretion, and resistance to apoptosis for prolonged survival, in eorts to generate super-producer cell lines that can sustain a continuous culture [62–68]. Gene and expression vector design and development are speciﬁcally tailored to the expression system to maximize mAb expression and cell line stabilization through host cell codon optimization and the addition of highly ecient transcription, secretion, selection, and integration elements [69–73]. Vectors can contain a single site for gene insertion for the subsequent co-transfection of vectors that carry the antibody heavy and light chains, or both chains can be cloned into a dual expression vector. In more elaborately designed recombinant mAb drug formats that require the expression of several genes, vector technologies have implemented multicistronic expression to enhance the eciency of the vector system. The stable or transient transfection of vectors is performed on highly viable cell cultures with high cell density. Antibody heavy and light chain ratios may require adjustment in transfection for optimizing expression and secretion. A reporter vector that expresses green ﬂuorescent protein is typically included Antibodies 2019, 8, 36 9 of 23 to observe transfection eciency during optimization [74,75]. Liposome-mediated transfection is preferential over all other mammalian transfection strategies and is induced chemically through the complexing of DNA to a cationic lipid prior to or during addition to a culture. Polyethylenimine is the most prevalently used transfection reagent despite the development of superior reagents such as Lipofectamine— 2000 and Freestyle— MAX (Thermo Fisher Scientiﬁc, Waltham, MA, USA), LyoVec— (InvivoGen, San Diego, CA, USA), FuGENE 6— (Promega, Madison, WI, USA), and TransIT-PRO (Mirus, Madison, WI, USA), owing to its lower cost and relative eciency [73,75–78]. The commercial manufacture of mAbs has transitioned to serum-free, chemically deﬁned, and animal-free media which has removed a source of expression variability and has been driven particularly from the advent of mad cow disease [50,79]. Several media supplements such as plant and yeast digests (peptones/hydrolysates), surfactants (e.g., Pluronic F-68), DNA methyltransferase (azacytidine), and histone deacetylase (sodium butyrate, valproic acid) inhibitors have been found to support cell viability and enhance mAb expression [79–85]. Mild hypothermia of the culture has also demonstrated to reduce cell expansion, support prolonged cell viability, and enhance mAb expression [73,86–90]. Further to this, continuous supplementation in expression cultures of uridine, manganese chloride, and galactose demonstrated successful the glycan engineering of expressed mAb, where mAb hyper-galactosylation was promoted to enhance complement-dependent cytotoxicity (CDC) activity [91]. In the manufacturing process of fed-batch, perfusion, and continuous culture systems, media is continuously fed in the culture system, whilst parameters such as cell viability, cell density, and metabolite levels are monitored in real time until the parameters indicate that it is the optimal time to harvest the expressed mAb from the culture supernatant. In perfusion and continuous culture systems, a feed is removed from the culture simultaneously to the media addition, the dierence being that the cell dilution rate in continuous culture is optimized to remain equal or higher than the cell growth rate, which allows the perpetuation of mAb expression and requires the continuous harvest of the supernatant [92–94]. The harvest of the supernatant requires clariﬁcation technologies such as the use of precipitants/ﬂocculants (e.g., polyethylene glycol, diethylaminoethyl dextran, caprylic acid, and polyethylenimine), high throughput centrifugation, and ﬁltration methods to remove cells and biological debris, for the lowering the loading burden in the lead up to mAb capture through anity chromatography [19,95–100]. The mechanical stresses from centrifugation and ﬁltration are unavoidable, although they can induce mild mAb product loss through fragmentation and aggregation. Protein A-ligand-based anity chromatography is the most robust, ecient, and prevalently used mAb capture technology, owing to its selectivity and high anity to various human Fc, as well as its ecient dissociation of captured mAbs at a low pH for reuse. Fragment mAbs and recombinant formats based on Fabs and single chain variable fragments (scFv) are unable to take advantage of protein A anity capture, and alternatives have been developed, such as capturing proteins G, M, and L, which bind at dierent mAb epitopes; ion exchange chromatography; and polyhistidine tagged capture through immobilized metal chromatography [100–102]. A further advantage of anity chromatography in manufacture is the integration of a viral inactivation step by holding the low pH-eluted mAb product prior to further puriﬁcation steps; however, this applies the mAb product to low pH at high concentrations, which can induce mild mAb instability, aggregation, and the formation of acidic variants [5,22,100,103,104]. Post mAb capture, further polishing steps are required to remove further contaminants such as host cell proteins and DNA, as well as leached anity chromatography ligand- and mAb-degradation products such as fragments, aggregates, and ionic variants. The polishing steps are designed based on the speciﬁc properties of the mAb product, such as the isoelectric point and molecular weight (MW), to implement appropriate chromatographic technologies such as anion and cation exchanges, hydrophobic interaction, and multimodal and size exclusion that will separate the mAb product from the manufacture-introduced impurities [22,100,103]. The additional chromatographic steps unavoidably apply the mAb product to buers of varying ionic strength and high concentrations, which can again induce mild mAb instability and aggregation. Post polishing steps, the puriﬁed Antibodies 2019, 8, 36 10 of 23 mAb product undergoes a further viral removal step, such as ﬁltration, and then it undergoes buer exchange and concentration through ultraﬁltration or diaﬁltration methods to prepare the bulk mAb product for formulation [100,105]. 4. Formulation Strategies and Considerations Strategies to improve the formulation of mAbs and mAb-based therapies continues to be an ongoing challenge, as is faced with all biotherapeutics. Firstly, whole therapeutic mAbs are unsuitable for non-invasive oral, nasal, or pulmonary routes of administration as they are susceptible to chemical and enzymatic degradation in the gastrointestinal tract. The bioavailability of mAbs through these routes is poor, owing to mAbs’ polar surface charge and relatively large MW, limiting transport through mucosal membranes. Fragment mAb platforms, together with excipient and PEGylation technologies, have been developed to help circumvent transportation limitations for pulmonary delivery, speciﬁcally. However, physical stresses applied to the mAb (e.g., shear stresses from the aerosolization of liquid formulations for a pressurized meter dose and a nebulization delivery, or from the production of dry powder for inhalation) pose further challenges of mAb instability, leading to degradation and reduced ecacy. [8,26,28]. Few biologics have been successfully approved for pulmonary delivery, most notably insulin formulation Afrezza (MannKind, Westlake Village, CA, USA), and, currently, an erythropoietin-Fc fusion and a nanobody-targeting respiratory syncytial virus are in clinical trials, showing promise for the further development of this administration strategy for both the systemic and localized delivery of mAb-based therapeutics [5,28]. Several drug carrier technologies such as microencapsulation, liposome, and nanoparticle formulations inherently enhance the stability and control the release of mAbs, which can prolong their half-life. These nanocarrier formulation strategies are of intense interest, as they hold promise for developing less invasive inhaled formulations of mAb-based therapeutics with superior attributes to currently established formulations [41,106–109]. The majority of currently approved mAb therapeutics are formulated for IV, although commercial interest has directed technologies to develop injectable mAb formulations which has seen success in many approved mAb therapies thus far, primarily SC for systemic delivery, along with a few intravitreal and intramuscular formulations for tissue-speciﬁc indications. Amongst others, anti-HER2 antibody trastuzumab was originally developed as an IV formulation and was successfully repurposed as a SC formulation [110–112], whereas next generation therapies have directly moved towards SC formulation, such as with anti-TNF- antibody adalimumab [113]. Injectable administrations oer several advantages over IV administration, especially in the treatment of chronic diseases in regards to a reduced burden to allied health services, patient tolerance, and adherence to treatment; however, the intrinsic physicochemical properties of mAbs may be undesirable for injectable formulation. In considering the low volume, injection pressure, and the typically high (>100 mg) eective dose of mAb for injectable delivery, the viscosity and aggregation propensity of mAbs become key formulation challenges to address, as they are dependent on mAb concentration. Certain mAb therapies are suitable, and others are not based on their solubility, viscosity, self-association, intrinsic stability, aggregation, and precipitation proﬁles. Injectable mAb formulations can be further improved with the use of excipients to increase solubility, reduce viscosity, and enhance the stability of mAbs. Excipients are considered for a formulation based on their physicochemical properties, pharmacokinetics, and safety. For example, polysorbates are commonly used in biologics as a stabilizing agent; however, their addition in high concentrations can denature proteins and cause adverse side eects such as injection site reactions [114–116]. Injectable mAb formulations are co-formulated with recombinant human hyaluronidase, speciﬁcally as a permeation enhancer for more ecient absorption into tissue, although the inclusion of this additional biologic adds further burden to the formulation’s viscosity and propensity to aggregate [5,8,25–28,117–123]. Antibody therapies for IV administration are prepared as Antibodies 2019, 8, 36 11 of 23 lyophilised powder for reconstitution and further dilution, and injectable administrations are prepared as liquid-based formulations in pre-ﬁlled syringes. Liquid formulations of mAbs are more susceptible to physiochemical degradation, are less stable, and have a reduced shelf-life as compared to lyophilised formulations. However, drying technologies to produce lyophilised formulations apply the mAb to physical stresses that induce instability and degradation, leading to reduced ecacy [124–126]. Long-term stability predictions are elucidated from formulation screening in accelerated aggregation studies, as part of the preliminary screening process of generated mAb libraries [127]. The stability proﬁles of mAbs can be greatly aected by the amino acid composition, structure, and potential glycosylation in their variable region CDRs that are isolated through the screening and maturation process. Elucidating the causes for reduced solubility or increased aggregation propensity has to be considered together with the molecular interactions between the mAb and the biological target as to not compromise anity. Computational tools have been developed to elucidate amino acids within the mAb CDR structure that have a high propensity to aggregate, and strategies for improving solubility and aggregation proﬁles have been developed through direct amino acid substitutions and the strategic addition of glycosylation sites [128–132]. The characterization of mAb stability and target interaction for optimization is considered a fundamental step as part of preliminary drug discovery, as the applicability for development, manufacture, and formulation of the mAb therapeutic is governed by the mAbs stability proﬁle. 5. Improving mAb Tissue Penetration for Cancer Treatment Antibody therapies are currently restricted to invasive parenteral routes of administration for maximum bioavailability and systemic distribution; however, delivery to the speciﬁc target tissue, such as tumors for cancer treatments, continues to be a challenge. Penetration to tissue from blood vessels is again poor, owing to mAbs’ polar surface charge and relatively large MW, limiting transport through physiological barriers. The eciency of tissue penetration is further inﬂuenced by systemic and local mAb clearance rates. Enhancement to mAbs’ anity to their biological target through maturation and strategic mutation technologies have led to faster diusion rates of mAb to target for increased ecacy. However, for tumourous tissue speciﬁcally, penetration through tumors is restricted by the tumor ’s binding site barrier, in which antigens expressed on the tumors periphery capture the majority of the mAb released to surrounding tissue; this eect is exaggerated with overexpressed antigen. The enhancement of whole mAbs’ anity beyond 1 nM has speciﬁcally shown that there is no further improvement of tumor diusion rates, tissue penetration, and accumulation [42,43,133]. An increased anity of mAbs to cellular targets also leads to the increased uptake, internalization, and catabolism of mAbs, which reduces ADCC and increases the mAb clearance rate. This mechanism is exploited through the development of high anity ADCs to target the delivery and release of potent drugs, inducing targeted cell death [43–45]. Fragment mAb platforms have shown better tissue penetration and biodistribution than whole mAb therapeutics; however, a pitfall of smaller peptides lacking an Fc region is a highly reduced in vivo half-life and poor retention times. Technologies to improve the half-life of mAb fragments have been primarily through PEGylation, along with strategic Fc mutation and glycosylation engineering to enhance neonatal Fc receptor (FcRn) recycling. Furthermore, hyper-glycosylation technology has been successfully applied in other biotherapeutics, such as with glycosylated erythropoietin Aranesp (Amgen, Thousand Oaks, CA, USA). Conversely, nanocarrier platforms that are relatively larger as compared to whole mAbs have demonstrated superior pharmacokinetics and tumor retention, as well as previously mentioned enhanced stability and the sustained, controlled release of mAbs [41]. These enhanced properties have generated considerable interest in developing the systemic delivery of mAb-nanoparticle platforms for superior tumor penetration, as well as targeted and sustained therapeutic drug delivery. Further to nanocarriers, additional formulation strategies developed for the controlled release of mAbs include hydrogels and crystalline antibodies, which have shown success as stable, injectable formulations for development [5,8,15,41–43,134–136]. Antibodies 2019, 8, 36 12 of 23 In addition, to further enhance the stability and half-life of other biotherapeutics, recombinant technologies allowed their fusion to mAb Fc as to introduce FcRn recycling as a protection mechanism, which has innovatively expanded the applicability of mAb-based therapeutic platforms. Notable examples include the TNF Receptor–Fc fusion protein Enbrel (Amgen, Thousand Oaks, CA, USA), which acts as an inhibitor to overexpressed TNF- in autoimmune diseases, and the Factor IX–Fc fusion protein Alprolix (Biogen, Cambridge, MA, USA), which is a blood factor supplement for hemophiliacs [11,46,133]. 6. Strategic Modulation of mAb Immune Eector Functions The modulation of mAbs eector functions through isotype switching, glycoengineering, and strategic mutations have proved advantageous for the development of more eective mAb treatment strategies. Antibody binding to Cq1 promotes the complement cascade, Fc R1A/B, Fc R2A, and Fc R3A/B receptors to activate immune eector functions; Fc R2B counter-balances the eector response, and FcRn prolongs mAb half-life. Binding to these receptors is primarily done through sites in the C , C 2, and the conserved glycosylation region in the Fc (N297). hinge H IgG3 does not eciently bind to FcRn, which reduces its half-life to approximately seven days, as opposed to a 21 day half-life for the other IgG isotypes. In most instances of mAb design, an extended half-life is preferred as to prolong the eective dose of a mAb in serum [15]. IgG2 and IgG4 mAbs have a reduced eector function as compared to IgG1 and are thus used in instances where minimal engagement to the immune system is warranted to increase safety of the mAb therapy—with ADCs reducing o-target cytotoxicity, for instance. Eculizumab (Soliris , Alexion Pharmaceuticals, New Haven, CT, USA) is the ﬁrst (and thus far only) approved recombinant IgG 2(C 1/C )–4(C 2/C 3) H hinge H H hybrid whole mAb. Further cross-sub-class variant mAb therapies are in development, with key amino acid substitutions from the IgG2 and IgG4 sub-classes introduced for intentionally suppressed eector functions [4,13]. The removal of the conserved glycosylation site through amino acid substitution of N297 or T299 (aglycosylation) has also demonstrated reduced eector function; however, this happens at the expense of mAb stability and is therefore not a suitable strategy for whole mAb therapeutics [11,14,15,137–140]. On the other hand, enhancing ADCC and CDC is useful for engaging the immune system to tumor tissues in the absence of a conjugated cytotoxic drug. Certain glycoengineered modiﬁcations to the conserved glycosylation region in the Fc, such as deﬁciency in core fucose (afucosylation) and hyper-galactosylation, have demonstrated enhanced mAb binding to Fc R3A and Cq1, speciﬁcally for an enhanced ADCC and CDC eect [13,43]. Afucosylated mAbs benralizumab (Fasenra—, AstraZeneca, London, UK) and mogamulizumab (Poteligeo , Kyowa Hakko Kirin, Tokyo, Japan), as well as low fucose content mAb obinutuzumab (Gazyva , Genentech, San Francisco, CA, USA), are currently approved therapies, with several further in development (along with clinical trials), which demonstrates the commercial interest and applicability of this technology in improving mAb therapeutics through enhancing ADCC. However, the signiﬁcance of manipulating sialylation in mAb therapeutics is a controversial topic, with several reports demonstrating a reduction in ADCC and CDC and others reporting no observable dierence. This highlights a clear need to characterize and deﬁne the in vivo ecacy of such manipulation [11,13,14,43]. No currently approved mAb therapies contain amino acid substitutions for an improved half-life through modulated binding properties to FcRn, improved CDC through binding to complement factor Cq1, or improved ADCC through enhanced binding anity to Fc R1A and 3A. However, several mutations have been reported, patented, and are in clinical development, demonstrating the commercial interest in this technology for improving mAb therapeutics [6,11,13,15,133,137,140–143]. For a more comprehensive understanding of mAb engineering strategies for modulated immune eector functions, we direct the reader to the following reviews [13–15,140]. Antibodies 2019, 8, 36 13 of 23 7. Computational Approaches for Aggregation Prediction and Rational Design of mAbs The advancement of in silico analysis of mAb peptide sequences, structures, conformation, and their associated biological interaction has been integral to the development of various computational tools for characterizing, designing, and optimizing mAb-based therapeutics. The generation and continued pursuit of mAb structural data for in silico analysis has led to the development of several integral databases, notably IMGT , serving as a key resource for data mining [144–146]. Molecular dynamic (MD) simulation analysis has driven the computational analysis of the discovery and characterization of relevant molecular interactions within the mAb molecule, mAb binding to biological targets and mAb surface association with the surrounding environment. These interactions can infer intrinsic stability, target binding associations, solubility and aggregation propensity of the mAb. MD simulations and free energy calculations from crystal structures remain key for the highly speciﬁc elucidation of mAb-target intermolecular interactions that correlate to binding anity, as signiﬁcant interactions such as hydrogen-bond formation can be predicted and determine the strength of the molecular associations [147]. However, elucidation of mAb self-association, solubility, and aggregation propensity have been driven by the development of computational modelling and simulation tools that simultaneously analyses mAb topography and surface polarity. Notably, AGGRESCAN3D, TANGO, and PASTA are the most prominent tools for predicting the site speciﬁc aggregation propensity of mAbs [35,148]. The preliminary elucidation of mAb structural data—that being amino acid sequences and higher order structure (HOS)—is experimentally derived to produce crystal structures that model the solid-state 3D structure of the mAb. Mass spectrometry technologies have come of age to produce high throughput and orthogonal analysis to elucidate mAb peptide sequences, oxidation, deamidation, and glycosylation heterogeneity, as well as, more recently, for native, destabilized, and aggregated HOS elucidation [149,150]. X-ray crystallography technologies have been the underlying workhorse for elucidating the crystal structure of mAbs. Producing crystalline mAbs is challenging, owing to the complexity of mAb HOS and the degree of mAb conformational heterogeneity. However, several complementing technologies have evolved in recent years to ascertain structure and interactions, including circular dichroism (CD), infrared (IR) and raman spectroscopy, cryogenic-electron microscopy, and nuclear magnetic resonance (NMR) spectroscopy [148,150–157]. Of the technologies available for HOS elucidation, 2D-NMR and X-Ray crystallography (followed by MS) provide the highest sensitivity and local speciﬁcity. In comparison, CD, IR, and raman are high-throughput methods, although they have much lower sensitivity [150,157]. Thus far, only four whole IgG antibodies have a successfully determined crystal structure (PDB ID: 1HZH, 1IGT, 1IGY, and 5DK3), notably 1HZH as a wholly human IgG1 and 5DK3 as a humanized IgG4/, both of which have been used as model structures for MD simulations [158]. However, fragment mAbs yield much higher success with producing crystal structures, which has led to much pursuit and contribution in generating mAb fragments and targetting complexing crystal structure libraries for data mining and in silico analysis [159,160]. Upon obtaining relevant crystal structures for a candidate mAb, detailed crystal structure and MD simulation analysis has been extensively used to elucidate the mAbs intramolecular interactions that confer intrinsic stability and intermolecular interactions to confer interactions to biological targets and self-association. The particular binding interface of interest is analyzed, and amino acids in the mAb structure are identiﬁed for substitution that can disrupt molecular interactions in the interface in the instance of diminishing target binding and identify potentially unutilized bonding sites and polarity mismatches in the interface that can be improved in the instance of enhancing target binding. MD simulations are then carried out for the native and substituted mAb structures to elucidate any relevant bond formations, interactions, and more favorable binding free energy produced by the substituted structure, of which promising candidates can be synthesized and validated through in vitro analysis. This predictive approach has been successfully applied to identify mutations in mAbs for modulating eector functions, target binding, optimizing anity capture for manufacturing, and, more Antibodies 2019, 8, 36 14 of 23 recently, for designing antibodies de novo from targets of interest [11,161–165]. Mutations have been speciﬁcally identiﬁed for enhanced binding to Fc R2A, Fc R3A, and Cq1, as well as a reduced binding to Fc R2B for a more pronounced ADCC and CDC eect, an enhanced binding to FcRn for an extended half-life, and a reduced binding to Fc Rs and Cq1 for a diminished ADCC and CDC eect, all of which are transferrable to mAbs of the same isotype sub-class [137,141,142]. Further to this, the molecular interactions of glycoengineered mAb variants to FcRs have been characterized through MD simulations to corroborate predictions with the observed modulated eector functions [13,15,137,141,142]. Self-association, solubility, and aggregation propensity are further evaluated by computational modelling tools that speciﬁcally characterize the topography and surface polarity of mAbs, concurrently analyzing the local spatial arrangement of amino acids in the mAb structure, solvent accessibility, and local surface charge [5]. In particular, surface exposed hydrophobicity is sought as the lead mechanism for protein self-association driving aggregation propensity, in which aggregation-prone regions (APRs) are identiﬁed in the mAb structure. The substitution of identiﬁed APRs to gatekeeper (i.e., polar) amino acids has experimentally demonstrated resistance to aggregation and improved solubility, which validates this strategy for improving mAb stability [36–40,166–170]. The majority of APRs identiﬁed are located in biologically relevant mAb regions—those being the CDRs and the Fc. Speciﬁcally targeting these APRs through mutation may disrupt important biological functions and mAb structure. Analyses of the mAbs intermolecular interactions are necessary to validate the intrinsic stability of substituted mAb variants; if conformational ﬂuctuations are elucidated, then the biologically active interface is likely disrupted. Interestingly, mutations for enhancing biological functions have demonstrated a negative impact on mAb solubility and stability, further suggesting that the self-association proﬁle of mAbs is linked to its biological activity [36,128,171]. Aside from direct residue substitution in mAb structures, other strategies reported to profoundly interfere with the eects of APRs include isotype switching and the strategic addition of N-linked glycans [39,128,172,173]. Additionally, N-linked glycosylation in the CDR regions of mAbs, although uncommon, has demonstrated improved solubility and stability proﬁles of mAbs without impacting their target anity, as compared to the same mAbs with removed CDR glycans [128,174]. The rationale behind strategic glycan addition is based on the election of N-linked glycosylation sites that have apparent spacial proximity to APRs, with the introduced glycan therefore sterically hindering the APR from self-association interactions. The beneﬁts of extending mAb half-life with strategic glycan addition, as seen with hyper-glycosylated biotherapeutics (as well as improving mAb solubility and stability for improved formulation strategies) has yet to be realized and suggests a very intriguing and highly relevant technology for perusal. 8. Concluding Remarks Therapeutic antibodies have come of age as continuing to be a key, dominant technology in the biopharmaceutical industry. The repurpose of antibodies to many formats have made them versatile to design and tailor highly specialized treatments, including ADCs as a targeted drug delivery system, bispeciﬁc and fragment mAb platforms for tailored engagement and increased bioavailability, and recombinant Fc-fusion proteins for an increased half-life and introduced immunological engagement. Further advancements include the modulation of Fc eector functions through manipulations of the Fc, either through isotype switching, glycoengineering, or strategic mutations in the Fc region, along with the PEGylation of fragment mAbs for enhanced half-life. Advancements in discovery, manufacture, and formulation technologies have further propelled the success of therapeutic antibodies, notably through expression system development and the transition from IV to SC formulations. Human-based expression systems have been extensively used in mAb development and are becoming an accepted manufacturing platform for mAb therapeutics. Furthermore, cell-free synthesis technology is giving rise to the potential for higher eciency in the manufacture process. Though developments for further generation antibody therapies have led to great strides in producing improved therapeutic outcomes, many facets of the manufacture process and formulation Antibodies 2019, 8, 36 15 of 23 development strategy pose as challenges to be considered. Antibody-based therapies are susceptible to chemical and enzymatic degradation through oral, nasal, or pulmonary routes of administration and are therefore currently restricted IV or SC delivery. Despite achieving maximum bioavailability through IV/SC administration, tissue penetration of mAb-based therapies is poor, which limits their local bioavailability, requiring high concentrations to achieve an eective dose. The stability of mAbs-based therapeutics is a highly pronounced and recurring challenge to be considered, as it aects manufacture yield and formulation considerations. Several strategies are in development to improve the stability of mAbs in order to potentially produce formulations for pulmonary or oral administration. Notably, computational tools have come of age, complementing experimental techniques to derive antibody structure and aggregation prediction. Through these methods, the stability and aggregation propensity of mAb-based therapies have demonstrated improvement through rational mutation and glycosylation within the framework region, which is potentially translatable to all mAbs within the same isotype. Furthermore, nanocarrier technologies have been shown to enhance the stability and potentially control the release of mAbs. The reﬁnement of rational mAb design coupled with nanocarrier technologies has the potential to overcome these challenges, to develop superior treatment strategies, and ultimately to formulate for non-invasive administration routes such as pulmonary delivery. Author Contributions: V.S. revised the ﬁgure and prepared the scope, literature review, and original manuscript draft. E.C prepared the original ﬁgure and reviewed the manuscript. B.E. and V.K. equally reviewed and edited the manuscript. Funding: This research received no external funding. Conﬂicts of Interest: The authors declare no conﬂict of interest. References 1. Urquhart, L. Top drugs and companies by sales in 2017. Nat. Rev. Drug Discov. 2018, 17, 232. [CrossRef] [PubMed] 2. Ecker, D.; Dana Jones, S.; Levine, H.L. The therapeutic monoclonal antibody market. mAbs 2015, 7, 9–14. [CrossRef] [PubMed] 3. An, Z. “Magic bullets” at the center stage of immune therapy: A special issue on therapeutic antibodies. Protein Cell 2018, 9, 1–2. [CrossRef] [PubMed] 4. Santos, M.L.d.; Quintilio, W.; Manieri, T.M.; Tsuruta, L.R.; Moro, A.M. Advances and challenges in therapeutic monoclonal antibodies drug development. Bras. J. Pharm. Sci. 2018, 54. [CrossRef] 5. Elgundi, Z.; Reslan, M.; Cruz, E.; Sifniotis, V.; Kayser, V. The state-of-play and future of antibody therapeutics. Adv. Drug Del. Rev. 2017, 122, 2–19. [CrossRef] 6. Almagro, J.C.; Daniels-Wells, T.R.; Perez-Tapia, S.M.; Penichet, M.L. Progress and challenges in the design and clinical development of antibodies for cancer therapy. Front. Immunol. 2018, 8, 1751. [CrossRef] [PubMed] 7. Liu, B.N.; Luo, J.H. Research and development of innovative antibody-based drugs. Yaoxue Xuebao 2017, 52, 1811–1819. 8. Awwad, S.; Angkawinitwong, U. Overview of antibody drug delivery. Pharmaceutics 2018, 10, 83. [CrossRef] [PubMed] 9. Klein, C. Special issue: Monoclonal antibodies. Antibodies 2018, 7, 17. [CrossRef] 10. Wang, C.; Xu, P.; Zhang, L.; Huang, J.; Zhu, K.; Luo, C. Current strategies and applications for precision drug design. Front. Pharmacol. 2018, 9, 787. [CrossRef] [PubMed] 11. Strohl, W.R. Current progress in innovative engineered antibodies. Protein Cell 2018, 9, 86–120. [CrossRef] [PubMed] 12. Homann, R.M.; Coumbe, B.G.T.; Josephs, D.H.; Mele, S.; Ilieva, K.M.; Cheung, A.; Tutt, A.N.; Spicer, J.F.; Thurston, D.E.; Crescioli, S.; et al. Antibody structure and engineering considerations for the design and function of antibody drug conjugates (adcs). OncoImmunology 2018, 7, e1395127. [CrossRef] [PubMed] 13. Wang, X.; Mathieu, M.; Brezski, R.J. Igg fc engineering to modulate antibody eector functions. Protein Cell 2018, 9, 63–73. [CrossRef] [PubMed] Antibodies 2019, 8, 36 16 of 23 14. Pawlowski, J.W.; Bajardi-Taccioli, A.; Houde, D.; Feschenko, M.; Carlage, T.; Kaltashov, I.A. Inﬂuence of glycan modiﬁcation on igg1 biochemical and biophysical properties. J. Pharm. Biomed. Anal. 2018, 151, 133–144. [CrossRef] [PubMed] 15. Fonseca, M.H.G.; Furtado, G.P.; Bezerra, M.R.L.; Pontes, L.Q.; Fernandes, C.F.C. Boosting half-life and eector functions of therapeutic antibodies by fc-engineering: An interaction-function review. Int. J. Biol. Macromol. 2018, 119, 306–311. [CrossRef] [PubMed] 16. Mizukami, A.; Caron, A.L.; Picanço-Castro, V.; Swiech, K. Platforms for recombinant therapeutic glycoprotein production. In Recombinant Glycoprotein Production; Humana Press Inc.: New York, NY, USA, 2018; Volume 1674, pp. 1–14. 17. Thomson, C.A. Igg structure and function. In Encyclopedia of Immunobiology; Elsevier Inc.: Amsterdam, The Netherlands, 2016; Volume 2, pp. 15–22. 18. Kayser, V.; Chennamsetty, N.; Voynov, V.; Forrer, K.; Helk, B.; Trout, B.L. Glycosylation inﬂuences on the aggregation propensity of therapeutic monoclonal antibodies. Biotechnol. J. 2011, 6, 38–44. [CrossRef] 19. Pieracci, J.P.; Armando, J.W.; Westoby, M.; Thommes, J. Chapter 9—Industry review of cell separation and product harvesting methods. In Biopharmaceutical Processing; Jagschies, G., Lindskog, E., Łacki, ˛ K., Galliher, P., Eds.; Elsevier: Amsterdam, The Netherlands, 2018; pp. 165–206. 20. Huang, C.-J.; Lin, H.; Yang, X. Industrial production of recombinant therapeutics in escherichia coli and its recent advancements. J. Ind. Microbiol. Biotechnol. 2012, 39, 383–399. [CrossRef] 21. Behme, S. Manufacturing of Pharmaceutical Proteins: From Technology to Economy: Second, Revised and Expanded Edition; Wiley: Hoboken, NJ, USA, 2015; pp. 1–427. 22. Ulmer, N.; Vogg, S.; Müller-Späth, T.; Morbidelli, M. Chapter 7—Puriﬁcation of human monoclonal antibodies and their fragments. In Human monoclonal Antibodies: Methods and Protocols; Steinitz, M., Ed.; Springer: New York, NY, USA, 2019; pp. 163–188. 23. Arosio, P.; Barolo, G.; Müller-Späth, T.; Wu, H.; Morbidelli, M. Aggregation stability of a monoclonal antibody during downstream processing. Pharm. Res. 2011, 28, 1884–1894. [CrossRef] 24. Kayser, V.; Chennamsetty, N.; Voynov, V.; Helk, B.; Trout, B.L. Conformational stability and aggregation of therapeutic monoclonal antibodies studied with ans and thioﬂavin t binding. mAbs 2011, 3, 408–411. [CrossRef] 25. Bittner, B.; Richter, W.; Schmidt, J. Subcutaneous administration of biotherapeutics: An overview of current challenges and opportunities. Biodrugs 2018, 32, 425–440. [CrossRef] 26. Viola, M.; Sequeira, J.; Seiça, R.; Veiga, F.; Serra, J.; Santos, A.C.; Ribeiro, A.J. Subcutaneous delivery of monoclonal antibodies: How do we get there? J. Control. Release 2018, 286, 301–314. [CrossRef] [PubMed] 27. Otvos, L. 6.05—Basic principles of formulation for biotherapeutics: Approaches to alternative drug delivery. In Comprehensive Medicinal Chemistry III; Chackalamannil, S., Rotella, D., Ward, S.E., Eds.; Elsevier: Oxford, UK, 2017; pp. 131–156. 28. Morales, J.O.; Fathe, K.R.; Brunaugh, A.; Ferrati, S.; Li, S.; Montenegro-Nicolini, M.; Mousavikhamene, Z.; McConville, J.T.; Prausnitz, M.R.; Smyth, H.D.C. Challenges and future prospects for the delivery of biologics: Oral mucosal, pulmonary, and transdermal routes. AAPS J. 2017, 19, 652–668. [CrossRef] [PubMed] 29. Muheem, A.; Shakeel, F.; Jahangir, M.A.; Anwar, M.; Mallick, N.; Jain, G.K.; Warsi, M.H.; Ahmad, F.J. A review on the strategies for oral delivery of proteins and peptides and their clinical perspectives. Saudi Pharm. J. 2016, 24, 413–428. [CrossRef] [PubMed] 30. Hunter, M.; Yuan, P.; Vavilala, D.; Fox, M. Optimization of protein expression in mammalian cells. Curr. Protoc. Protein Sci. 2019, 95, e77. [CrossRef] [PubMed] 31. Lindskog, E.K.; Fischer, S.; Wenger, T.; Schulz, P. Chapter 6—Host cells. In Biopharmaceutical Processing; Jagschies, G., Lindskog, E., Łacki, ˛ K., Galliher, P., Eds.; Elsevier: Amsterdam, The Netherlands, 2018; pp. 111–130. 32. Fliedl, L.; Grillari, J.; Grillari-Voglauer, R. Human cell lines for the production of recombinant proteins: On the horizon. New Biotechnol. 2015, 32, 673–679. [CrossRef] 33. Matsuda, T.; Ito, T.; Takemoto, C.; Katsura, K.; Ikeda, M.; Wakiyama, M.; Kukimoto-Niino, M.; Yokoyama, S.; Kurosawa, Y.; Shirouzu, M. Cell-free synthesis of functional antibody fragments to provide a structural basis for antibody–antigen interaction. PLoS ONE 2018, 13, e0193158. [CrossRef] 34. Stech, M.; Kubick, S. Cell-free synthesis meets antibody production: A review. Antibodies 2015, 4, 12. [CrossRef] Antibodies 2019, 8, 36 17 of 23 35. Pujols, J.; Peña-Díaz, S.; Ventura, S. Aggrescan3d: Toward the prediction of the aggregation propensities of protein structures. In Methods Mol. Biol.; Humana Press Inc.: New York, NY, USA, 2018; Volume 1762, pp. 427–443. 36. Van der Kant, R.; Karow-Zwick, A.R.; Van Durme, J.; Blech, M.; Gallardo, R.; Seeliger, D.; Aßfalg, K.; Baatsen, P.; Compernolle, G.; Gils, A.; et al. Prediction and reduction of the aggregation of monoclonal antibodies. J. Mol. Biol. 2017, 429, 1244–1261. [CrossRef] 37. Lerch, T.F.; Sharpe, P.; Mayclin, S.J.; Edwards, T.E.; Lee, E.; Conlon, H.D.; Polleck, S.; Rouse, J.C.; Luo, Y.; Zou, Q. Inﬂiximab crystal structures reveal insights into self-association. mAbs 2017, 9, 874–883. [CrossRef] 38. Dobson, C.L.; Devine, P.W.A.; Phillips, J.J.; Higazi, D.R.; Lloyd, C.; Popovic, B.; Arnold, J.; Buchanan, A.; Lewis, A.; Goodman, J.; et al. Engineering the surface properties of a human monoclonal antibody prevents self-association and rapid clearance in vivo. Sci. Rep. 2016, 6, 38644. [CrossRef] 39. Courtois, F.; Agrawal, N.J.; Lauer, T.M.; Trout, B.L. Rational design of therapeutic mabs against aggregation through protein engineering and incorporation of glycosylation motifs applied to bevacizumab. mAbs 2016, 8, 99–112. [CrossRef] 40. Courtois, F.; Schneider, C.P.; Agrawal, N.J.; Trout, B.L. Rational design of biobetters with enhanced stability. J. Pharm. Sci. 2015, 104, 2433–2440. [CrossRef] 41. Yu, M.; Wu, J.; Shi, J.; Farokhzad, O.C. Nanotechnology for protein delivery: Overview and perspectives. J. Control. Release 2016, 240, 24–37. [CrossRef] 42. Shi, J.; Kanto, P.W.; Wooster, R.; Farokhzad, O.C. Cancer nanomedicine: Progress, challenges and opportunities. Nat. Rev. Cancer 2016, 17, 20. [CrossRef] 43. Dalziel, M.; Beers, S.A.; Cragg, M.S.; Crispin, M. Through the barricades: Overcoming the barriers to eective antibody-based cancer therapeutics. Glycobiology 2018, 28, 697–712. [CrossRef] 44. Lambert, J.M.; Berkenblit, A. Antibody-drug conjugates for cancer treatment. Annu. Rev. Med. 2018, 69, 191–207. [CrossRef] 45. Lambert, J.M.; Morris, C.Q. Antibody–drug conjugates (adcs) for personalized treatment of solid tumors: A review. Adv. Ther. 2017, 34, 1015–1035. [CrossRef] 46. Levin, D.; Golding, B.; Strome, S.E.; Sauna, Z.E. Fc fusion as a platform technology: Potential for modulating immunogenicity. Trends Biotechnol. 2015, 33, 27–34. [CrossRef] 47. Henry, K.A. Next-generation DNA sequencing of vh/vl repertoires: A primer and guide to applications in single-domain antibody discovery. In Phage Display: Methods and Protocols; Hust, M., Lim, T.S., Eds.; Springer: New York, NY, USA, 2018; pp. 425–446. 48. Frenzel, A.; Roskos, L.; Klakamp, S.; Liang, M.; Arends, R.; Green, L. Antibody anity. In Handbook of Therapeutic Antibodies, 2nd ed.; Wiley Blackwell: Hoboken, NJ, USA, 2014; Volume 1–4, pp. 115–140. 49. Hu, J.; Han, J.; Li, H.; Zhang, X.; Liu, L.; Chen, F.; Zeng, B. Human embryonic kidney 293 cells: A vehicle for biopharmaceutical manufacturing, structural biology, and electrophysiology. Cells Tissues Organs 2018, 205, 1–8. [CrossRef] 50. Jacobi, A.; Enenkel, B.; Garidel, P.; Eckermann, C.; Knappenberger, M.; Presser, I.; Kaufmann, H. Process development and manufacturing of therapeutic antibodies. In Handbook of Therapeutic Antibodies, 2nd ed.; Wiley Blackwell: Hoboken, NJ, USA, 2014; Volume 2–4, pp. 601–664. 51. Kayser, V.; Chennamsetty, N.; Voynov, V.; Helk, B.; Forrer, K.; Trout, B.L. A screening tool for therapeutic monoclonal antibodies: Identifying the most stable protein and its best formulation based on thioﬂavin t binding. Biotechnol. J. 2012, 7, 127–132. [CrossRef] 52. Tada, M.; Tatematsu, K.-I.; Ishii-Watabe, A.; Harazono, A.; Takakura, D.; Hashii, N.; Sezutsu, H.; Kawasaki, N. Characterization of anti-cd20 monoclonal antibody produced by transgenic silkworms (bombyx mori). mAbs 2015, 7, 1138–1150. [CrossRef] 53. Maccani, A.; Landes, N.; Stadlmayr, G.; Maresch, D.; Leitner, C.; Maurer, M.; Gasser, B.; Ernst, W.; Kunert, R.; Mattanovich, D. Pichia pastoris secretes recombinant proteins less eciently than chinese hamster ovary cells but allows higher space-time yields for less complex proteins. Biotechnol. J. 2014, 9, 526–537. [CrossRef] 54. Frenzel, A.; Hust, M.; Schirrmann, T. Expression of recombinant antibodies. Front. Immunol. 2013, 4, 217. [CrossRef] 55. Thoring, L.; Kubick, S. Versatile cell-free protein synthesis systems based on chinese hamster ovary cells. In Recombinant Protein Expression in Mammalian Cells: Methods and Protocols; Hacker, D.L., Ed.; Springer: New York, NY, USA, 2018; pp. 289–308. Antibodies 2019, 8, 36 18 of 23 56. Tran, K.; Gurramkonda, C.; Cooper, M.A.; Pilli, M.; Taris, J.E.; Selock, N.; Han, T.-C.; Tolosa, M.; Zuber, A.; Peñalber-Johnstone, C.; et al. Cell-free production of a therapeutic protein: Expression, puriﬁcation, and characterization of recombinant streptokinase using a cho lysate. Biotechnol. Bioeng. 2018, 115, 92–102. [CrossRef] 57. Stech, M.; Nikolaeva, O.; Thoring, L.; Stöcklein, W.F.M.; Wüstenhagen, D.A.; Hust, M.; Dübel, S.; Kubick, S. Cell-free synthesis of functional antibodies using a coupled in vitro transcription-translation system based on cho cell lysates. Sci. Rep. 2017, 7, 12030. [CrossRef] 58. Li, J.; Wang, H.; Kwon, Y.-C.; Jewett, M.C. Establishing a high yielding streptomyces-based cell-free protein synthesis system. Biotechnol. Bioeng. 2017, 114, 1343–1353. [CrossRef] 59. Jaroentomeechai, T.; Stark, J.C.; Natarajan, A.; Glasscock, C.J.; Yates, L.E.; Hsu, K.J.; Mrksich, M.; Jewett, M.C.; DeLisa, M.P. Single-pot glycoprotein biosynthesis using a cell-free transcription-translation system enriched with glycosylation machinery. Nat. Commun. 2018, 9, 2686. [CrossRef] 60. Gurramkonda, C.; Rao, A.; Borhani, S.; Pilli, M.; Deldari, S.; Ge, X.; Pezeshk, N.; Han, T.-C.; Tolosa, M.; Kostov, Y.; et al. Improving the recombinant human erythropoietin glycosylation using microsome supplementation in cho cell-free system. Biotechnol. Bioeng. 2018, 115, 1253–1264. [CrossRef] 61. Nakamura, T.; Omasa, T. Optimization of cell line development in the gs-cho expression system using a high-throughput, single cell-based clone selection system. J. Biosci. Bioeng. 2015, 120, 323–329. [CrossRef] 62. Zhou, Y.; Raju, R.; Alves, C.; Gilbert, A. Debottlenecking protein secretion and reducing protein aggregation in the cellular host. Curr. Opin. Biotechnol. 2018, 53, 151–157. [CrossRef] 63. Inwood, S.; Betenbaugh, M.J.; Lal, M.; Shiloach, J. Genome-wide high-throughput rnai screening for identiﬁcation of genes involved in protein production. In Recombinant Protein Expression in Mammalian Cells: Methods and Protocols; Hacker, D.L., Ed.; Springer: New York, NY, USA, 2018; pp. 209–219. 64. Dangi, A.K.; Sinha, R.; Dwivedi, S.; Gupta, S.K.; Shukla, P. Cell line techniques and gene editing tools for antibody production: A review. Front. Pharmacol. 2018, 9, 630. [CrossRef] 65. Delic, M.; Göngrich, R.; Mattanovich, D.; Gasser, B. Engineering of protein folding and secretion—Strategies to overcome bottlenecks for ecient production of recombinant proteins. Antioxid. Redox Signal. 2014, 21, 414–437. [CrossRef] 66. Zhong, X.; Wright, J.F. Biological insights into therapeutic protein modiﬁcations throughout tracking and their biopharmaceutical applications. Int. J. Cell Biol. 2013, 2013, 19. [CrossRef] 67. Altamirano, C.; Berrios, J.; Vergara, M.; Becerra, S. Advances in improving mammalian cells metabolism for recombinant protein production. Electron. J. Biotechnol. 2013, 16. [CrossRef] 68. Parola, C.; Mason, D.M.; Zingg, A.; Neumeier, D.; Reddy, S.T. Genome engineering of hybridomas to generate stable cell lines for antibody expression. In Recombinant Protein Expression in Mammalian Cells: Methods and Protocols; Hacker, D.L., Ed.; Springer: New York, NY, USA, 2018; pp. 79–111. 69. You, M.; Yang, Y.; Zhong, C.; Chen, F.; Wang, X.; Jia, T.; Chen, Y.; Zhou, B.; Mi, Q.; Zhao, Q.; et al. Ecient mab production in cho cells with optimized signal peptide, codon, and utr. Appl. Microbiol. Biotechnol. 2018, 102, 5953–5964. [CrossRef] 70. Mauro, V.P.; Chappell, S.A. Considerations in the use of codon optimization for recombinant protein expression. In Recombinant Protein Expression in Mammalian Cells: Methods and Protocols; Hacker, D.L., Ed.; Springer: New York, NY, USA, 2018; pp. 275–288. 71. Michael, I.P.; Nagy, A. Inducible protein production in 293 cells using the piggybac transposon system. In Recombinant Protein Expression in Mammalian Cells: Methods and Protocols; Hacker, D.L., Ed.; Springer: New York, NY, USA, 2018; pp. 57–68. 72. Balasubramanian, S. Recombinant cho cell pool generation using piggybac transposon system. In Recombinant Protein Expression in Mammalian Cells: Methods and Protocols; Hacker, D.L., Ed.; Springer: New York, NY, USA, 2018; pp. 69–78. 73. Jäger, V.; Büssow, K.; Schirrmann, T. Transient recombinant protein expression in mammalian cells. In Animal Cell Culture; Al-Rubeai, M., Ed.; Springer International Publishing: Cham, Switzerland, 2015; pp. 27–64. 74. Wijesuriya, S.D.; Pongo, E.; Tomic, M.; Zhang, F.; Garcia-Rodriquez, C.; Conrad, F.; Farr-Jones, S.; Marks, J.D.; Horwitz, A.H. Antibody engineering to improve manufacturability. Protein Expression Purif. 2018, 149, 75–83. [CrossRef] Antibodies 2019, 8, 36 19 of 23 75. L’Abbé, D.; Bisson, L.; Gervais, C.; Grazzini, E.; Durocher, Y. Transient gene expression in suspension hek293-ebna1 cells. In Recombinant Protein Expression in Mammalian Cells: Methods and Protocols; Hacker, D.L., Ed.; Springer: New York, NY, USA, 2018; pp. 1–16. 76. Arena, T.A.; Harms, P.D.; Wong, A.W. High throughput transfection of hek293 cells for transient protein production. In Recombinant Protein Expression in Mammalian Cells: Methods and Protocols; Hacker, D.L., Ed.; Springer: New York, NY, USA, 2018; pp. 179–187. 77. Agrawal, V.; Yu, B.; Pagila, R.; Yang, B.; Simonsen, C.; Beske, O. A high-yielding, cho-k1-based transient transfection system. BioProcess Int. 2013, 11, 28–35. 78. Hacker, D.L.; Kiseljak, D.; Rajendra, Y.; Thurnheer, S.; Baldi, L.; Wurm, F.M. Polyethyleneimine-based transient gene expression processes for suspension-adapted hek-293e and cho-dg44 cells. Protein Expression Purif. 2013, 92, 67–76. [CrossRef] 79. Ritacco, F.V.; Wu, Y.; Khetan, A. Cell culture media for recombinant protein expression in chinese hamster ovary (cho) cells: History, key components, and optimization strategies. Biotechnol. Prog. 2018, 34, 1407–1426. [CrossRef] 80. Whitford, W.G.; Lundgren, M.; Fairbank, A. Chapter 8—Cell culture media in bioprocessing. In Biopharmaceutical Processing; Jagschies, G., Lindskog, E., Łacki, ˛ K., Galliher, P., Eds.; Elsevier: Amsterdam, The Netherlands, 2018; pp. 147–162. 81. Davami, F.; Eghbalpour, F.; Barkhordari, F.; Mahboudi, F. Eect of peptone feeding on transient gene expression process in cho dg44. Avicenna J. Med. Biotechnol. 2014, 6, 147–155. 82. Mahboudi, F.; Abolhassan, M.R.; Azarpanah, A.; Aghajani-Lazarjani, H.; Sadeghi-Haskoo, M.A.; Maleknia, S.; Vaziri, B. The role of dierent supplements in expression level of monoclonal antibody against human cd20. Avicenna J. Med. Biotechnol. 2013, 5, 140–147. 83. You, M.; Liu, Y.; Chen, Y.; Guo, J.; Wu, J.; Fu, Y.; Shen, R.; Qi, R.; Luo, W.; Xia, N. Maximizing antibody production in suspension-cultured mammalian cells by the customized transient gene expression method. Biosci. Biotechnol. Biochem. 2013, 77, 1207–1213. [CrossRef] 84. Backliwal, G.; Hildinger, M.; Kuettel, I.; Delegrange, F.; Hacker, D.L.; Wurm, F.M. Valproic acid: A viable alternative to sodium butyrate for enhancing protein expression in mammalian cell cultures. Biotechnol. Bioeng. 2008, 101, 182–189. [CrossRef] 85. Elgundi, Z.; Sifniotis, V.; Reslan, M.; Cruz, E.; Kayser, V. Laboratory scale production and puriﬁcation of a therapeutic antibody. JoVE 2017. [CrossRef] 86. Kim, S.J.; Ha, G.S.; Lee, G.; Lim, S.I.; Lee, C.M.; Yang, Y.H.; Lee, J.; Kim, J.E.; Lee, J.H.; Shin, Y.; et al. Enhanced expression of soluble antibody fragments by low-temperature and overdosing with a nitrogen source. Enzyme Microb. Technol. 2018, 115, 9–15. [CrossRef] 87. Lalonde, M.-E.; Durocher, Y. Therapeutic glycoprotein production in mammalian cells. J. Biotechnol. 2017, 251, 128–140. [CrossRef] 88. Rajendra, Y.; Kiseljak, D.; Baldi, L.; Hacker, D.L.; Wurm, F.M. A simple high-yielding process for transient gene expression in cho cells. J. Biotechnol. 2011, 153, 22–26. [CrossRef] 89. Codamo, J.; Munro, T.P.; Hughes, B.S.; Song, M.; Gray, P.P. Enhanced cho cell-based transient gene expression with the epi-cho expression system. Mol. Biotechnol. 2011, 48, 109–115. [CrossRef] 90. Codamo, J.; Hou, J.J.C.; Hughes, B.S.; Gray, P.P.; Munro, T.P. Ecient mab production in cho cells incorporating pei-mediated transfection, mild hypothermia and the co-expression of xbp-1. J. Chem. Technol. Biotechnol. 2011, 86, 923–934. [CrossRef] 91. Castan, A.; Schulz, P.; Wenger, T.; Fischer, S. Chapter 7—Cell line development. In Biopharmaceutical Processing; Jagschies, G., Lindskog, E., Łacki, ˛ K., Galliher, P., Eds.; Elsevier: Amsterdam, The Netherlands, 2018; pp. 131–146. 92. Lindskog, E.K. Chapter 31—The upstream process: Principal modes of operation. In Biopharmaceutical Processing; Jagschies, G., Lindskog, E., Łacki, ˛ K., Galliher, P., Eds.; Elsevier: Amsterdam, The Netherlands, 2018; pp. 625–635. 93. Fisher, A.C.; Kamga, M.-H.; Agarabi, C.; Brorson, K.; Lee, S.L.; Yoon, S. The current scientiﬁc and regulatory landscape in advancing integrated continuous biopharmaceutical manufacturing. Trends Biotechnol. 2018, 37, 253–267. [CrossRef] 94. Rathore, A.S.; Agarwal, H.; Sharma, A.K.; Pathak, M.; Muthukumar, S. Continuous processing for production of biopharmaceuticals. Prep. Biochem. Biotechnol. 2015, 45, 836–849. [CrossRef] Antibodies 2019, 8, 36 20 of 23 95. Shukla, A.A.; Suda, E. Chapter 3—Harvest and recovery of monoclonal antibodies: Cell removal and clariﬁcation. In Process Scale Puriﬁcation of Antibodies; Gottschalk, U., Ed.; John Wiley & Sons, Inc.: Hoboken, NJ, USA, 2017; pp. 55–79. 96. Thömmes, J.; Twyman, R.M.; Gottschalk, U. Chapter 10—Alternatives to packed-bed chromatography for antibody extraction and puriﬁcation. In Process Scale Puriﬁcation of Antibodies; Gottschalk, U., Ed.; John Wiley & Sons: Hoboken, NJ, USA, 2017; pp. 215–231. 97. Glynn, J. Chapter 11—Process-scale precipitation of impurities in mammalian cell culture broth. In Process Scale Puriﬁcation of Antibodies; Gottschalk, U., Ed.; John Wiley & Sons, Inc.: Hoboken, NJ, USA, 2017; pp. 233–246. 98. Singh, N.; Arunkumar, A.; Chollangi, S.; Tan, Z.G.; Borys, M.; Li, Z.J. Clariﬁcation technologies for monoclonal antibody manufacturing processes: Current state and future perspectives. Biotechnol. Bioeng. 2016, 113, 698–716. [CrossRef] 99. Singh, N.; Chollangi, S. Chapter 4—Next-generation clariﬁcation technologies for the downstream processing of antibodies. In Process scale Puriﬁcation of Antibodies; Gottschalk, U., Ed.; John Wiley & Sons, Inc.: Hoboken, NJ, USA, 2017; pp. 81–112. 100. Kelley, B. Chapter 1—Downstream processing of monoclonal antibodies: Current practices and future opportunities. In Process Scale Puriﬁcation of Antibodies; Gottschalk, U., Ed.; John Wiley & Sons, Inc.: Hoboken, NJ, USA, 2017; pp. 1–21. 101. Danielsson, Å. Chapter 17—Anity chromatography. In Biopharmaceutical Processing; Jagschies, G., Lindskog, E., Łacki, ˛ K., Galliher, P., Eds.; Elsevier: Amsterdam, The Netherlands, 2018; pp. 367–378. 102. Kinna, A.; Tolner, B.; Rota, E.M.; Titchener-Hooker, N.; Nesbeth, D.; Chester, K. Imac capture of recombinant protein from unclariﬁed mammalian cell feed streams. Biotechnol. Bioeng. 2016, 113, 130–140. [CrossRef] 103. Ghose, S.; Jin, M.; Liu, J.; Hickey, J.; Lee, S. Chapter 14—Integrated polishing steps for monoclonal antibody puriﬁcation. In Process Scale Puriﬁcation of Antibodies; Gottschalk, U., Ed.; John Wiley & Sons, Inc.: Hoboken, NJ, USA, 2017; pp. 303–323. 104. Joshi, V.; Shivach, T.; Kumar, V.; Yadav, N.; Rathore, A. Avoiding antibody aggregation during processing: Establishing hold times. Biotechnol. J. 2014, 9, 1195–1205. [CrossRef] 105. Liderfelt, J.; Royce, J. Chapter 23—Filtration methods for use in puriﬁcation processes (concentration and buer exchange). In Biopharmaceutical Processing; Jagschies, G., Lindskog, E., Łacki, ˛ K., Galliher, P., Eds.; Elsevier: Amsterdam, The Netherlands, 2018; pp. 441–453. 106. Anselmo, A.C.; Gokarn, Y.; Mitragotri, S. Non-invasive delivery strategies for biologics. Nat. Rev. Drug Discov. 2018, 18, 19. [CrossRef] 107. Sousa, D.; Ferreira, D.; Rodrigues, J.L.; Rodrigues, L.R. Chapter 14—Nanotechnology in targeted drug delivery and therapeutics. In Applications of Targeted Nano Drugs and Delivery Systems; Mohapatra, S.S., Ranjan, S., Dasgupta, N., Mishra, R.K., Thomas, S., Eds.; Elsevier: Amsterdam, The Netherlands, 2019; pp. 357–409. 108. Jani, R.; Krupa, G.; Rupal, J. Active targeting of nanoparticles: An innovative technology for drug delivery in cancer therapeutics. J. Drug Deliv. Ther. 2019. [CrossRef] 109. Abdelaziz, H.M.; Gaber, M.; Abd-Elwakil, M.M.; Mabrouk, M.T.; Elgohary, M.M.; Kamel, N.M.; Kabary, D.M.; Freag, M.S.; Samaha, M.W.; Mortada, S.M.; et al. Inhalable particulate drug delivery systems for lung cancer therapy: Nanoparticles, microparticles, nanocomposites and nanoaggregates. J. Control. Release 2018, 269, 374–392. [CrossRef] 110. Jackisch, C.; Kim, S.B.; Semiglazov, V.; Melichar, B.; Pivot, X.; Hillenbach, C.; Stroyakovskiy, D.; Lum, B.L.; Elliott, R.; Weber, H.A.; et al. Subcutaneous versus intravenous formulation of trastuzumab for her2-positive early breast cancer: Updated results from the phase iii hannah study. Ann. Oncol. 2015, 26, 320–325. [CrossRef] 111. Lambertini, M.; Pondé, N.F.; Solinas, C.; de Azambuja, E. Adjuvant trastuzumab: A 10-year overview of its beneﬁt. Expert Rev. Anticancer Ther. 2017, 17, 61–74. [CrossRef] 112. Sanford, M. Subcutaneous trastuzumab: A review of its use in her2-positive breast cancer. Target. Oncol. 2014, 9, 85–94. [CrossRef] 113. Reichert, J.M. Adalimumab (humira ). In Handbook of Therapeutic Antibodies, 2nd ed.; Wiley Blackwell: Hoboken, NJ, USA, 2014; Volume 3–4, pp. 1309–1322. Antibodies 2019, 8, 36 21 of 23 114. Crommelin, D.J.A.; Hawe, A.; Jiskoot, W. Formulation of biologics including biopharmaceutical considerations. In Pharmaceutical Biotechnology: Fundamentals and Applications; Crommelin, D.J.A., Sindelar, R.D., Meibohm, B., Eds.; Springer International Publishing: Cham, Switzerland, 2019; pp. 83–103. 115. Singh, S.K.; Mahler, H.-C.; Hartman, C.; Stark, C.A. Are injection site reactions in monoclonal antibody therapies caused by polysorbate excipient degradants? J. Pharm. Sci. 2018, 107, 2735–2741. [CrossRef] 116. Rayaprolu, B.M.; Strawser, J.J.; Anyarambhatla, G. Excipients in parenteral formulations: Selection considerations and eective utilization with small molecules and biologics. Drug Dev. Ind. Pharm. 2018, 44, 1565–1571. [CrossRef] 117. Pimentel, F.F.; Morgan, G.; Tiezzi, D.G.; de Andrade, J.M. Development of new formulations of biologics: Expectations, immunogenicity, and safety for subcutaneous trastuzumab. Pharmaceut. Med. 2018, 32, 319–325. [CrossRef] 118. Garidel, P.; Kuhn, A.B.; Schäfer, L.V.; Karow-Zwick, A.R.; Blech, M. High-concentration protein formulations: How high is high? Eur. J. Pharm. Biopharm. 2017, 119, 353–360. [CrossRef] 119. Kemter, K.; Altrichter, J.; Derwand, R.; Kriehuber, T.; Reinauer, E.; Scholz, M. Amino acid-based advanced liquid formulation development for highly concentrated therapeutic antibodies balances physical and chemical stability and low viscosity. Biotechnol. J. 2018, 13, e1700523. [CrossRef] 120. Mandal, A.; Pal, D.; Agrahari, V.; Trinh, H.M.; Joseph, M.; Mitra, A.K. Ocular delivery of proteins and peptides: Challenges and novel formulation approaches. Adv. Drug Del. Rev. 2018, 126, 67–95. [CrossRef] 121. Reslan, M.; Kayser, V. The eect of deuterium oxide on the conformational stability and aggregation of bovine serum albumin. Pharm. Dev. Technol. 2016, 1–7. [CrossRef] 122. Reslan, M.; Ranganathan, V.; Macfarlane, D.R.; Kayser, V. Choline ionic liquid enhances the stability of herceptin(r) (trastuzumab). Chem. Commun. (Camb.) 2018, 54, 10622–10625. [CrossRef] 123. Reslan, M.; Kayser, V. Ionic liquids as biocompatible stabilizers of proteins. Biophys. Rev. 2018, 10, 781–793. [CrossRef] 124. Emami, F.; Vatanara, A.; Park, E.J.; Na, D.H. Drying technologies for the stability and bioavailability of biopharmaceuticals. Pharmaceutics 2018, 10, 131. [CrossRef] 125. Izutsu, K.I. Applications of freezing and freeze-drying in pharmaceutical formulations. In Adv. Exp. Med. Biol.; Springer: New York, NY, USA, 2018; Volume 1081, pp. 371–383. 126. Schermeyer, M.T.; Wöll, A.K.; Kokke, B.; Eppink, M.; Hubbuch, J. Characterization of highly concentrated antibody solution—A toolbox for the description of protein long-term solution stability. mAbs 2017, 9, 1169–1185. [CrossRef] 127. Kayser, V.; Chennamsetty, N.; Voynov, V.; Helk, B.; Forrer, K.; Trout, B.L. Evaluation of a non-arrhenius model for therapeutic monoclonal antibody aggregation. J. Pharm. Sci. 2011, 100, 2526–2542. [CrossRef] 128. Rabia, L.A.; Desai, A.A.; Jhajj, H.S.; Tessier, P.M. Understanding and overcoming trade-os between antibody anity, speciﬁcity, stability and solubility. Biochem. Eng. J. 2018, 137, 365–374. [CrossRef] 129. Van de Bovenkamp, F.S.; Derksen, N.I.L.; van Breemen, M.J.; de Taeye, S.W.; Ooijevaar-de Heer, P.; Sanders, R.W.; Rispens, T. Variable domain n-linked glycans acquired during antigen-speciﬁc immune responses can contribute to immunoglobulin g antibody stability. Front. Immunol. 2018, 9, 740. [CrossRef] 130. Kuhn, A.B.; Kube, S.; Karow-Zwick, A.R.; Seeliger, D.; Garidel, P.; Blech, M.; Schäfer, L.V. Improved solution-state properties of monoclonal antibodies by targeted mutations. J. Phys. Chem. B 2017, 121, 10818–10827. [CrossRef] 131. Singh, S.N.; Yadav, S.; Shire, S.J.; Kalonia, D.S. Dipole-dipole interaction in antibody solutions: Correlation with viscosity behavior at high concentration. Pharm. Res. 2014, 31, 2549–2558. [CrossRef] 132. Ionescu, R.M.; Vlasak, J.; Price, C.; Kirchmeier, M. Contribution of variable domains to the stability of humanized igg1 monoclonal antibodies. J. Pharm. Sci. 2008, 97, 1414–1426. [CrossRef] 133. Liu, L. Pharmacokinetics of monoclonal antibodies and fc-fusion proteins. Protein Cell 2018, 9, 15–32. [CrossRef] 134. Schweizer, D.; Serno, T.; Goepferich, A. Controlled release of therapeutic antibody formats. Eur. J. Pharm. Biopharm. 2014, 88, 291–309. [CrossRef] 135. Rudnick, S.I.; Adams, G.P. Anity and avidity in antibody-based tumor targeting. Cancer Biother. Radiopharm. 2009, 24, 155–161. [CrossRef] 136. Ickenstein, L.M.; Garidel, P. Hydrogel formulations for biologicals: Current spotlight from a commercial perspective. Ther. Deliv. 2018, 9, 221–230. [CrossRef] Antibodies 2019, 8, 36 22 of 23 137. Saxena, A.; Wu, D. Advances in therapeutic fc engineering—Modulation of igg-associated eector functions and serum half-life. Front. Immunol. 2016, 7, 580. [CrossRef] 138. Jeeris, R. Glycosylation of antibody molecules. In Handbook of Therapeutic Antibodies, 2nd ed.; Wiley Blackwell: Hoboken, NJ, USA, 2014; Volume 1–4, pp. 171–200. 139. Zheng, K.; Bantog, C.; Bayer, R. The impact of glycosylation on monoclonal antibody conformation and stability. mAbs 2011, 3, 568–576. [CrossRef] 140. Lei, C.; Gong, R.; Ying, T. Editorial: Antibody fc engineering: Towards better therapeutics. Front. Immunol. 2018, 9, 2450. [CrossRef] 141. Booth, B.J.; Ramakrishnan, B.; Narayan, K.; Wollacott, A.M.; Babcock, G.J.; Shriver, Z.; Viswanathan, K. Extending human igg half-life using structure-guided design. mAbs 2018, 10, 1098–1110. [CrossRef] 142. Kellner, C.; Otte, A.; Cappuzzello, E.; Klausz, K.; Peipp, M. Modulating cytotoxic eector functions by fc engineering to improve cancer therapy. Transfus. Med. Hemoth. 2017, 44, 327–336. [CrossRef] 143. Wang, Q.; Chen, Y.; Pelletier, M.; Cvitkovic, R.; Bonnell, J.; Chang, C.Y.; Koksal, A.C.; O’Connor, E.; Gao, X.; Yu, X.Q.; et al. Enhancement of antibody functions through fc multiplications. mAbs 2017, 9, 393–403. [CrossRef] 144. Lefranc, M.P.; Ehrenmann, F.; Kossida, S.; Giudicelli, V.; Duroux, P. Use of imgt databases and tools for antibody engineering and humanization. In Methods Mol. Biol.; Humana Press Inc.: New York, NY, USA, 2018; Volume 1827, pp. 35–69. 145. Lefranc, M.-P.; Giudicelli, V.; Duroux, P.; Jabado-Michaloud, J.; Folch, G.; Aouinti, S.; Carillon, E.; Duvergey, H.; ® ® Houles, A.; Paysan-Lafosse, T.; et al. Imgt( ), the international immunogenetics information system( ) 25 years on. Nucleic Acids Res. 2015, 43, D413–D422. [CrossRef] 146. Martin, A.C.R.; Allen, J. Bioinformatics tools for analysis of antibodies. In Handbook of Therapeutic Antibodies, 2nd ed.; Wiley Blackwell: Hoboken, NJ, USA, 2014; Volume 1–4, pp. 201–228. 147. Sledz, ´ P.; Caﬂisch, A. Protein structure-based drug design: From docking to molecular dynamics. Curr. Opin. Struct. Biol. 2018, 48, 93–102. [CrossRef] 148. Pandya, A.; Howard, M.J.; Zloh, M.; Dalby, P.A. An evaluation of the potential of nmr spectroscopy and computational modelling methods to inform biopharmaceutical formulations. Pharmaceutics 2018, 10, 1–24. [CrossRef] 149. Rathore, D.; Faustino, A.; Schiel, J.; Pang, E.; Boyne, M.; Rogstad, S. The role of mass spectrometry in the characterization of biologic protein products. Expert Rev. Proteomic. 2018, 15, 431–449. [CrossRef] 150. Wang, X.; An, Z.; Luo, W.; Xia, N.; Zhao, Q. Molecular and functional analysis of monoclonal antibodies in support of biologics development. Protein Cell 2018, 9, 74–85. [CrossRef] 151. Baker Edward, N. Crystallography and the development of therapeutic medicines. IUCrJ 2018, 5, 118–119. [CrossRef] 152. Vénien-Bryan, C.; Li, Z.; Vuillard, L.; Boutin, J.A. Cryo-electron microscopy and X-ray crystallography: Complementary approaches to structural biology and drug discovery. Acta Crystallogr. Sect. F Struct. Biol. Commun. 2017, 73, 174–183. [CrossRef] 153. Brader, M.L.; Baker, E.N.; Dunn, M.F.; Laue, T.M.; Carpenter, J.F. Using x-ray crystallography to simplify and accelerate biologics drug development. J. Pharm. Sci. 2017, 106, 477–494. [CrossRef] 154. Nero, T.L.; Parker, M.W.; Morton, C.J. Protein structure and computational drug discovery. Biochem. Soc. Trans. 2018, 46, 1367–1379. [CrossRef] 155. Blaert, J.; Haeri, H.H.; Blech, M.; Hinderberger, D.; Garidel, P. Spectroscopic methods for assessing the molecular origins of macroscopic solution properties of highly concentrated liquid protein solutions. Anal. Biochem. 2018, 561–562, 70–88. [CrossRef] 156. Brinson, R.G.; Marino, J.P.; Delaglio, F.; Arbogast, L.W.; Evans, R.M.; Kearsley, A.; Gingras, G.; Ghasriani, H.; Aubin, Y.; Pierens, G.K.; et al. Enabling adoption of 2d-nmr for the higher order structure assessment of monoclonal antibody therapeutics. mAbs 2019, 11, 94–105. [CrossRef] 157. Young, J.A.; Gabrielson, J.P. Higher order structure methods for similarity assessment. In Biosimilars: Regulatory, Clinical, and Biopharmaceutical Development; Gutka, H.J., Yang, H., Kakar, S., Eds.; Springer International Publishing: Cham, Switzerland, 2018; pp. 321–337. 158. Kumar, S.; Plotnikov, N.V.; Rouse, J.C.; Singh, S.K. Biopharmaceutical informatics: Supporting biologic drug development via molecular modelling and informatics. J. Pharm. Pharmacol. 2018, 70, 595–608. [CrossRef] Antibodies 2019, 8, 36 23 of 23 159. Westbrook, J.D.; Burley, S.K. How structural biologists and the protein data bank contributed to recent fda new drug approvals. Structure 2018, 27, 211–217. [CrossRef] 160. Burley, S.K.; Berman, H.M.; Christie, C.; Duarte, J.M.; Feng, Z.; Westbrook, J.; Young, J.; Zardecki, C. Rcsb protein data bank: Sustaining a living digital data resource that enables breakthroughs in scientiﬁc research and biomedical education. Protein Sci. 2018, 27, 316–330. [CrossRef] 161. Kuroda, D.; Tsumoto, K. Antibody anity maturation by computational design. In Methods Mol. Biol.; Humana Press Inc.: New York, NY, USA, 2018; Volume 1827, pp. 15–34. 162. Fischman, S.; Ofran, Y. Computational design of antibodies. Curr. Opin. Struct. Biol. 2018, 51, 156–162. [CrossRef] 163. Adolf-Bryfogle, J.; Kalyuzhniy, O.; Kubitz, M.; Weitzner, B.D.; Hu, X.; Adachi, Y.; Schief, W.R.; Dunbrack, R.L., Jr. Rosettaantibodydesign (rabd): A general framework for computational antibody design. PLoS Comp. Biol. 2018, 14, e1006112. [CrossRef] 164. Branco, R.J.; Dias, A.M.; Roque, A.C. Understanding the molecular recognition between antibody fragments and protein a biomimetic ligand. J. Chromatogr. A 2012, 1244, 106–115. [CrossRef] 165. Huang, B.; Liu, F.F.; Dong, X.Y.; Sun, Y. Molecular mechanism of the anity interactions between protein a and human immunoglobulin g1 revealed by molecular simulations. J. Phys. Chem. B 2011, 115, 4168–4176. [CrossRef] 166. Van der Kant, R.; van Durme, J.; Rousseau, F.; Schymkowitz, J. Solubis: Optimizing protein solubility by minimal point mutations. In Methods Mol. Biol.; Humana Press Inc.: New York, NY, USA, 2019; Volume 1873, pp. 317–333. 167. Gil-Garcia, M.; Banó-Polo, M.; Varejao, N.; Jamroz, M.; Kuriata, A.; Díaz-Caballero, M.; Lascorz, J.; Morel, B.; Navarro, S.; Reverter, D.; et al. Combining structural aggregation propensity and stability predictions to redesign protein solubility. Mol. Pharm. 2018, 15, 3846–3859. [CrossRef] 168. Seeliger, D.; Schulz, P.; Litzenburger, T.; Spitz, J.; Hoerer, S.; Blech, M.; Enenkel, B.; Studts, J.M.; Garidel, P.; Karow, A.R. Boosting antibody developability through rational sequence optimization. mAbs 2015, 7, 505–515. [CrossRef] 169. Chennamsetty, N.; Voynov, V.; Kayser, V.; Helk, B.; Trout, B.L. Prediction of aggregation prone regions of therapeutic proteins. J. Phys. Chem. B 2010, 114, 6614–6624. [CrossRef] 170. Chennamsetty, N.; Voynov, V.; Kayser, V.; Helk, B.; Trout, B.L. Design of therapeutic proteins with enhanced stability. Proc. Natl. Acad. Sci. USA 2009, 106, 11937–11942. [CrossRef] 171. Majumder, S.; Jones, M.T.; Kimmel, M.; Alphonse Ignatius, A. Probing conformational diversity of fc domains in aggregation-prone monoclonal antibodies. Pharm. Res. 2018, 35, 220. [CrossRef] 172. Nakamura, H.; Oda-Ueda, N.; Ueda, T.; Ohkuri, T. Introduction of a glycosylation site in the constant region decreases the aggregation of adalimumab fab. Biochem. Biophys. Res. Commun. 2018, 503, 752–756. [CrossRef] 173. Pepinsky, R.B.; Silvian, L.; Berkowitz, S.A.; Farrington, G.; Lugovskoy, A.; Walus, L.; Eldredge, J.; Capili, A.; Mi, S.; Gra, C.; et al. Improving the solubility of anti-lingo-1 monoclonal antibody li33 by isotype switching and targeted mutagenesis. Protein Sci. 2010, 19, 954–966. [CrossRef] 174. Wu, S.-J.; Luo, J.; O’Neil, K.T.; Kang, J.; Lacy, E.R.; Canziani, G.; Baker, A.; Huang, M.; Tang, Q.M.; Raju, T.S.; et al. Structure-based engineering of a monoclonal antibody for improved solubility. Protein Eng. Des. Sel. 2010, 23, 643–651. [CrossRef] © 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

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Current Advancements in Addressing Key Challenges of Therapeutic Antibody Design, Manufacture, and Formulation

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Current Advancements in Addressing Key Challenges of Therapeutic Antibody Design, Manufacture, and Formulation

Current Advancements in Addressing Key Challenges of Therapeutic Antibody Design, Manufacture, and Formulation

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