Advances inthe Development. of Therapeutic

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1 Advances inthe Development of Therapeutic Monoclonal Antibodies Susan Dana Jones, Francisco J. Castillo, Howard L. Levine ABSTRACT Monoclonal antibodies (MAbs) and related products are a dominant component of the biopharmaceutical market, generating revenues of several billion dollars. While MAbs have proven to be valuable therapeutic products, the typical doses of these products required for treatment are significantly higher than those required for most other biologic products, resulting in the need for large-scale production and efficient, costeffective manufacturing processes. In the past few years, improvements have been made in critical areas, such as cell line generation and large-scale cell culture production, to maximize productivity. These advances, coupled with improvements in cell culture media and optimized bioreactor processes, have made large-scale production of MAbs economically viable. However, the increasing production requirements and the drive to reduce the cost to develop these expensive medicines continue to present challenges to the industry to further improve the overall efficiency of manufacturing processes. This article presents a historical review of the discovery, development, and production of therapeutic antibodies. Susan Dana Jones, PhD, is a senior consultant, Francisco J.Castillo, PhD, is a senior consultant, and Howard L. Levine, PhD, is a principal consultant, all at BioProcess Technology Consultants, Inc., Acton, MA, , sjones@bioprocessconsultants.com. Listen to a podcast interview with Howard Levine at biopharminternational.com/biopharmnow The first therapeutic monoclonal antibody (MAb) product entered the market in 1986, but it took another decade before the potential of this new class of biologic products began to be realized. From the mid 1990s until today, almost 30 therapeutic monoclonal antibodies (MAbs) have been approved throughout the world along with several antibody-related products (e.g., Fc-fusion proteins) making MAbs and related products a dominant component of the biopharmaceutical market, generating revenues of several billion dollars. The first approved MAb was a murine antibody. This was followed by several chimeric MAbs containing a mix of murine and human regions. These early antibody products posed a moderate risk of immunogenicity to patients from their residual murine components, somewhat limiting the development of MAb products. To address this issue, new technologies for creating MAbs that were predominately or entirely of human origin were developed. Today, almost all antibody products currently in development are humanized or fully human. While MAbs have proven to be valuable therapeutic products, the typical doses of these products required for treatment are significantly higher than those required for most other biologics, resulting in the need for large-scale production and efficient, cost-effective manufacturing processes. In the past few years, improvements have been made in critical areas, such as cell line generation and large-scale cell culture production, to maximize specific antibody productivity from a given cell line and improve overall productivity in bioreactors. These advances include the use of new expression vectors and transfection technology 96 BioPharm International October 2007

2 Figure 1. Annual approval of recombinant biologic products and monoclonal antibody products. 2,3 The total number of biologics, including MAb products, approved by FDA for market each year since 1982 is shown in green. MAb product approvals only are shown in black. Antibody-related products such as Fc fusions, engineered antibody fragments, or other products derived from antibodies but not containing an antibody binding region are not included in the MAb figures. However, those products are included in the total product figures Total biologics including MAbs MAbs to improve cell line generation; novel parental cell lines that have been selected or designed to grow to maximum density and productivity under standard bioreactor conditions; and high-throughput, robust screening technologies to select the highest producing clones rapidly and more effectively. As a result, the production of cell lines expressing multigram quantities of antibody per liter of culture medium is now routine. These advances, coupled with improvements in cell culture media and greatly optimized bioreactor processes, have made the large-scale production of MAbs economically viable. However, the increasing production requirements and the drive to reduce the cost to develop these expensive medicines continue to present challenges to the industry to further improve the overall efficiency of manufacturing processes. These challenges include the need to streamline downstream processing to enable the processing of increased product quantities; the implementation of Quality by Design (QbD) and other new regulatory concepts to reduce the cost and development timelines for MAb products without adversely affecting their quality; the need for high-concentration product formulations with sufficient stability to address the increasing doses of antibody products; and the development of alternative delivery systems. DISCOVERY OF ANTIBODY THERAPEUTICS In 1984, Kohler and Milstein received the Nobel Prize in Medicine for their pioneering work on the production of MAbs. 1 One of the most significant advantages of this new technology over traditional techniques for producing antibodies was the development of an immortalized cell line creating a continuous source of the same antibody with a single antigen specificity. This enabled the development of highly specific antibodies directed toward a single epitope on the target antigen. Initially, MAbs were used as laboratory reagents, but they were quickly adopted as clinical diagnostic reagents, and eventually as therapeutic agents. The development of therapeutic MAbs commenced in the early 1980s and by 1986 the first monoclonal antibody for human use Orthoclone OKT3 (Ortho Pharmaceuticals) was approved for the prevention of kidney transplant rejection. Following the approval of OKT3, the enthusiasm for MAbs as therapeutic products grew with the next wave of antibody products generally being developed as anticancer agents. Several of these products were approved in the US and Europe in the mid to late 1990s, a trend that continues to grow today. Since the commercialization of the first therapeutic MAbs, these products have become a dominant component of the biopharmaceutical market, representing approximately 20% of all biologic products, with combined revenues of over $20 billion in The growth of MAb products over the past 25 years, as shown in Figure 1, confirms the importance of these products and also shows that MAbs represent a significant subset of all biopharmaceuticals on the market and in development. With over 300 antibody products currently in development, this unique and effective category of therapeutic compounds is poised to grow significantly in the coming years. MOLECULAR STRUCTURES OF ANTIBODIES:THEN AND NOW Murine Antibodies The initial technology for producing MAbs 98 BioPharm International October 2007

3 involved fusing individual antibody-secreting spleen cells from immunized mice with a murine myeloma cell line to generate immortalized cell lines that secreted individual, or monoclonal, antibodies. Hence, the first MAbs developed for use as potential human therapeutics were murine antibodies. While initial interest in these murine MAbs was high and several companies began developing products based on this technology, OKT3 was the only murine monoclonal antibody that was approved for human therapeutic use. Despite the fact that OKT3 has been moderately successful in the market, the use of murine MAbs as therapeutic agents quickly ran into many roadblocks. One of the potential advantages of MAbs as therapeutic agents is their long circulating half-life, allowing them to provide a therapeutic effect in patients over several days. However, when murine MAbs were repeatedly administered to humans during clinical trials, it was observed that the half-life decreased and the products became less effective with each injection. This was because of the immunogenicity of murine proteins in humans and the rapid development of a human antimurine antibody (HAMA) response in the patients. This HAMA response neutralized the effectiveness of the murine antibodies and resulted in their rapid clearance from the body. For example, it has been reported that OKT3 can elicit a HAMA response in up to 86% of patients treated, leading to some limitations in its efficacy. 5 Chimeric Antibodies To overcome the HAMA responses occuring from the usage of murine MAbs as therapeutics, several approaches were developed in an attempt to make MAbs more human-like and less immunogenic. In the early 1990s, molecular biology techniques enabled the creation of chimeric antibodies by linking the murine genes encoding the antigenbinding portion of the antibody (the variable region) to the genes encoding the constant region of human immunoglobulin light and heavy chains. Because over 75% of the protein sequence of the resulting chimeric antibodies was of human origin, these chimeric MAbs elicited much lower HAMA responses in patients. Moreover, because the antibody Future therapeutic monoclonal antibody products will be predominantly humanized or fully human. constant region in these chimeric antibodies is human, it is capable of activating other components of the human immune system to potentially create more effective therapeutic agents. Many of the MAbs approved for commercialization in the 1990s and early 2000s were chimeric antibodies, including the highly successful anticancer antibodies Rituxan (approved in 1997) and Erbitux (approved in 2004), as well as the antiinflammatory product Remicade (approved in 1998). Chimeric antibody products are superior to murine antibody products but they still pose a moderate risk of immunogenicity to patients from their residual murine components. Therefore, antibody engineering approaches that further reduce the murine component or that remove immunogenic portions of the chimeric antibody, have been developed and used to generate fully humanized antibody products. Humanized Antibodies In 1991, Protein Design Labs (PDL) developed and patented the first technology for successfully humanizing MAbs. 6 The antigen binding specificity of any antibody is determined by the amino acids present in three distinct highly variable regions per antibody chain, referred to as complementarity determining regions (CDRs), and located in a more conserved framework sequence in the variable regions. Therefore, PDL scientists developed methods for engineering an antibody gene in which the CDRs of a human antibody gene were replaced by those from the CDR of a murine MAb gene. The resulting humanized antibody has the same antigen binding properties as the original murine antibody but contains minimal murine sequences and, therefore, elicits a lower HAMA response in patients. The CDR-grafted human antibody can be used as is or, in cases where affinity of the chimeric antibody is slightly reduced from the original murine antibody, additional BioPharm International October

4 Table 1. Comparison of sales for antibody-based anti-inflammatory products Product Company Year approved 2006 sales worldwide ($ million) Market share Humira Abbott , % Remicade Johnson & Johnson , % Enbrel Amgen , % changes can be made in the antibody sequence to regain or enhance its binding properties. Like chimeric antibodies, humanized antibodies can activate other parts of the immune system to create a more effective product. Several humanized antibody products are currently on the market, including Synagis (approved in 1998), Herceptin (approved in 1998), Mylotarg (approved in 2000), Xolair (approved in 2003), and Avastin (approved in 2004). In addition to the production of chimeric and humanized antibodies, other technologies have been developed to help minimize the HAMA response in patients. These include human engineering or deimmunization, in which amino acids on the surface of the murine variable region that are known to be effective immunogenic sequences are changed to their non-immunogenic human counterpart, leaving the other non-immunogenic murine sequences unchanged. 7 The advantage of this approach is that the structural integrity of the variable region is better maintained and reduction of affinity for the target is minimized. Fully Human Antibodies The latest advancement in creating less immunogenic therapeutic antibody products is the ability to generate fully human MAbs. Several technologies exist to develop fully human antibodies, each falling into one of the two general classes in vivo approaches using a murine system in which the immunoglobulin genes have been replaced by their human counterparts or in vitro approaches using libraries containing millions of variations of antibody sequences coupled with a mechanism to express and screen these antibodies in vitro. Humira (approved in 2004) is the first fully human antibody to be approved. This anti-tnf-α antibody was first identified by scientists at Cambridge Antibody Technology (CAT, now part of AstraZeneca) using an in vitro molecular engineering technology known as phage display. In the marketplace, this human MAb competes with Enbrel, an Fc fusion protein, and Remicade, a chimeric antibody. The power of the fully human antibody platform can be seen in the sales figures for these three products. Although Humira was approved four years later than the other products, it has successfully taken a significant market share from them, garnering almost 16% market share in Worldwide sales in 2006 for all three products are shown in Table 1. Many antibody products currently in early clinical development are fully human, because the technologies that enable the generation of human antibodies are now accessible through partnerships or licensing from the companies that have developed these approaches. Moreover, the expectation in the medical and regulatory community is that companies will use the best approach for their product to achieve humanization. There will be exceptions to this generalization, for example when a short half life is desired or when a toxic or radioactive payload is linked to the antibody, but for unmodified therapeutic antibody products the industry standard has changed; most future antibody products will be humanized or fully human antibodies. Most MAb products are naked antibodies, which rely on either blocking an important biological function or on activating the immune system, to elicit a therapeutic effect. However, antibodies are also well suited as targeting agents to deliver potent chemo- or radioactive agents specifically to target cells. For example, Mylotarg contains a cytotoxic compound conjugated to a monoclonal antibody. This immunoconjugate product is designed to deliver the potent cytotoxic compound selectively to cancer cells. The radio-immunoconjugate products Zevalin and Bexxar (both anti-cd20 MAbs), deliver radioisotopes for the treatment of lymphoma. Both these products are murine antibodies because the human or humanized forms of these products would bind to and target not only the CD20 positive target cells but also those cells that contain the IgG 100 BioPharm International October 2007

5 receptors that function to enable antibodies to recruit additional immune system components to the site of a foreign antigen. By inadvertently targeting these cells, human antibody-based radio-immunoconjugates could do more harm to nontarget cell types than to the targeted cancer cell. All of the above technologies now allow the generation of better designed antibody products with fully human sequences and optimized function. Combining in vivo and in vitro discovery and molecular engineering technologies allows exquisite control of the antibody sequences and properties that was not possible 20 years ago. New approaches for the rapid production of cell lines suitable for large-scale commercial production have enabled the development of MAb therapies to treat myriad diseases and made these products available to an increasing number of patients. In addition to enabling more efficient and economic production of MAbs, the above antibody engineering technologies, coupled with advances in cell culture production discussed below, have greatly increased our ability to control or alter the properties of the resulting antibodies. For example, the extent of glycosylation, which can increase effector function and thereby increase product efficacy, can be controlled by both cell line engineering and cell culture technologies. In the future, human cell lines may replace CHO and other mammalian cell lines for the production of MAbs. MARKET DEMANDS AND CELL LINE PRODUCTIVITY One challenging feature of most therapeutic antibody products is that the doses required for these products are much higher than for other biologic products. To meet the large annual production requirements for these products, companies have made substantial progress in developing more efficient and costeffective methods for manufacturing antibody products. When antibody products were first developed and approved, expression levels of MAbs were typically on the order of milligrams per liter. Even as recently as five years ago, antibody titers in excess of 1 g/l were not common and many MAb products were launched using production cell lines and manufacturing processes that produced approximately g/l antibody. 8 As MAb products became successful in the marketplace and as the demands for new products increased, newer methods of generating highexpressing antibody production cell lines and of culturing these cell lines for maximum productivity have been developed. Today s technologies are enabling antibody production in the bioreactor of 5 g/l or more. 9 Advances in cell line generation over the past decade include new expression vectors and transfection technology to introduce the genes into cells; novel parental cell lines that have been selected or designed to grow to maximum density; and robust screening technologies that in combination can enable rapid generation of production cell lines. ADVANCES IN THE GENERATION OF PRODUCTION CELL LINES Today s MAbs must be manufactured using reliable production cell lines capable of producing sufficient quantities of product to meet the market demand. For most products, this means that antibody titers in the bioreactor must be greater than 1 g/l in a fedbatch process initially and 3 5 g/l following process optimization. To achieve these levels of productivity, it is necessary to quickly develop a cell line expressing reasonably high quantities of antibody for early preclinical, formulation, and analytical validation studies that can be further optimized to achieve the desired productivity levels. If the productivity of the initial cell line is high enough, it can even be used to support initial clinical development of the product. Once the initial cell line is established, a production cell line exhibiting the highest possible level of production of functional antibody and capable of supporting commercial production at a reasonable cost can be developed. In today s highly competitive market, it is important to complete the initial stages of cell line development as quickly and efficiently as possible to enable early entry into human clinical trials but equally important is to devote sufficient time and resources to the full development and optimization of the commercial cell line so that BioPharm International October

6 The use of parental cell lines adapted to grow in suspension and serum-free media can reduce development times. a suitable cell line is available for commercial production as soon as possible. High-Expressing Cell Lines To create a production cell line for a specific antibody, expression vectors containing the heavy- and light-chain genes under control of strong mammalian promoters are introduced into the parental cell line. Usually, a selectable marker is also included so that cells containing the gene can be easily selected by adding a drug or substance to the culture that causes the cell to require the activity of the selectable marker. The driving factors behind the selection of a particular cell clone during cell line generation is the expression level of the recombinant protein, which is measured independently of the selection, and the time that it takes to obtain a cell line that expresses enough product to enable nonclinical and clinical development. Technologies that increase the percentage of transfectants with high expression levels will reduce the time needed to identify a production cell line because the high-expressing clones will be easier to select without having to screen thousands of individual clones. Recent advances in cell line generation include technologies that increase this percentage, as well as sophisticated and automated approaches to screening that enable more individual transfectants to be screened for expression levels. 10,12,15,16 Production levels in the bioreactor are a function of specific productivity the density to which the cells can grow and the longevity of the culture. Before actual testing in the bioreactor, expression levels are determined in small culture vessels, from multiwell plates to shake flasks. Levels of picograms of antibody/cell/day (pcd) are considered appropriate for initial transfectants, with greater productivity arising from optimized cell culture conditions, secondary transfections, or amplification of the transfected antibody genes using selective pressure. Using parental cell lines adapted to grow in suspension and serum-free media reduces development times and increases the likelihood of reaching high cell densities during manufacturing and high product yields in the grams-per-liter level. Selection Systems One of the earliest effective methods for transfection, selection, and amplification of foreign genes in mammalian cells was developed in 1981 by scientists at Columbia University using dihydrofolate reductase (DHFR) selection. In this method, a parental mammalian cell line deficient in the enzyme DHFR is transfected with an expression vector containing the DHFR gene under control of a relatively weak promoter and the antibody (or other protein) genes under control of a strong promoter. 11 By performing multiple rounds of amplification and selection of cells in the presence of the folate analog methotrexate (MTX), a potent inhibitor of DHFR, production cell lines with relatively high levels of expression of the foreign genes can be obtained. The original patents for this technology have now expired but it is still widely used to generate antibody production cell lines. However, because each amplification cycle requires 12 weeks to complete and up to five cycles or more, about one year total may be necessary to obtain a clone with acceptably high expression levels. Nevertheless, the DHFR system is effective and has been used in conjunction with other aspects of cell line development to achieve multigram-per-liter expression levels of MAb. Also, alternative systems requiring less time to reach maximal expression have been developed. For example, the glutamine synthetase selection system, developed by scientists at Celltech (now Lonza), can achieve production clones with higher levels of antibody or protein expression in 4 6 months. 12 Glutamine synthetase (GS) is the enzyme responsible for the biosynthesis of glutamine from glutamate and ammonia. This enzymatic reaction provides the only pathway for glutamine formation in a mammalian cell. Therefore, in the absence of glutamine in the growth medium, the GS enzyme is essential for the survival of the mammalian cells in culture. Some mammalian cell lines, such as the murine cell 104 BioPharm International October 2007

7 Figure 2. In matrix attachment region (MAR) technology, MAR elements are inserted into expression vectors surrounding the desired transgene and impose an open chromatin configuration on the nearby chromatin. This open structure allows RNA polymerase and other transcription factors to access the transcriptional promoters and enhancers found within the expression vector and thereby enables greater levels of transcription. This leads to increased productspecific translation and a higher yield in a greater percentage of transfected cells. Figure provided courtesy of Selexis SA. Closed chromatin Promoters/ enhancers Open chromatin chromatin Closed MAR lines NSO or SP2/0 widely used for antibody production, do not express sufficient GS to survive without added glutamine. With these cell lines, a transfected GS gene can function as a selectable marker by permitting growth in a glutamine-free medium. Chinese hamster ovary (CHO) cells, also widely used for antibody and other recombinant protein production, contain sufficient active GS to survive without exogenous glutamine. 13 In these cases the specific GS inhibitor, methionine sulphoximine (MSX), can be used to inhibit endogenous GS activity such that only transfectants with additional GS activity can survive. GS selection can be used to select high-expressing cell lines without amplification, which reduces the time compared to the DHFR selection approach. The GS system has enabled the rapid identification and selection of production cell lines that express up to pcd and multiple grams per liter of product as part of an overall cell culture process development effort. According to Lonza, more than 85 global pharmaceutical companies are currently using this technology to create production cell lines and five products using the GS system have been approved for commercial sale, including Synagis and Zenapax. The GS technology is available for licensing from Lonza for the use in research and commercial applications, making it widely available for the development of MAb products. 14 Improving Gene Expression Another recent approach to improve expression of antibody genes in the initially transfected cells is to ensure that the genes are integrated into regions of the chromatin, which are easily available to the enzymes that transcribe the gene into RNA, thereby increasing the rate of transcription. The transfection of a mammalian cell generally results in the integration of the DNA into the chromatin in one or more random locations. Because most of the genome is not transcriptionally active, there is a high likelihood that integration will occur in regions that are not able to transcribe high levels of the antibody genes. Targeting the expression plasmid to locations on the chromatin that are known to be transcriptionally active and accessible to the necessary enzymes would increase the expression of all genes integrated at these sites. Although this is an excellent concept in theory, homologous recombination or targeted integration has not been widely adapted in practice because of the lack of information about which sites are good locations for integration and the need to have unique plasmids and cell lines that are able to perform the recombination. Rather than targeting a specific site in the chromatin for integration, an alternative approach is to include elements on the expression plasmid. This will cause the random integration site to become transcriptionally active and available to the enzymes that transcribe the genes. There have been several reports of such genetic elements that enable the integrated plasmid to create a transcriptionally active region at any integration location on the chromosome and to enable higher transcription levels in a higher percentage of transfectants. Two types of elements that function to create a region of transcriptionally active chromatin are the ubiquitous chromatin opening elements (UCOE) and the matrix attachment regions (MAR) elements. 15,16 These genetic elements have different mechanisms of action but both work to increase the expression levels 106 BioPharm International October 2007

8 of linked genes that are transfected on the same plasmid as the MAR or UCOE. The use of MAR elements for improving expression has been commercialized by Selexis. The company has developed a set of expression vectors and transfection technologies (the MARtech technology) that use these elements to increase the percentage of cells expressing the desired gene. As shown schematically in Figure 2, the MAR elements are inserted into an expression vector such that the gene for the desired product is surrounded by these elements to impose an open chromatin configuration, thereby allowing RNA polymerase and other transcription factors to access the transcriptional promoters and enhancers found in the expression vector. For this reason, MARtech increases the number of independently transformed cells that express the desired protein and enables expression levels in the initial transfectants of as much as pcd. Selexis claims that MARtech allows for generation of clonal mammalian production cell lines in about 10 weeks. Many companies have begun exploring the use of MARtech to enable rapid generation of high producing cell lines for their antibody products. Later this year the first product using this technology will enter clinical trials. 17 UCOE technology, now available through Millipore Corporation, provides an approach to increasing gene expression similar to that of the MARtech technology. The UCOE elements are functionally similar to MAR elements although their composition and structure are different. 16 UCOE consists of regions that are rich in the sequence CpG, and that increase the accessibility of the surrounding chromatin. Therefore, a single UCOE element can be included on an expression vector and can increase the expression levels of linked genes. There is less commercial experience with UCOE elements than with MAR elements, but the intent is to offer the technology to companies for use in research and in commercial production cell line generation. ADVANCES IN CELL CULTURE TECHNOLOGY Host cell lines currently used to produce commercial MAb products include murine hybridoma and myeloma cell lines, CHO cell lines, and one human cell line (Table 2). Table 2. Host cell types used in the manufacture of commercial MAbs Cell line Species Number of products Hybridoma Murine 5 SP2/0 myeloma Murine 5 NS0 myeloma Murine 3 Other myeloma Murine 1 Chinese hamster ovary (CHO) Hamster 10 EBV-transformed B cell Human 1 E. coli Microbial 1 Those antibody products produced in hybridoma cell lines generally have lower dose requirements than others and are also older than those produced using highly engineered systems such as CHO, NSO, or SP2/0. The single product produced in a human cell line may represent a trend in coming years as others develop human cell lines capable of producing antibody products at high levels. While the use of murine cell lines still prevails in commercial processes, the use of CHO cells for producing commercial products is growing and most antibody products currently in development are produced from CHO or human cell lines. Hybridoma Technology MAbs were first produced from hybridomas consisting of a murine B cell producing a specific antibody fused to an immortal murine lymphoid cell line. Initially, MAbs were produced by injecting a hybridoma cell line into the abdomen of pristane-primed mice, in which the cells could grow to a significant level. As the hybridoma cells grow in the abdomen, MAb-rich ascites fluid accumulates. The ascites fluid can then be collected by withdrawing it with needles at several day intervals. The collected ascites fluid is very complex in composition and highly contaminated, but frequently contains antibody concentrations approaching 1 g/l or greater. This process is widely used for the production of small to moderate amounts of antibodies for multiple applications and one commercial antibody product is produced today using this technology. The limitations of large-scale production in the abdomens of mice were quickly realized and scientists turned their efforts to use BioPharm International October

9 Fed-batch processes are readily scaled-up to commercial volumes and represent the primary method in use today. in vitro culture as an alternative to replace in vivo production in ascites. These initial efforts focused on growing hybridomas in culture, under conditions enabling the same high level of antibody expression as seen in the ascites fluid. Initial studies characterized and compared the growth of hybridomas and production of antibodies in either batch suspension cultures using stirred tanks and airlift fermentors or in perfusion cultures using a variety of methods for cell retention. From simple batch cultures, the use of controlled feeding, also know as fed-batch, evolved as extremely successful in increasing maximum cell concentrations, culture longevities, and corresponding product titers. Fed-batch is the primary mode of biopharmaceutical production used today, both for antibodies and other recombinant protein products. Hybridoma technology enabled the creation and production of MAbs for research, analytical use, and as limited-dose therapeutic products. However, these cell lines are generally difficult to engineer for high levels of protein expression and usually grow to only moderate densities in bioreactors. Hence, although these cells are designed to produce antibodies, in many cases they do so at levels that are too low to be optimal for manufacturing today s MAbs. Using CHO Cells as Production Hosts To circumvent the limitations of hybridomas for MAb production, scientists began experimenting with alternative production hosts that could be grown to higher densities and transfected with the antibody genes to enable higher cellular productivity. The murine myeloma cell lines NSO and SP2/0 were among the first used to produce recombinant MAbs. At the same time, others began examining CHO cell lines. The CHO cell lines proved to be a suitable production host for antibodies. Today, the vast majority of biologic products made in mammalian cells are produced using a CHO host cell line. Because of the widespread adoption of this host cell, the growth characteristics, metabolism, behavior in bioreactors, virulence factors, and the likely host-cell related impurities that might be in a process or product are well understood. Moreover, because there is a strong regulatory history of CHO cells, more and more products in development are now made using CHO cells. Human Cell Lines While the use of CHO cells as production hosts continues, other cell lines, especially human cell lines, are being developed as alternative hosts. For example, the PER.C6 cell line developed by Crucell, has been shown to produce antibodies at levels similar to or even greater than CHO cell lines. 18 One potential advantage of products produced in these cell lines is that the glycosylation patterns and other post-translational modifications of antibodies produced in them may be more similar to human antibodies. Therefore, the PER.C6 cell line and other human cell lines may prove to be reliable, safe, scalable, and economical alternatives to the CHO cell lines currently in use for the production of MAbs. Chemically Defined Media Current regulatory requirements strongly discourage or ban the use of any products in the culture media that are derived from animals, especially from bovine sources. Therefore, the use of bovine serum, commonly used earlier in mammalian cell culture, has been discontinued and significant efforts have been directed towards the development of cell culture media, that is free from animal-derived products. There is a growing trend toward the use of chemically defined media. In such media, recombinant proteins such as IGF-1, transferrin, insulin, or others may be included to provide the necessary signals for cell growth. When used, the recombinant human versions of these proteins are preferred. To further minimize the risk associated with the addition of animal-derived components, CHO and other production host cell lines used for antibody production are now selected for their ability to grow and produce product at high levels in chemically defined media. 110 BioPharm International October 2007

10 Significant efforts are being devoted to the continuous improvement in the safety and quality of MAbs. Several different chemically defined media are now commercially available from a variety of vendors. However, most companies involved in the development of MAb products today have developed proprietary cell culture media and growth conditions suitable for production of their particular monoclonal antibody at high titers. Along with improvements and refinements in expression systems and cell lines for MAb production, there have also been significant advances in cell culture conditions over the past 20 years to further optimize antibody production. 19,20 The optimization of fed-batch processes has increased antibody titers in culture orders of magnitude so that expression levels of greater than 1 g/l are frequently achieved. Perfusion Technology One initial approach to increase the yield of antibody products from a single bioreactor was the use of perfusion technology in which the media is continuously removed from the bioreactor and replaced with fresh media. Perfusion technology is based on the rationale that cells in culture could continue to produce antibody over several weeks if the conditioned media, containing the antibody product along with potentially growth limiting metabolites, were replaced regularly with fresh media and growth factors. Years of comparative work have shown that perfusion cultures can achieve higher volumetric productivities than fed-batch cultures at the expense of lower product titers per liter of medium consumed. Moreover, the continuously changing media conditions and long culture times required for perfusion production frequently lead to inconsistent processes, variable glycosylation, and other post-translational modifications in the product over time in culture. The risk of contamination also increases. Perfusion operations tend to be complex, difficult to scale up, and generally less robust than fed-batch processes. 21,22 Therefore, fed-batch culture is now the method of choice for robust, reproducible, and reliable manufacturing processes. While the capital investments in a manufacturing facility using fed-batch culture are higher than those for a perfusion-based facility, the overall cost of goods for fed-batch and perfusion processes are similar. While both culture technologies are successfully used today by commercial manufacturers, the biopharmaceutical industry is converging on the use of fed-batch suspension cultures in stirred-tank bioreactors with controlled feeding. FUTURE CHALLENGES IN ANTIBODY MANUFACTURING The advances in cell line generation and cell culture described above have enabled companies to produce monoclonal antibodies at very high expression levels. As a result, early concerns that the industry would not be able to meet the growing production demands of MAbs have subsided. While these significant improvements in upstream production have resulted in the ability to express MAbs at levels approaching 10 g/l, the capacity and ability of downstream processes to handle these high quantities of antibody has been strained. The competing demands of growing production requirements and reduced cost to the patient present challenges to the industry to make manufacturing processes even more efficient. Improvements in chromatography media for antibody purification have resulted in media with higher capacities, faster throughput, and improved contaminant clearance. Significant efforts are currently being devoted to developing alternative techniques to improve downstream processing to enable the efficient processing of high levels of antibody, enhance process robustness and yields, and reduce overall manufacturing costs. Companies today are striving to incorporate Quality by Design and other new regulatory concepts into the development of MAb products to further reduce the cost and development timelines for these products. The manufacturers are also striving to develop final product formulations containing high concentrations of antibody with sufficient stability to address the increasing doses of antibody products without adversely impacting the quality of these products. 112 BioPharm International October 2007

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J Biotechnol. 2006;123(1): How Cell Culture Became King... and May be Usurped David Estell, vice president of technology at Genencor International, , dave.estell@danisco.com In the early 1980s, most recombinant protein production was carried out in E. coli. The disadvantage of this method was that the proteins were produced intracellularly and had to be refolded to obtain active protein. As a result, at Genentech we were looking for ways to produce properly folded proteins in other cell systems. By 1981, Art Levinson s group had developed techniques to allow selectable, stable expression in mammalian cells. These methods were initially applied to our hepatitis B surface antigen and tissue plasminogen activator (t-pa) expression. The resulting proteins were efficiently expressed in a properly folded form. Meanwhile, James Stramondo s group had developed large-scale cell culture processes to improve performance. The biggest concern in using transformed cells was that DNA or viruses could be carried into the final product. The hepatitis B surface antigen assembled into 22-nm particles that were similar in size and shape to some viruses, which made the problem particularly difficult. So the team proceeded to work toward FDA approval. My group was Bacterial expression systems can secrete large amounts of protein in fermentations that take only a few days per batch. responsible for creating the initial recovery process and for demonstrating viral clearance and DNA removal. Several other research and development groups also put in a tremendous amount of work to develop other aspects of the new mammalian-cell-based processes. In the end, it paid off. Within a few years, both the hepatitis B vaccine and the t-pa processes were validated and approved by the FDA. This new expression technology rapidly spread through the industry to become the standard production system for recombinant proteins. Thus, cell culture became king. The fact that most human proteins are secreted efficiently in properly folded form by mammalian cells means that the production of test quantities of a new pharmaceutical protein is now straightforward, and many production processes have become highly standardized. Cell culture may not always keep its crown, however. Mammalian cell expression is highly efficient on a per cell basis, but creating the initial working cell banks and production trains requires long lead times and is expensive, leading to costs of $500 $1,000 per gram of protein. The system s effectiveness, however, has made the industry reluctant to investigate other options, such as bacillus and fungal expression systems. These alternative systems have been demonstrated to secrete extremely large amounts of protein in fermentations that take only a few days per batch, and produce several metric tons of protein per year. Because these microbial systems can be created in weeks and produce protein at 1/10,000th of the cost of mammalian cells, they may replace some of the mammalian cell capacity for high volume, lower-cost pharmaceutical proteins in the future. So watch out, cell culture. A microbial coup may be in the making. 114 BioPharm International October 2007