The AAPS Journal, Vol. 18, No. 4, July 2016 ( 2016) DOI: 10.1208/s12248-016-9935-9 Mini-Review Theme: Next Generation Formulation Design: Innovations in Material Selection and Functionality Guest Editors: Otilia M. Koo, Panayiotis P. Constantinides, Lavinia M. Lewis, and Joseph Reo 1,2 Fouad Atouf Received 6 February 2016; accepted 17 May 2016; published online 27 May 2016 Abstract. Cell-based therapy is the fastest growing segment of regenerative medicine, a ﬁeld that promises to cure diseases not treated by other small molecules or biological drugs. The use of living cells as the active medicinal ingredient present great opportunities to deliver treatment that can trigger the body’s own capacity to regenerate damaged or diseased tissue. Some of the challenges in controlling the quality of the ﬁnished cell-therapy product relate to the use of a variety of raw materials including excipients, process aids, and growth promotion factors. The quality of these materials is critical for ensuring the safety and quality of the ﬁnished therapeutic products. This review will discuss some of the challenges and opportunities associated with the qualiﬁcation of excipients as well as that of the ancillary materials used in manufacturing. KEYWORDS: ancillary material; cell-based therapy; excipient; formulation. INTRODUCTION that may remain in the ﬁnal product. The goal of this review is to provide an overview of the types of excipients used in Cell-therapy products offer new solutions to treat unmet cell-therapy formulations, as well as discuss risk assessment medical needs by providing therapeutic products based on strategies to ensure their quality, and how to mitigate their living cells that can promote the body’s own regenerative impact on the critical quality attributes of ﬁnished cell- capacity. The cell-therapy market is one of the most therapy products. diversiﬁed segments of the biotech industry; it includes human somatic and embryonic cells. A patient’sown Unintended Components (autologous) cells as well as donated (allogenic) cells or tissues can be used to manufacture cell-therapy products. In In the USA, the U.S. Food and Drug Administration both cases, the development and manufacture of the ﬁnished (FDA) Guidance on Content and Review of Chemistry, product can require either minimal manipulation or extensive Manufacturing, and Control (CMC) Information for Human processing that can expose the cells to additional components. Somatic Cell Therapy Investigational New Drug Applications Major challenges in cell-therapy formulations are potential (INDs) (2) states that Ban excipient is any component that is interactions between the components used in manufacturing intended to be part of the ﬁnal product, such as human serum and formulation, and the living cells. As with other biologics, albumin or Dimethyl Sulfoxide (DMSO).^ The US code of cell-based therapy formulations include a variety of excipients federal regulations require that all excipients used during the (buffers, salts, polymers, proteins, and preservatives) that are manufacture of products that are intended to be present in added to stabilize the cells or to provide physiological the ﬁnal product must be listed in the Investigational New osmolality. The manufacture of cell-based therapies often Drug (IND) Application (21 CFR 312.23(a) (7) (iv) (b)). The requires multiple steps where the cells are exposed to a qualiﬁcation of intended excipients present in the ﬁnished variety of cell culture supplements not intended for inclusion therapeutic product is required per 21 CFR 211.84(a). The in the ﬁnal product. These cell culture supplements are United States Pharmacopeia’s book of public standards referred to as process aids, ancillary reagents, or ancillary (referred to as a Bcompendia^), U.S. Pharmacopeia–National materials (1); the ﬁnal harvest of cells prior to formulation Formulary (USP-NF), deﬁnes excipients under General and patient use may carry residual amounts of these Chapter <1078> as follows: BAny substances, other than the materials. In this review, the term Bexcipients^ will refer to active drug or product, that have been appropriately evalu- components used in the formulation and to ancillary materials ated for safety and are included in a drug delivery system to either aid the processing of the drug delivery system during its manufacture, protect, support or enhance stability, bio- United States Pharmacopeial Convention, Rockville, Maryland, availability, or patient acceptability, assist in product identiﬁ- USA. To whom correspondence should be addressed. (e-mail: firstname.lastname@example.org) cation, or enhance any other attribute of the overall safety 1550-7416/16/0400-0844/0 2020 The Author(s) 844 Cell-Based Therapies Formulations: Unintended Components 845 and effectiveness of the drug delivery system during storage support of the use of a new excipient. Additional information or use.^ While excipients that are intended to be used in cell- on regulatory requirements can be found in the International therapy formulations that meet the deﬁnition will be qualiﬁed Conference on Harmonisation of Technical Requirements for and tested against compendial monographs when they exist, Registration of Pharmaceuticals for Human Use (ICH) Q8 some of the materials used in manufacturing other than the Pharmaceutical Development (R2) (5) where it states that excipients may end up in the ﬁnished products. These Bthe ability of excipients to provide their intended function- ancillary materials are used as process aids for the manufac- ality, and to perform throughout the intended drug product ture of the cell-based therapies and for which removal from shelf life, should also be demonstrated.^ An example of ﬁnished products may not be consistently effective. repurposing an established excipient for cell therapies is Sakamoto et al.(3) reported antibody responses in the use of poloxamer based hydrogels as cell carriers, patients treated with cell-based therapies cultured in the which has gained attraction with some of the cell-based presence of fetal calf serum and that bovine apolipopro- regenerative medicine products under development (e.g., tein B-100 is the predominant target of the antibody therapies to treat bone defects). Poloxamers have been response. This observation suggests that the washing steps established as pharmaceutical excipients in a variety of leading to the ﬁnal harvest of a cell culture-based product topical dosage formations including dental and burn may not be effective in clearing the cell suspension from dressing applications (6)Somepoloxamersare water- proteins or other components in the media, which can be soluble and will form a gel at higher temperatures (e.g., considered Bunintended^ components. Ancillary materials body temperature) and can be used to deliver cells into should go through the same risk assessment for inclusion the human body as an alternative to solid scaffolds. as the excipients used in the product. Risk based During development of such products, the safety proﬁle approaches should take into account supplier qualiﬁca- of and experience with these types of gels may be used tions, control of supply chain and proper documentation, and extrapolated to cell- and tissue-based therapies; and testing strategies. Such approaches will allow the however, the impact of these materials on living cells identiﬁcation of critical ancillary materials so that they can (and vice versa) needs to be evaluated. be qualiﬁed and sourced as for the excipients. Ancillary materials used in large quantities, materials used down- Risk Assessment Strategies and Qualification of Components stream of the process that may come in contact with the cells prior to ﬁnal harvest, and complex mixtures such as The use of established excipients is the preferred animal-derived materials are examples of high-risk mate- formulation approach. However, this is not always an option rials that are more critical. and developers of cell-therapy products will often use non- pharmaceutical grade materials. In these situations, strategies Quality of Components and Requirements for Novel need to be in place to control the safety and quality of all raw Formulations materials used in manufacturing. Risk assessment must address the complexity and multicomponent nature of the The excipients and other materials intended for use in raw materials. Animal-derived materials are considered to be cell-based formulations may have been qualiﬁed for use in a higher risk because of the potential of contamination with other drug and biologic formulations. Table I shows examples adventitious agents, and justiﬁcation is needed for the use of of US approved cell-therapy products with a list of reported such materials, as well as testing to conﬁrm absence of inactive ingredients (excipients) in addition to other ingredi- adventitious agents. Additionally, information on the country ents used as ancillary materials with the potential to be of origin is required per Good Manufacturing Practices present in the ﬁnal formulation at residual levels. The (GMPs) regulations when using materials that carry the risk reported inactive ingredients are known excipients with of transmissible spongiform encephalopathy. The use of established safety proﬁles. However, applying formulations human-derived materials in cell-based therapies carries the and excipients shown to be successful for existing dosage risk of transmission of communicable diseases. In line with forms to new ﬁnished cell-therapy products requires a the quality risk management described in ICH Q9, a risk scientiﬁc justiﬁcation for the use of each excipient in addition mitigation strategy should include having controls in place to to toxicity studies to demonstrate that the excipient does not assess the impact of using animal- or human-derived excipi- have deleterious effects on living cells. The use of established ents. While pharmaceutical-grade excipients are considered excipients in cell-therapy formulations will require evaluating low risk because of their established safety proﬁle, it is the potential toxicity of the excipient on the cells, if the important to note that suitability of the excipient and its excipient is used a higher amount or in a new route of impact on the therapeutic effect of the cells need to be administration and will also require bridging safety and established. A strong qualiﬁcation program is required to toxicological studies. Additionally, the potential impact of ensure the quality of excipients and other components used in the cells and cell culture byproducts on the functionality of the manufacture of cell-therapy formulations. Proper speciﬁ- the excipient needs to be addressed. A scientiﬁc rationale cations are required to determine the suitability of these needs to be provided for the introduction of novel excipients, components during manufacture and in the ﬁnal formulation. demonstrating how the novel excipient improves the stability, The manufacturing history of these components as well as the safety, or efﬁcacy of the ﬁnished product. The FDA Guidance vendor (preferably the manufacturer) needs to be docu- for Industry on Nonclinical Studies for the Safety Evaluation mented. The relationship between the cell density and the of Pharmaceutical Excipients (4) provides guidelines on the ﬁnal concentration of the excipient needs to support the toxicity information that must be submitted to the FDA in intended use of the excipient. The stability of the excipients 846 Atouf Table I. Examples of Excipients and Formulations in the US Licensed Cell Therapy Products Product name Manufacturer Active ingredient and Description Additional product reported inactive cell type and description and a a ingredients formulation potential ingredients that may end up in the product Provenge ® Dendreon Sipuleucel-T CD54+ cells activated with Peripheral blood mononuclear (autologous) Inactive Ingredients: prostatic acid phosphatase cells, including antigen Calcium chloride (PAP) linked to granulocyte- presenting cells (APCs), Potassium chloride macrophage colony-stimulating activated with a recombinant Sodium chloride factor (PAP-GM-CSF) in human protein, PAP-GM-CSF, Sodium lactate Lactated Ringer’s Injection, Water USP Laviv ™ Fibrocell Technologies, Azficel-t Cultured dermal ﬁbroblasts Cells are expanded and then (autologous) Inc. suspended in Dulbecco’s cryopreserved in a protein-free Modiﬁed Eagle Medium solution containing DMSO. without Phenol Red. Cells are thawed, washed, and shipped to the clinic. Carticel® Genzyme Biosurgery Autologous cultured Cultured chondrocytes cells Cells expanded in media (autologous) chondrocytes aseptically processed containing 50 μg/mL and suspended in 0.4 mL gentamicin. Residual of sterile, buffered quantities of gentamicin up Dulbecco’s Modiﬁed Eagles to 5 μg/mL may be present Medium (DMEM). in the Carticel product Gintuit Organogenesis Foreskin fibroblast, Cultured keratinocytes and Cells shipped in an agarose (allogeneic) bovine type I collagen, and ﬁbroblasts in bovine collagen gel medium to maintain foreskin keratinocyte for topical application product potency and (neonatal) in the oral cavity. therefore may contain low amounts of inactive components present from the media. These include agarose type IV HI EEO, L-glutamine, hydrocortisone, full-chain human recombinant insulin, ethanolamine, O-phosphorylethanolamine, adenine, selenious acid, Dulbecco’s Modiﬁed Eagle Medium (DMEM) nutrients, Ham’s F-12 nutrients, sodium bicarbonate, calcium chloride, and water for injection. ALLOCORD SSM Cardinal Glennon Human cord blood The active ingredient is When prepared for infusion (allogeneic) Children’s Medical Hematopoietic hematopoietic progenitor according to instructions, Center St. Louis Cord progenitor cell cells which express the infusate contains the Blood Bank Inactive ingredients: the cell surface marker following inactive ingredients: Dimethyl sulfoxide (DMSO) CD34. The cellular PrepaCyte-CB separation Dextran 40 composition depends solution, citrate-phosphate- on the composition of cells in dextrose, Dextran 40, human the blood recovered from the serum albumin, and residual umbilical cord and placenta DMSO. of the donor. Source of information: www.dailymed.nlm.nih.gov should be assessed in the presence and absence of the actual ﬁnished cell-therapy products, residual levels of ancillary cell-therapy product given that the established stability materials may remain due to ineffective washing steps or proﬁle of the excipient by itself may not be relevant once because of uptake or binding to the cellular components. it is added to the formulation. It is also critical to The development of robust assays to measure levels of determine if potential degradation of the excipient might ancillary materials in the ﬁnal formulation is critical to have deleterious effects on the ﬁnished product. Overall, show the effectiveness of their removal. If residual the stability and viability the cells and expression of amounts of the ancillary materials are present in the therapeutic entity needs to be established. While the ﬁnished product, they should be qualiﬁed including intent is to remove the non-cellular components from toxicity assessment in animal models or other systems. Cell-Based Therapies Formulations: Unintended Components 847 Cryopreservation and Formulations of Cell-Therapy Products metabolites, cytokines, and other growth factors in the media. This may increase the possibility for excipients to interact Cell-therapy products prepared for speciﬁc patients (e.g., with media components, and cells and their functions may be autologous bone marrow derived cells) can be maintained in impacted because of these interactions. It is therefore critical liquid suspensions or formulations until administration. For other that a risk assessment strategy for the qualiﬁcation of cell-therapy applications, the timing between cell collection/ excipients used in cell-based therapies encompass elements processing and administration to the patient may require an beyond standard approaches for excipients used in drugs and additional hold time. In these situations, cryopreservation may be other biologics. Cell-therapy manufacturers need to address a suitable approach to maintain product stability during storage the functionality and stability of the excipients used in these and shipping steps (7). Cryopreservation may also be applicable to applications. The functionality of an excipient established preserve the starting cell-source material and intermediate within a formulation for another drug or biologic may or may products before further processing to manufacture the ﬁnal not be extrapolated to a cell-therapy formulation and may product. In all applications, the goal is to ensure preservation of require testing of multiple batches of the excipient in multiple product characteristics and quality attributes. Additionally, for batches of the cell-therapy product. Another challenge is large-scale manufacturing of allogenic cell-therapy products, multisource suppliers of the same grade of excipient which cryopreservation facilitates the establishment and implementation can lead to batch-to-batch or supplier-supplier variability and, of quality systems and workﬂow optimization allowing timely lot- potentially, inequivalent performance of the excipient. The release testing, management of a product inventory, transport of successful manufacture of a robust product requires the use of the product to the clinical site, and coordination of product well-deﬁned excipients and processes that together yield a administration (8). While cryopreservation is critical for stabilizing consistent product (USP <1078> (10), section of GMP cell-therapy products until administration to the patient, the principles). Among the factors that contribute to quality of ﬁnished products are the following: adherence to cGMPs; components of a cryopreservation medium may impact the safety process control and validation; and appropriate testing and and efﬁcacy of the ﬁnished products. Serum-free and protein-free cryopreservation media formulations are commonly used. While quality of components added to manufacturing. For cell- cell product washing methods prior to administration must be based therapies, the latter is even more critical as the validated to ensure adequate recovery and cell functions and components used in manufacturing come in intimate contact removal of cryopreservation components, it is important to note with the active ingredients—the cells—whether these are that for some of the applications, the product is delivered to the process materials or elements of ﬁnal formulations. A robust patient in the freeze media to minimize manipulation of the qualiﬁcation program for starting materials, ingredients, cryopreserved cells. Cell washing does not guarantee complete excipients, and other raw materials is important to ensure a removal of cryopreservation components and residual levels may successful manufacturing process and the safety and efﬁcacy be detected in the ﬁnished product; their levels should be of ﬁnished products. Excipients and cell culture media estimated or measured. In both scenarios, the cryopreservation components need to be carefully selected especially for media components may be accounted for as excipients and complex and not well-deﬁned substances (e.g., material of qualiﬁed as such. Dimethyl sulfoxide (DMSO) is commonly used animal origin). Cell therapies are manufactured under aseptic in cryopreservation media for starting cellular materials such as conditions and generally are intended to be delivered through hematopoietic stem cells derived from cord blood, which are used injection or infusion of cells. Like any other therapeutic to manufacture autologous or allogenic cell products to treat some produced by aseptic processing, cell-based therapies can be genetic disorders that affect the immune system. High levels of contaminated through components including microorganisms DMSO may be given to patients who receive multiple doses of or or endotoxins. The starting material, water for injection, multiple cryopreserved cell-therapy products. Developers of these excipients, and other raw materials are all potential sources of products will use compendial-grade DMSO as there are mono- contamination. It is important to characterize the bioburden and measure the endotoxin levels of each component and graphs for DMSO in both the US Pharmacopeia and European establish appropriate acceptance limits. Sterility assurance Pharmacopoeia. In addition to using DMSO manufactured under and freedom from endotoxins are critical for cell- and tissue- a Good Manufacturing Practice (GMP) environment and that based products more than other biologics because these meet compendial standards, cell-therapy manufacturers can products are not subject to treatments such as terminal reference a drug master ﬁle for DMSO ﬁled with the US FDA. sterilization and ﬁltration. It is important to highlight that in addition to ensuring the quality of DMSO itself,special attentionneedstobepaidtothe packaging material used for cryopreservation and formulation of CONCLUSIONS the cell products. DMSO interacts with many polymeric materials, and only high-density polypropylene and polytetraﬂuoroethylene As cell-therapy applications are increasingly introduced materials are suitable for packaging a product containing high to the market, qualiﬁcation of ancillary materials used in levels of DMSO (9). Package testing and compatibility with manufacturing should be addressed as well as excipients DMSO should be built into the risk assessment strategy. because of the potential for residual ancillary material in the ﬁnished products. Regardless of whether the material is an Excipients and Quality of Finished Products excipient or an ancillary material, the functionality of the material needs to be addressed in the context of the Among biologic drugs, cell and tissue therapies are formulation as these materials may interact with the living unique in the sense that their Bactive ingredients^ may be cells impacting their function (and vice versa) and that this living cells capable of continuous growth and they can secrete impact may be further ampliﬁed by the batch-to-batch or 848 Atouf supplier-supplier variability. The introduction of novel excipi- REFERENCES ents or other elements in the formulation of cell-therapy products must take into account and address the possible impact 1. <1043> Ancillary materials for cell, gene, and tissue engineered products. In: USP38-NF33. Rockville, MD: US Pharmacopeial on the quality of the ﬁnished products. 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"The AAPS Journal" – Springer Journals
Published: May 27, 2016
Keywords: ancillary material; cell-based therapy; excipient; formulation