150 Chinese Hamster Ovary (CHO) cells are a valuable tool for manufacturing recombinant proteins. Genetic engineers can modify CHO cells to produce proteins with "mammalian" post-translational modifications. Two gene amplification systems used in CHO cells are methotrexate (MTX) resistance and glutamine synthetase (GS) systemusing methionine sulfoxamine (MSX) resistance.
150 Chinese Hamster Ovary (CHO) cells are a valuable tool for manufacturing recombinant proteins. Genetic engineers can modify CHO cells to produce proteins with "mammalian" post-translational modifications. Two gene amplification systems used in CHO cells are methotrexate (MTX) resistance and glutamine synthetase (GS) systemusing methionine sulfoxamine (MSX) resistance.
150 Chinese Hamster Ovary (CHO) cells are a valuable tool for manufacturing recombinant proteins. Genetic engineers can modify CHO cells to produce proteins with "mammalian" post-translational modifications. Two gene amplification systems used in CHO cells are methotrexate (MTX) resistance and glutamine synthetase (GS) systemusing methionine sulfoxamine (MSX) resistance.
valuable tool for producing recombinant proteins with "native" mammalian glycosylation patterns. Following their introduction in the 1950s, CHO cell lines have become a workhorse for manufacturing recombinant proteins. Genetic engineers can modify CHO cells to produce proteins with "mammalian" post-translational modications, and through gene amplication can selectively produce high levels of recombinant proteins. Process development, using CHO cell lines, focuses on achieving the maximumamount of active product. Optimization of the amount of active product can be achieved in at least two basic ways. The rst way is to increase the specic productivity (i.e., the product per cell) through cell line development. Cell line development may include both sub-cloning the cell line to select higher producing clones and use of gene amplication. Drug resistance has been the tool of choice in the biopharmaceutical industry to induce gene amplication. By combining the gene of interest with a selectable gene, increased production levels can be accomplished. Using productivity-per-cell approaches, scientists have increased expression levels more than 1,000-fold (Schimke, 1982). Two gene amplication systems used in CHO cells are the dihydrofolate reductase (DHFR) systemusing methotrexate (MTX) resistance, and the glutamine synthetase (GS) systemusing methionine sulfoxamine (MSX) resistance. The DHFR enzyme catalyzes the conversion of folate to tetrahydrofolate (Figure 1). This precursor is necessary for the de novo synthesis of purines, pyrimidines, and glycine (Goeddel, 1990). Methotrexate is a drug which is similar (i.e., an analog) to folate. MTX binds to DHFR, thereby inhibiting the production of tetrahydrofolate. With insufcient levels of DHFR, cells are deprived of nucleoside precursors (hypoxanthine and thymidine) and die. Gene amplication is a technique used by molecular biologists. They transform cells with recombinant DNA consisting of the gene of interest closely linked to the gene for DHFR. Then during gene amplication the cells are cultured in increasingly higher levels of MTX. Those CHO cells that have increased copies of the DHFR gene, and therefore higher levels of the enzyme, are selected. The GS/MSX systemis similar to the DHFR/MTX system. The GS enzyme catalyzes the production of glutamine fromglutamate and ammonia (Figure 2). Methionine sulfoxamine binds to the GS enzyme and prevents the production of glutamine. Gene amplication occurs when cells are subjected to increasing concentrations of MSX. A second way to achieve higher recombinant protein yields is to increase the cell yield (i.e., cells per volume) of the process. This may be accomplished through process development (e.g., batch, fed-batch, perfusion, etc.) and medium development. By increasing the cells per volume per day, higher levels of product may be produced. To demonstrate the utility of this protein production system, we used CHO cell line (ATCC
CRL-10154 CHO 5/9 M! 3-18), which
produces human macrophage colony stimulating factor (hm-CSF). This cell line was produced using a DHFR-CHO DG44 cell clone as the parental line. Gene amplication was performed using the DHFR/MTX system. The cell growth and protein production kinetics of this cell line were studied using a powdered medium developed for CHO cells by Themo Scientic HyClone. Standard culturing procedures for CHO cells were used for these evaluations (Table 1). Initial evaluations compared commercially available serum-free and protein-free media to Themo Scientic HyClone PF-CHO MPSq(SH30333) using the CHO 5/9 M! 3-18 cell line. Representative results obtained in these comparisons are shown in Table 2 and Figure 3. Table 1: Evaluation Parameters for use with Shaker and Spinner Cultures Temperature 37C CO2 Atmosphere 5% Agitation Shaker 100130 rpm Spinner 6080 rpm Oxygen Atmospheric Buffering 2 g/L Sodium Bicarbonate Table 2: Comparison of SH30333 vs Competitor Medium Parameter SH30333 Maximum Cell Density (Mean) 212.1% Higher Maximum Population Doubling Time (Mean) 19.6% Higher Maximum Protein Production (Mean) 74.6% Higher Application-Specic Technical InformationApplication Notes Chinese Hamster Ovary Cells for the Production of Recombinant Glycoproteins Camire, Joseph. Art to Science, Vol. 19, No. 1; Logan, UT, 2000 Analysis of growth promotion and hm-CSF productivity kinetics showed a maximum specic productivity (Figure 4) of 1.26 pg/cell/day. These data indicate that increased protein expression with this cell clone is cell-growth associated; that is, the maximum specic productivity is seen during log-phase growth. Additional studies analyzed extended cell performance (Figure 5). In the case of this clone, cells were cultured for more than 70 days with approximately 68 cumulative population doublings (CPD). The CPD was linear over the course of the study indicating good stability of the cell line in this medium. Following these evaluations, cultures were scaled up to increase the total protein yield by increasing the cell population density. PF-CHO MPSqmedium is a two-part powder, protein-free medium designed for suspension growth of CHO cell lines. The medium was designed to be "regulatory friendly" by limiting animal-derived components to just two, i.e., cod liver oil and cholesterol. This product comes in a range of standard packaging congurations (Table 3). HyClone_150:Layout 1 12/27/2007 3:16 PM Page 150 Some typical components that may be added at the time of liquid preparation are: sodium bicarbonate, L-glutamine (for cell clones which require this component for growth), and Pluronic F68e. L-glutamine supplementation is recommended for DHFR systems. We anticipate that users will evaluate the medium prior to scale-up procedures. We recommend using cells that have been previously adapted to the medium to eliminate carryover variables from prior sera or media. Through customer collaborations, we have successfully optimized PF-CHO MPS to specic CHO clones. Contact your sales representative to inquire about availability of these services. We anticipate that users will evaluate the medium prior to scale-up procedures. We recommend using cells that have been previously adapted to the medium. References: 1. Schimke, R.T. (ed.). 1982. Gene Amplication Coldspring Harbor Laboratory, Cold Spring Harbor, N.Y. 2. Goeddel, D.V. (ed.). 1990. Methods in Enzymology, Vol. 185 pp. 543-551. Pluronic, F68eis a registed trademark of BASF Corporation The medium is designed to be used with a wide range of CHO cell lines and production processes, from straight batch to fed-batch to continuous perfusion cultures. The formulation, which is decient in nucleic acids and precursors (i.e., hypoxanthine and thymidine), is designed for use with the dihydrofolate reductase (DHFR) selection system and methotrexate (MTX) induced gene amplication. The medium is also decient in L-glutamine for use with the glutamine synthetase (GS) selection system and the methionine sulfoxamine (MSX) gene amplication system. PF-CHO MPS can be purchased in lot sizes as large as 400,000 L. This reduces QA/QC cost; by reducing the number of lots needing tests. 151 T e c h n i c a l I n f o r m a t i o n Table 3. Standard Packaging Conguration Catalog Number Package Size SH30333.01 5 L SH30333.02 10 L SH30333.03 50 L SH30333.04 100 L SH30333.05 1,000 L Figure 3. Comparison of commercially available CHO medium and PF-CHO MPSq (SH30333), using CHO 5/9 Ma 3-18 cells. Figure 4. Cell growth performance of CHO 5/9 Ma 3-18 cells using PF-CHO MPSq. Specic productivity appears to be strongly growth- associated with this cell clone. Figure 5. Adaptation of cell clones to a medium often results in decreased cell growth over multiple passages. The cell growth performance of CHO 5/9 M! 3-18 was observed to be consistent over an extended period of greater than 70 days when using PF-CHO MPSq. HyClone_151-152:Layout 1 12/27/2007 3:17 PM Page 151