Stephen F. Gorfien

Growth of NS0 Cells in Protein-Free, Chemically Defined Medium

Many hybridoma and recombinant myeloma cell lines have been successfully adapted to growth in protein‐free media. Compared with serum‐supplemented media, use of protein‐free media promotes superior cell growth and protein expression and facilitates downstream purification of the expressed product. Owing to its sterol auxotrophy, the NS0 myeloma is normally grown in either a serum‐supplemented medium or a serum‐free medium supplemented with an animal‐derived lipoprotein. CD Hybridoma Medium (a protein‐free, chemically defined formulation) grows many cell lines that do not exhibit lipid dependence, but this medium does not support growth of NS0 cells without further lipid supplementation. We tested several commercially available lipid supplements in CD Hybridoma Medium, including bovine EX‐CYTE VLE. None of the tested supplements supported long‐term growth of NS0 cells in CD Hybridoma Medium. Sustained long‐term growth of NS0 cells was achieved in CD Hybridoma Medium supplemented with various animal‐ or plant‐derived lipids complexed with cyclodextrin. NS0 cells adapted to CD Hybridoma Medium supplemented with cyclodextrin‐lipid complex reached peak cell densities that were more than double those observed in serum‐supplemented medium and were cultured for more than 15 passages. These cultures were also successfully cryopreserved and recovered in this defined medium. Through the use of cyclodextrin‐based additives to CD Hybridoma Medium, it is possible to solubilize significant quantities of sterols and other lipids and to maintain a protein‐free, chemically defined cultivation environment for NS0 cells. The culture system can be kept entirely free of animal‐derived components if the supplement is made with plant‐derived or synthetic lipids.

Recombinant Protein Production by CHO Cells Cultured in a Chemically Defined Medium

Serum-free culture of Chinese hamster ovary (CHO) cells has become increasingly common as a way of obtaining high levels of expression of recombinant proteins while simplifying recovery and downstream processing of the product. However, serum-free media may still contain one or more of a variety of animal-derived components including albumin, fetuin, various hormones and other proteins. We have demonstrated that it is possible to eliminate animal-derived proteins from a CHO medium formulation. Plasma protein fractions like albumin and fetuin may be replaced by plant-derived hydrolysates, resulting in medium that is protein-free but still undefined (CHO III PFM). CD CHO Medium is a chemically defined formulation which contains no protein or hydrolysates of either plant or animal origin. Peak cell densities and recombinant protein expression in CD CHO cultures compared favorably to expression in other media, although the maximal cell density and the highest levels of expression were observed at later time points. We were able to successfully supplement the culture with sodium butyrate to increase expression levels at the expense of peak cell density, so for recombinant cell lines showing an inverse relationship between growth and expression of recombinant product, strategies which limit the peak cell density may be useful for increasing expression.

Screening and Optimization of Chemically Defined Media and Feeds with Integrated and Statistical Approaches

The majority of therapeutic proteins are expressed in mammalian cells, predominantly in Chinese Hamster Ovary cells. While cell culture media and feed supplements are crucial to protein productivity, medium optimization can be labor intensive and time-consuming. In this chapter, we describe some basic concepts in medium development and introduce a rational and rapid workflow to screen and optimize media and feeds. The major goal of medium screening is to select a base formulation as the foundation for further optimization, but ironically, the most conventional screening method may actually rule out ideal chemically defined medium candidates. Appropriate cell adaptation is the key to identifying an optimal base medium, particularly when cells were originally cultured in serum-free medium containing recombinant proteins and/or undefined hydrolysates. The efficient workflow described herein integrates the optimization of both medium and feed simultaneously using a Design-of-Experiment (DOE) approach. The feasibility of the workflow is then demonstrated with a case study, in which chemically defined medium and feed were optimized in a single fed-batch study using a high-throughput microbioreactor system (SimCell™), which resulted in improving protein titers three- to sixfold.

Optimized Feeding Strategy for NS0 Cells

Growth of NS0 cells in a protein-free, chemically defined culture system has been made possible by the development of cyclodextrin-based lipid additives. Batch cultures of NS0 cells in this system demonstrated growth and expression levels equal to or better than levels obtained in serum supplemented media. To improve the total yield and the efficiency of recombinant product expression in chemically defined culture, several nutrient supplementation strategies were tested in cultures of NS0 cells. Recombinant NS0 (rNS0) cells expressing IgG were adapted to growth in a proteinfree, chemically defined medium (CD Hybridoma Medium). Liquid Media Concentrate (LMC) technology was used to segregate nutrient components into groupings that favor complete solubility and selective component addition to a culture. Two pre-solubilized feed subgroups were combined immediately prior to culture addition resulting in a low salt, concentrated supplement containing selected nutrients (except L-glutamine). The effects of nutrient feeding to standard CD Hybridoma Medium, and to a modified (reduced glucose and glutamine) version of CD Hybridoma Medium were studied. Spent medium analysis was employed to identify rate-limiting nutrients, which were combined to form a simplified feed supplement. Other variables included addition of lipid-based feed supplements and comparison of nutrient supplementation in bioreactors versus shake flask cultures. Results indicate potential for inhibitory effects of non-critical nutrients and different nutrient utilization profiles in the controlled environment of a bioreactor versus less controlled shake flask cultures. INTRODUCTION NS0 is a non-immunoglobulin secreting, non-light chain synthesizing subclone of NS-1. NS0 cells lack the glutamine synthetase (GS) enzyme, making them useful for recombinant expression systems utilizing GS as a selectable marker (1). Cholesterol auxotrophy of NS0 cells was demonstrated by Keen and Steward (2). They also showed that NS0 cells can be adapted to cholesterol independence and grown to high density in a protein-free medium. This protein-free medium would not support growth of the original NS0 cells unless further supplemented with cholesterol, phosphatidyl choline and β cyclodextrin. Once adapted to the modified medium, NS0 cells were reported to reach peak viable cell densities of about 1.2 x 106/mL, although the lipids precipitated after one week in the medium. The low peak cell density and precipitation problems encountered by Keen and Steward led us to investigate alternate means of sterol supplementation. We have previously described development of cyclodextrin-based lipid supplements, which when added to a proteinfree hybridoma culture medium made it possible to grow NS0 cells in a completely protein-free culture system lacking animal derived components (3). Peak cell density in this small scale system exceeded previously reported results obtained in systems which contained animal derived components. Large-scale production of recombinant proteins is often performed using batch culture systems, which have the advantages of simplicity and reproducibility over more complex perfusion culture systems. However, productivity may be limited by nutrient depletion and/or build-up of toxic metabolites in batch culture systems. We have recently described development of a model culture system in Chinese Hamster Ovary (CHO) cells for optimization of recombinant protein expression through nutrient supplementation (4). The goal of our present work was to take a similar approach to improve productivity of an rNS0 line. Several approaches to provide ratelimiting nutrients to the cultures have been tested and indicate that feeding amino acids and lipids together improve productivity in these cells. Differences in performance between shake flask and bioreactor environments suggest a need to conduct optimization studies under conditions of controlled pH and dissolved oxygen. MATERIALS AND METHODS Cells and Base Media A proprietary rNS0 cell line that expresses IgG was used in bioreactor experiments. This rNS0 line does not utilize the Glutamine Synthetase (GS) expression system. Liquid Media Concentrate (LMC) technology was used to prepare catalog and low glucose/low glutamine CD Hybridoma Medium (5). All components were added aseptically. CD Hybridoma 50X Acid Solubles (GIBCO PL000686) were added to sterile distilled, deionized H2O at a 1:50 dilution. Next, CD Hybridoma 25X Base Solubles (GIBCO PL000687) were added at a 1:25 dilution and CD Hybridoma 50X Salts (GIBCO PL000717) were added at a 1:50 dilution. Glutamine was supplemented at 2 mM (low glucose/low glutamine) or 8 mM (catalog) using 200 mM L-glutamine (GIBCO 25030-081). The 250X Cholesterol Lipid Concentrate (GIBCO 12531-018) was added at a 1:250 dilution. For bioreactor work, Penicillin-Streptomycin (GIBCO 15140-122) was added at a 1:100 dilution. Glucose levels were adjusted using a 30% glucose solution. Amino Acid and Lipid Supplements The CD Hybridoma Medium Partial Nutrient Supplement (PNS) was prepared by mixing equal volumes of CD Hybridoma Medium Partial Nutrient Supplement Acid Solubles (GIBCO 00-0336DG) and CD Hybridoma Medium Partial Nutrient Supplement Base Solubles (GIBCO 00-0337DG) within 10 minutes of use. The Acid Solubles contain all the amino acids in CD Hybridoma Medium except L-glutamine. The PNS was added at a 1:100 dilution in amino acid feeding strategies. CD Hybridoma Medium 5 Amino Acid (5AA) Solution of cystine, leucine, methionine, tyrosine, and valine was prepared to reduce the number of amino acids in the PNS feed. A 1:100 dilution of 5AA Solution was mixed with a basic solution within 10 minutes of use to neutralize the pH. Cholesterol Lipid Concentrate (CLC), 1000X Aqueous Liquid (SKU # 01-0025DG) was used in lipid feeding strategies. This cyclodextrin-based lipid concentrate was added to bioreactors at a 1:1000 dilution.

Profiling of glycosylation gene expression in CHO fed-batch cultures in response to glycosylation-enhancing medium components

Characterization of the glycosylation profile of a recombinant protein product is an important part of defining product quality in the bioproduction industry. Development of a protein with desired characteristics would require the capacity to modify and target specific glycosylation patterns as well as an understanding of the implications of changes to these glycosylation profiles. Previous cell culture studies have demonstrated the ability to modulate glycan profiles without negative impact to culture growth and product titer through the addition of glycosylation-enhancing medium components. With new methods, including increased measurement sensitivity and new capabilities in RNA-Seq technology, it is possible to develop a glycosylation gene expression profile for CHO cells. Specific glycosylation genes can then be tracked to ensure that the addition of these compounds will not negatively impact gene expression. Analyses comparing growth and titer, glycan distribution, and transcriptome differences can present us with potential insight into what changes are taking place on a genetic level in the cell in response to changes in medium and culture conditions.

Chinese Hamster Ovary (CHO) Cell Growth and Recombinant Protein Production in Serum-Free Media

CHO cells have become increasingly important for recombinant gene expression, owing to their low rate of spontaneous transformation and biomanufacture of recombinant products that structurally and functionally resemble the native molecules. We recently developed a low protein (< 100 µg/ml), low endotoxin (<0.25 EU/ml) serum-free medium (CHO-S-SFM II) formulated to support the growth of CHO cells and the production of recombinant proteins in suspension culture. Both wild type and recombinant CHO cells were adapted, maintained, cryopreserved and recovered in CHO-S-SFM II. Cells cultured in this serum-free medium out-perform parallel cultures in serum-supplemented medium, reaching peak densities of 3–4 X 106 viable cells/ml and producing over 1.0 µg/m1 of recombinant human chorionic gonadotropin. CHO-S-SFM II demonstrated superior growth performance compared to four commercial serum-free media for CHO cells. A prototype powdered form of CHO-S-SFM Il exhibited performance equivalent to liquid medium. Serum-free medium eliminates problems associated with serum usage, such as lot-to-lot performance variability, presence of adventitious agents and fluctuations in price and availability. The low protein content of CHO-S-SFM II facilitates downstream processing of recombinant proteins and reduces final product cost. Additionally, the low endotoxin level of this medium reduces regulatory concerns for the production of therapeutic proteins.

Enhancement of Biological Productivity in Serum-Free Culture

In recent years, recombinant DNA and hybridoma technologies have revolutionized mammalian and insect cell culture, making these processes suitable for the manufacture of various biologicals. Among these biologicals are monoclonal antibodies produced by hybridomas, recombinant proteins elaborated by genetically engineered Chinese hamster ovary (CHO) cells and a variety of recombinant products, including viral glycoproteins and chimeric antibodies produced by the baculovirus expression vector system (BEVS) in insect cell culture. These systems facilitate the highly efficient production of monoclonal antibodies and recombinant proteins possessing structures and biological activities that closely resemble those of the native proteins. However, to develop cost effective methods for production of medically and agriculturally important proteins, it is of paramount importance to use serum-free cell culture. Such culture techniques facilitate the less costly downstream processing necessary for economical manufacturing of product. To optimize production of monoclonal antibodies in hybridomas and expression of foreign genes in rCHO cells and BEVS, we have developed and commercialized specific serum-free media (SFM). The optimized production parameters established in spinner and suspension cultures were scaled up to commercially available Celligen™ (New Brunswick Scientific) cell culture bioreactors.

Bioreactor Process Strategies for rNS0 Cells

rNS0 cell line has been grown in protein-free, chemically-defined medium using stirred-tank bioreactors in batch and fed-batch mode. Various operating parameters, including pH and temperature, were investigated to assess their effect on growth and IgG production in a chemically defined, protein-free culture system. Spent medium analysis was used to determine the effect of bioreactor operating parameters on nutrient metabolism and IgG production. To increase the total yield, optimum bioreactor operating conditions found in batch culture were then applied to a fed-batch process. The effect of bioreactor conditions on nutrient utilization rates and feeding strategy will be discussed.

Technical Approaches to Minimize Regulatory Hurdles to Fed-Batch and Continuous Medium Supplementation

Mammalian cell culture biological production applications have traditionally implemented batch production techniques to minimize real or perceived obstacles to product approval by regulatory agencies. Analysis of the bioreactor environment during the course of a batch fermentation run demonstrates significant fluctuations in levels of critically-limiting nutrients, accumulation of catabolic products and increased medium osmolality. These factors can be demonstrated to impact specific productivity and to result in product microheterogeneity. Extended bioreactor campaigns may exhibit superior productivity and longevity by controlled nutrient delivery via fed batch or continuous perfusion of concentrated supplements. Regulations defining criteria for “well-characterized products” and international efforts to harmonize standards may minimize barriers to extended production campaigns and process modifications. This paper examines the evolution of these regulatory guidelines and their application to process changes which permit productivity enhancement through supplementation with salt-free nutrient concentrates.

Anchorage-Dependent Growth and Recombinant Protein Production by Chinese Hamster Ovary Cells in Serum-Free Medium

Chinese hamster ovary (CHO) cells are commonly used for the production of recombinant proteins owing to the ability of these cells to stably maintain the expression of foreign gene products which structurally and functionally resemble the naturally occurring human proteins. Serum-free culture of CHO cells is desirable since it facilitates downstream processing and recovery of products and minimizes problems associated with serum usage, such as lot-to-lot performance variability, presence of adventitious agents, and fluctuations in price and availability. We previously developed several serum-free media (SFM) formulations which support anchorage-independent growth and protein expression of CHO cells. While suspension culture of CHO cells is now an accepted method, there are many applications for which anchorage-dependent culture is desirable. Use of SFM optimized for suspension culture may result in suboptimal performance when used in anchorage-dependent culture systems. A recently developed prototype formulation, designated Adherent CHO-SFM, has-been specifically formulated to support growth and recombinant protein production using anchorage-dependent culture systems. This medium contains no bovine-derived components and has protein and endotoxin concentrations of <250 μg/ml and <1.0 EU/ml, respectively. The utility of this formulation has been demonstrated using small and larger-scale anchorage-dependent cell culture systems including tissue culture flasks, roller bottles, microcarriers and artificial capillary units. Cells cultured in Adherent CHO-SFM demonstrated biological performance which was superior to that obtained with serum-supplemented medium. Development of Adherent CHO-SFM complements our existing options for serum-free culture of CHO cells and offers the end user greater flexibility in choosing an appropriate cell culture system.