By James W. Brooks, et al
In addition to having the optimal cell line and process, it is crucial to have the optimal cell culture medium and feed to maximize performance potential.
FDA approval in 1986 of the first monoclonal antibody (mAb), Orthoclone OKT3, for clinical use in humans ushered in the era of mAb-based therapeutics (1). Being a murine-derived mAb, OKT3’s lack of appropriate post-translational modification (PTM) resulted in a short serum half-life. This short half-life necessitated its daily administration and consequent withdrawal by FDA (2). The ensuing development of chimeric, humanized, and fully human forms of mAbs through recombinant technologies dramatically improved the efficacy of mAb-based therapy. Today, mAbs are considered the cornerstone of modern therapeutics and are used to treat a wide range of illnesses, including multiple forms of cancer and autoimmune, cardiovascular, and neurodegenerative diseases. Global sales of mAb therapeutics are projected to reach $94 billion by 2017 and nearly $125 billion by 2020 (3)
Fueling the significant growth in mAb therapeutics is their innate ability to specifically bind target proteins and initiate immune responses such as antibody-dependent cellular cytotoxicity (ADCC). These effector functions, combined with their long serum persistence, make mAbs highly effective therapeutic agents. Their therapeutic efficacy is heavily dependent on appropriate quality characteristics. In the bioproduction process, appropriate mAb quality can take multiple forms, including correct sequence, proper glycosylation, and limited protein aggregation and charge heterogeneity.
Upstream processing challenges
During upstream cell culture bioprocessing, glycosylation and charge heterogeneity are two primary quality concerns, especially in the development of mAb biosimilars. Glycosylation is highly cell line specific and achieved through the action of a complex network of enzymes and transporters within the cell. Each of the two heavy chains of the mAb contains a single conserved N-linked glycan site at Asn297. Glycosylation at this site affects conformation, stability, solubility, and function (4). Additionally, charge heterogeneity is introduced during cell culture bioprocessing and exists in all marketed mAb therapeutics. Various chemical or enzymatic modifications can lead to the formation of acidic or basic charge variants, which can impact mAb stability and biological activity (5, 6).
In the bioproduction process, three primary drivers affect culture performance and mAb protein quality. The first is the bioproduction cell line, such as Chinese hamster ovary (CHO), NS/0, and HEK293. Additionally, there are a number of specialized selection and expression systems, including dihydrofolate reductase (DHFR) and glutamine synthetase (GS). Today, researchers are working to better understand and control cell systems through metabolomics and cell engineering. Glycoengineering is being employed to more consistently dictate the critical glycan profile of mAbs through the development of specific cell lines such as a FUT8 knockout CHO cell line producing an afucosylated and ADCC-enhanced mAb variant (7). The second key bioproduction driver is the cell culture medium and supplementation used for establishment and expansion of the cell line and generation of the desired mAb product. A number of media, supplements, and feeds are commercially available, but none are finely tuned and optimized for each specific bioproduction cell line. Finally, the physical, chemical, and biological process parameters are crucial to the performance of the production system. While much effort is dedicated to engineering and selecting the production cell line and defining process parameters, often too little focus is given to designing and selecting the optimal cell culture medium and supplementation to maximize the performance of the system.
Advancements in media
Cell culture media have evolved significantly during the past few decades, driven primarily by advancements in media supplementation. Prior to the 1990s, serum was the supplement of choice to achieve the highest possible titers while helping to get biotherapeutics to the market quickly. In the 1990s, bovine spongiform encephalopathy (BSE) risk reduction became a central theme in bioproduction. At this point, peptones were found to be an alternative supplementation solution to deliver high titers without detrimentally affecting protein quality and were successfully used for the development of many of today’s blockbuster mAb therapeutics. During the 2000s, the further push for consistency drove the desire for fully chemically defined (CD) media formulations.
Numerous CD formulations have been developed for cell culture-based bioproduction. Not all formulations have produced the desired results, however. In multiple cases, limitations in composition or the presence of contaminants, such as trace elements, in CD raw materials have impacted mAb production and/or quality (8–10). This has led to a rekindled interest in peptones as bioproduction supplements, especially for biosimilar mAb development. Although not fully CD, peptones are well characterized and long established cell culture supplements that can enhance culture performance along with achieving or maintaining desired protein quality. Peptones are a rich source of amino acids, peptides, vitamins, carbohydrates, nucleosides, minerals, and other components and, therefore, serve as an optimal nutritional source. Peptones have been shown to provide additional benefits to cell culture, including exhibiting anti-apoptotic effects (11, 12) and positively affecting cell cycle (13) and/or cell metabolism (14).
With the wide range of media and supplement options commercially available today, it is crucial to ensure those selected are optimized for the specific cell line of choice. While a platform process is often desired, the reality is that there is no universal cell culture medium. Cell types and cell lines are highly heterogeneous, with each having its own specific nutritional requirements. CHO cell lines, for example, exhibit an extensive amount of genotypic and phenotypic diversity (15). CHO cell lines derived from the same parental line and expressing the same mAb may have different nutritional requirements and need their own specialized media to perform optimally.
Cell culture media and supplement formulations have been shown to affect culture performance, specifically mAb glycosylation and charge variants. The addition of galactose may increase galactosylation of mAbs (16), whereas additional mannose in the medium has been shown to increase mAb high mannose glycoforms (17). Amino acids such as glutamine, asparagine, threonine, proline, and glycine have been shown to impact levels of sialyation and galactosylation (18–20). Certain basic amino acids, such as lysine and arginine, have been found to reduce acidic charge variants (21). Trace elements can function as enzymatic co-factors or modulators of membrane potential. Manganese has been found to modulate galactose and high mannose content on glycoforms (16, 22). Copper concentrations in media have been shown to affect levels of basic charge variants (8), while calcium, along with niacinamide, has been found to decrease acidic charge variants (21).
Improving culture performance
At BD, culture performance and/or protein quality has been improved in multiple customer projects through the optimization of media formulations, including both CD and peptone components. In one project, an existing peptone-containing base medium was optimized to correct the charge variant profile of a mAb biosimilar. The medium was originally developed for another cell line. When attempting to use it as a platform medium, however, the customer encountered issues with the charge variant profile compared to the originator molecule. Through spent media and biostatistical analysis, a number of components, both CD and peptone, were identified for optimization. Media variations with these components either removed or enhanced were evaluated, and several media met the cell growth and/or production criteria (Figure 1). Importantly, multiple media gave acceptable mAb charge variant profiles (Figure 2) comparable to the originator molecule. Through the removal of a peptone and CD component (Medium 4) or the addition of a single CD component (Medium 7), the targeted cell growth and mAb biosimilar production and charge variant profile were achieved. These data support the view that base medium optimization, whether through peptone or CD components, can be used to reach the desired performance and protein quality requirements. In another instance, a customer using a commercial CD medium wanted a custom CD-base medium and feed that would enhance mAb production at least two-fold, while retaining the appropriate glycosylation profile. An initial screen of the customer’s CHO cell line against BD’s proprietary CD media library identified several media that met the desired production target (Figure 3). Media 32 and 36 were selected for process optimization and feed evaluation in an ambr 15 microbioreactor system (Sartorius Stedim Biotech), where both media retained the improved cell growth and mAb production in batch culture (data not shown). While the addition of two proprietary CD feeds further increased performance, CD Feed 1 was found to have a more significant impact (data not shown). The Medium 36 and CD Feed 1 combination was scaled to benchtop bioreactors (Figure 4), where a six-fold increase in mAb titer was achieved and a satisfactory glycosylation profile was retained (Figure 5). In this case, pairing an optimized CD base medium with a properly matched CD feed significantly enhanced mAb production while retaining the desired protein quality.
These examples illustrate that, in addition to having the optimal cell line and process, it is crucial to have the optimal cell culture medium and feed to maximize performance potential. Cell culture media and supplements continue to be critical elements of the bioproduction process, helping to deliver significant improvements in titer and providing mechanisms for modulating quality. Whether supplementing with peptones or CD supplements, it is essential to understand the components of the culture system which have the most significant impact and drive performance. Only through this understanding can the potential of the entire cell culture bioproduction system be realized.
James W. Brooks is R&D manager, emily Gramiccioni is scientist II, Thomas O’Brien is scientist II, and Nuvjeevan S. Dosanjh is senior scientist, all at Bd Biosciences – advanced Bioprocessing.
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This article originally appeared in BioPharm International, Vol. 29, No. 11, pp. 24–28, 35 (November 2016).