Upstream Processing: Regulatory Considerations Regarding Quality Aspects of Monoclonal Antibodies

ABSTRACT

In the past two decades, more than 20 therapeutic monoclonal antibodies (MAbs) targeting a range of antigens (and working through a variety of mechanisms) have been approved for treatment of serious diseases. First to be approved were murine antibodies, followed by humanized molecules with superior efficacy, safety, and tolerance. Most of the licensed MAbs have been made by the hybridoma method and expressed in cell culture.^{2, 3} This review describes the next generation of MAbs, taking next generation antibodies into account, and suggests considering international regulatory guidelines for qualifying and characterizing monoclonal-antibody-producing source cells at different phases of development. In addition, in vivo and in vitro manufacture of MAbs is outlined. Adhering to the guidelines ensures a finished product of consistent pharmaceutical quality.

Atibodies (immunoglobulins) are glycoprotein molecules. They are made by foreign immunogen-stimulated B-cells and secreted into the blood to react with antigens present on soluble or cell-surface immunogens. Antibodies induce different immunological effects, such as neutralization of pathogens either by forming antibody-antigen complexes or by involving other immune cells.¹ Immortalized cell cultures of antibody-producing B cells can be propagated outside a living organism to maintain infinite supply. Antibodies constructed by this method are directed against single epitopes and are known as monoclonal antibodies (MAbs). Medicinal products containing MAbs as active substances have been developed in major therapeutic areas such as cancer, autoimmune and inflammatory diseases, and for several orphan drug indications.

HYBRIDOMA TECHNOLOGY

MAbs recognize and bind to receptors (antigens) of specific targets. These potential targets are proteins, which, unlike other cellular macromolecules (nucleic acids, polysaccharides, or lipids), have immunogenic properties. Drug development attempts to identify antibodies that affect protein–protein interactions by competing with endogenous molecules for binding sites.⁴ To rationally design potent therapeutic antibodies, the target's function should be well understood in the context of the immunopathologic pathway of the disease. It should also play a key role either as soluble factor (e.g., growth factor or cytokine) or as cell type implicated in tissue damage.

After being isolated and purified, target proteins are subjected to antibody screening procedures: Purified protein is used for the immunization of animals. From the natural antibody repertoire, antibody molecules with high specificity and affinity for the target are selected and their potencies defined in bioassays. The selected antibodies may work via interfering, blocking, or stimulating effects as required.⁵

The standard technology to immortalize activated B cells, which has been of fundamental importance in the monoclonal antibody history, is the hybridoma method. Most currently licensed MAbs were created this way. Developed in the mid 1970s by Köhler and Milstein, it is based on the fusion of rodent antibody-secreting B-cells with rodent tumor (myeloma) cells, followed by segregation steps to isolate single clones.⁶ The hybridoma method is outlined in Table 1.

Table 1. Description of hybridoma technology7

Because murine antibodies are immunogenic in humans, researchers have tried to limit the human antimouse immune reaction. One strategy has been to transfer mouse hybridoma technology to human cells. Ethical concerns associated with the immunization of human subjects, along with the lack of a suitable human myeloma cell line, have slowed the development of this approach. Nevertheless, it should be noted that human antibody-producing cells can be immortalized by infection with viruses. The small DNA Epstein-Barr herpes virus has been used to infect and immortalize human B cells. But current methods allow only inefficient immortalization rates and low antibody yields.⁸ Additionally, since immortalization involves the transfer of viral genes into the parent cell line, there is a risk of generating infectious viruses.

Humanized MAbs can alternatively be constructed using transgenic mice carrying human immunoglobulin genes, thereby minimizing the immunogenicity of resulting antibodies.^9,10,11 Although such animals express human antibodies, their production still requires standard hybridoma methods (immunization, B-cell isolation, and hybridoma formation).

Also, in mouse cell lines (used both for production purposes and as fusion partners for the preparation of hybridomas), recombinant DNA technologies facilitate the grafting of murine-antigen-recognizing DNA regions to human immunoglobulin genes integrated with mammalian expression vectors. This approach allows the stable expression of humanized antibodies in a range of cell lines such as Chinese hamster ovary (CHO) cells.¹²

REGULATORY CONSIDERATIONS REGARDING QUALITY ASPECTS OF MONOCLONALS

Once a clone has been identified, manufacturers should establish a seed-lot system to ensure a consistent supply of product with reproducible quality. This is achieved by means of creating a GMP-compliant source cell bank system, consisting of a master cell bank (MCB) and a working cell bank (WCB).^13,14,15,16

Typically, a MCB is sourced from a research cell bank, which may not be GMP-compliant. Thus, before introducing such cells into a GMP facility, minimal biosafety testing (e.g. sterility, mycoplasma, in vitro assay) is required. A MCB is defined as a "homogenous suspension of the cell line (producing the MAb), distributed in equal volumes in a single operation and usually cryopreserved in individual containers for storage."¹⁴ It usually consists of 100–200-vial lots.

A WCB is a "homogenous suspension of cells, derived from the MCB at a finite passage level."¹⁴ It is prepared using identical conditions and dispensed in identical volumes into cryopreserved vials for storage. Both the MCB and WCB should be stored under controlled (GMP) conditions; the method for preparation, as well as number of vials produced and used, should be recorded and storage conditions documented.

Along with MCB and WCB, post production cells, which are cells propagated up to or beyond the generation number used for routine production, should be tested and characterized.

PRODUCT CHARACTERIZATION

MAbs derived from the selected cell clone should be precisely analyzed with respect to structural integrity, specificity, and potency using a range of state-of-the-art chemical, physical, immunochemical, and biological tests. To assess biopotency, binding assays and functional in vitro/in vivo assays should be carried out.^14,15

When submitting a marketing authorization application, at least six months of stability data should be provided, complemented by further realtime data, which should be supplied as data become available.¹⁷

CHARACTERIZATION AND TESTING OF SOURCE CELLS

As part of their product analysis, manufacturers must test extensively the cell line used for production. The entire drug development program relies on thoroughly characterized source cells. It is recommended that manufacturers create source cells from nonmanipulated parental cell lines, for instance, via cell fusion or transfection. Based on characterized parental cell line, the presence of adventitious agents—ranging from bacteria, mycoplasma, yeast, and fungi to viruses and transmissible agents—should be addressed in the MCB and WCB.

Contamination of cells may result from infected source animals used for the original cell line.¹⁸ Viral contaminants may be introduced by failure of GMP during production or by contaminated excipients. Also, animal-derived raw materials, such as fetal bovine serum or trypsin, may be responsible for the transmission of animal (e.g., bovine or porcine) viruses.¹⁹ Regardless of how cells have been propagated, it is necessary to test for bovine and porcine viruses.

Table 2 summarizes methods for cell bank testing. Each assay is inoculated with a test sample, either a cell lysate or supernatant fluid from cell culture to test for contaminants such as species–specific viruses, adventitious viruses, and retroviruses.

Table 2. Summary of tests used for cell bank characterization18, 19

Species–specific viruses are usually detected by administrating the test sample via four different routes into appropriate virus and antibody-free test animals followed by immunological analyses of serum samples for a panel of animal specific viruses. For instance, a murine hybridoma is screened via the aforementioned mouse antibody production(MAP)test for 16 mouse viruses.¹⁵ In adventitious agent testing to detect introduced, nonobvious viral contaminants, the test sample is inoculated onto tissue cultures from various species susceptible to a wide range of viruses. Morphological evidence of cytopathology is sought after a suitable incubation period (14 days or 28 days). At the end of the observation period, tests for hemabsorbing and hemagglutination viruses are performed. Retrovirus screening is investigated by reverse transcriptase assay, infectivity assays, and detection and quantification of viral particles by electron microscopy.^16,20,21 More test methods are compiled in Table 2. MAbs are considered highly purified products not contaminated by cells; thus, tumorigenicity in athymic mice need not to be addressed.¹³

For hybridoma cell lines used as fusion partners, manufacturers must detail the following:

• source species, strain, and tissue

• feeder cell line

• immortalization procedures

• nature of the immunization antigen

• immunization procedure

• screening and selection processes

• cell cloning

• if applicable, adaptation to serum-free medium.

If a monoclonal is expressed in genetically engineered cells such as CHO, the origin, isolation, and cloning of antigen-recognizing DNA regions into the expression vector should be described.^{22, 23} The registration documentation should also contain the construction of the cell line, including vector and transgene nucleotide sequences, transfection procedures, cloning, amplification, and as the case maybe the change to serum-free medium should be described.

In addition, the genome structure should be investigated and stability of the cell line, copy number, and location of the transgene on chromosomes demonstrated. Species should be verified on the chromosome number or banding level by means of karyology.

It is important to note that after the cell substrate has been developed, it is not easy to switch to another cell line for production. If the cell line must be changed, for instance, to increase yield—comparability between the old and new cell banks has to be shown. This may not be limited to analytical data: depending on the development stage, nonclinical and clinical bridging studies may be required to show bioequivalence, safety, and efficacy.²⁴

Culture and Harvest

Cells can be cultured up to a defined number of passages, followed by recovery of product in a single harvest. Another option is multiple harvests during continuous growth of cells and cultivation for defined period, which requires careful surveillance of the cell line throughout its life span.

Each harvest of unprocessed bulk should be monitored by in-process testing for product potency, bioburden, endotoxin, and mycoplasma. Furthermore, for single harvest processes, representative samples of at least three lots of fermenter supernatant should be subjected to comprehensive biosafety testing¹⁴ as shown in Figure 1.

Figure 1

Purification

A downstream purification process is designed to enrich the product and to eliminate contaminating proteins, DNA, endotoxin, process-related components (media constituents, leachables), viruses, and prions. Process development hinges on finding the most efficient route from the starting material to pure, active final product, considering both yield and viral clearance. A characteristic purification process consists of a primary recovery step followed by multiple adsorption and chromatographic operations and final polishing steps (e.g., size-exclusion chromatography). In many cases, antibody is captured onto a Protein A matrix, followed by low-pH hold and elution, which is effective as a combined purification and virus inactivation step.²⁷

Viral Clearance

The purification process should include at least two robust viral clearance steps that inactivate or remove viruses based on different mechanisms (e.g., inactivation by low-pH treatment and nanofiltration). The capacity of a manufacturing process to reduce, inactivate, or eliminate viruses and other contaminants must be demonstrated by virus validation studies. In designing such studies, researchers take into account the nature of the cell line-in particular, the presence of cell-line-specific endogenous retroviruses and other relevant viruses.^{28, 29}

Final Bulk or Finished Product

Purified bulk product is formulated and aseptically filled into final container (e.g., prefilled syringe, infusion bottle). Normally, purified bulk itself is tested for sterility and residual DNA before formulation and preparation of finished product.

Because residual cellular DNA may be associated with malignant transformation activity, interference with normal gene expression, or production of infectious viruses, the amount of residual DNA in the final bulk product should be quantified. Accepted limits are in the range of 100 pg to 10 ng/dose. A more precise analysis examines not only the residual content, but also the molecular size of the residual DNA; smaller fragments (<500 nucleotides) are not likely to be transforming or oncogenic genes, and are therefore of less concern than larger fragments.^30,31

Likewise, the manufacturer needs to assess contaminating host cell proteins (HCPs) and process-related impurities. Risks associated with HCPs relate to adjuvant effects, transforming effects, and allergic and immune reactions. However, limits are not yet established for HCPs.

Other factors, such as route and frequency of administration, may play a role in the occurrence of adverse events.³² During early development, analyzing process impurities and HCPs by generic immunoassays (such as protein A or CHO) may be sufficient. Ultimately, however, in Phase 3 it is necessary to develop customized immunoassays using extracts from both nontransfected and transfected production cell lines.

The final product is subjected to a range of controls as shown in Figure 1. The overall qualification of cell banks and batch-specific safety testing requirements are summarized in the same figure. In Europe, quality specifications for MAbs have recently been integrated in the European Pharmacopoeia. Therefore, manufacturers must demonstrate compliance with Monograph 2031 concerning Monoclonal Antibodies For Human Use, Animal (2031). This does not apply to MAbs produced in vivo, which are governed by the requirements of the relevant authorities.

IN VIVO PRODUCTION

Fully tested and characterized MCB and WCB can be used as the cell substrate for routine production. In principle, any monoclonal can be produced in vitro by means of cell culture fermentation processes, or in vivo by injecting hybridomas into the abdominal cavity of mice (mouse ascites method). In practice however, certain cases require in vivo production,⁷ including the following:

• the production cell line cannot be adapted to in vitro growth conditions

• the antibody from in vitro culture is prone to denaturation during purification or concentration steps

• in vitro culture could affect the modification of the molecules (sugar residues) with consequences for the binding and biological activity of the antibody

Some companies have adopted the mouse ascites method for large-scale production of licensed first generation (mouse) antibodies. Table 3 summarizes information about several such murine monoclonals. It should be noted that in most European countries, the ascites method is restricted to certain conditions or even banned³⁶ due to animal welfare considerations.

Table 3. First generation antibodies10,37–38

If the ascites method is justified and authorized, the following regulatory information should be presented in the registration dossier⁷:

Source Animals

• information on animals used for production (age, sex, strain, confirmation of specific pathogen free status, source)

• information on animal health monitoring program (monitoring of stock, mouse-specific viruses, mycoplasma) and demonstration of meeting animal welfare requirements

• information on physiological and physical examination and testing of animals

Ascites Production

• information on priming with tumor-promoting compound

• information on inoculum, cell number, and timing

• information on ascites harvesting (number, frequency, and procedures)

• batch definition, storage conditions and duration

For large-scale in vivo production a high number of mice (up to several thousand) may have to be used.³⁸ Following priming and inoculating with selected hybridoma cells (e.g., WCB), mice need to be monitored daily as a process control measure. About 5 mL ascites per mouse is collected approximately two to four weeks post inoculation.³⁹ Once a batch is harvested, ascites are tested for a range of parameters (potency, bioburden, endotoxin, mycoplasma, murine viruses) and antibody is purified as described previously.

IN VITRO PRODUCTION

MAbs should preferably be manufactured by in vitro methods to avoid using animals. As indicated, both mouse cell lines and genetically engineered mammalian cell lines have been used in producing licensed antibodies. For stable expression of recombinant MAbs in continuous mammalian cell lines, such as CHO K1 or SP2/0 (a murine myeloma cell line), are generally preferred. Productivity of selected continuous cell lines (CCLs), which are either cultivated as attached cells on carrier materials or in suspension in bioreactors, is often further increased and maximized by vector amplification.^12,18,40 CCLs enable manufacturers to establish cell banks for a reliable production source based on standardized and characterized cells. Also, they are easier to cultivate than primary or diploid cells, require only simple culture media, and can be adapted to serum-free medium. Some cell substrate examples from licensed MAbs are given in Table 4.

Table 4. Examples of cell lines used for production of MAbs34–35, 41–43

For large-scale production, antibody product is usually enriched from cell-free supernatant from up to 15,000 L fermenters (e.g. Synagis: 10,000 L, Herceptin: 12,000 L,^35,43) and purified as described above.

BIOSAFETY ASPECTS

CCLs have been isolated by serial subcultivation of primary animal cells, transformation of normal cells with oncogenic virus or, as in the case of hybridomas, by fusion of tumor and B-cells.³¹ Concerns about the safety of products derived from such cell lines relate particularly to the presence of the following:

• human pathogenic viral contaminants and potential transmission of viruses to humans

• tumorigenic or mutagenic residual cellular DNA

• antigenic or unwanted catalytic (e.g., growth-promoting) HCPs

Despite these concerns, the safety of products derived from established CCLs (e.g., CHO) is broadly accepted, and well-characterized standard cell lines are available.³¹ In terms of a particular production cell line, biosafety is achieved by the following complementary approaches:^18,19

• Rigorous control of cell lines by testing or characterization and control of raw materials used during manufacture

• in-process and finished product testing

• virus clearance and reduction steps in the manufacturing process

• strict adherence to GMP

In order to minimize viral risks during propagation of CCLs, it is important to source serum from reliable suppliers who provide certificates of analysis demonstrating freedom from viruses in compliance with the relevant guidance (e.g., CPMP/BWP/1793/02). Furthermore, compliance with TSE guidance (e.g., 2004/C 24/03) must be shown. In addition to established cell lines (e.g. CHO, NSO), other cell lines of human (e.g., PerC6) or insect (e.g., Sf9) origin may be used pending a comprehensive biosafety assessment.

SUMMARY AND OUTLOOK

Hybridoma technology has achieved broad regulatory acceptance as evidenced by licensure of about 20 MAbs. However, the method is laborious and does not work for certain immunogens (e.g., toxins, highly conserved proteins, pharmacological active molecules) which are not suitable for obtaining high affinity antibodies.^5,11 An alternative to the hybridoma method is to produce engineered antibody molecules and fragments by in vitro methods. These methods are based on complex combinatorial libraries containing a large collection of variant antibody-like molecules. They involve the selection of candidate molecules by screening procedures. Several discovery platforms (e.g. phage, bacterial, yeast, and ribosome display) have been developed. Most advanced is phage display. This approach allows the insertion of human antibody genes into phage DNA and the production of combinatorial libraries containing random heavy- and light-chain pairings which are presented as variant antibody fragments on the surface of filamentous phages. In vitro screening assays can identify antibodies that bind to the target. After initial antibody isolation, the affinity of candidate molecules can be further increased by reiterative in vitro maturation processes.^11,44 Recently, a fully human phage-display engineered MAb, adalimumab (Humira) has been approved for treatment of rheumatoid arthritis.⁴² In the next several years, many more monoclonals prepared in vitro will enter the market.

Acknowledgment: I would like to thank Dr. Richard Peck for his critical review of the manuscript.

Manfred Kurz, PhD, is a regulatory affairs associate at CSL Behring, Bern, Switzerland, 41.0.31.344.5022, manfred.kurz@cslbehring.com

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