Partial Replacement of Chemically Defined Media with Plant-Derived

Protein HydrolysatesPlant-derived hydrolysates can be used as valuable and

practical tools to improve cell culture performance.

JAMES BABCOCK, CHRISTOPHER WILCOX, HANS HUTTINGA

ABSTRACT Protein hydrolysates are routinely used as cell culture sup-plements to enhance the overall performance of many biopharmaceutical production systems. This enhance-ment is subject to the additive effect of the native hydro-lysate components and the supplemented growth or production medium. Therefore, it is necessary to experi-mentally determine the proper hydrolysate dosage for a given hydrolysate medium combination that provides the desired optimization effect such as better growth pro-motion, enhanced cell viability, increased target protein production, or a combination of all three. In mammalian systems, hydrolysates have been used in combination with a variety of other supplements to help reduce or

eliminate serum requirements in systems using traditional basal media. Today, many high-performing, richly formu-lated chemically defined media have become available as stand-alone substrates for biopharmaceutical production. This article shows that these chemically defined media can benefit from the addition of hydrolysates and other supplements. It also demonstrates that in other cases, plant-derived hydrolysates can partially replace a significant por-tion of the active ingredients in these rich media.

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JAMES BABCOCK, PHD, is the global applications manager of cell culture at the Sheffield Bio-Science Center for Cell Culture Technology. CHRISTOPHER WILCOX, PHD, is the global market segment manager of cell culture and HANS HUTTINGA is the global business development director of cell nutrition, both at Sheffield Bio-Science, a Kerry Group Business, Beloit, WI, 800.833.8308, christopher.wilcox@kerry.com.

June 2010

Volume 23 Number 6

The Science & Business of Biopharmaceuticals

Optimizing the cul-t u r e me d iu m i s an integral part of upst ream process development, and is

essential for efficient biopharma-ceutical manufacturing. The aim is to design a robust, economi-cal, and reproducible system that enhances the overall performance of the specific cell line. Typically, cell culture performance is assessed using a number of parameters, including cell density and viability. However, the defining parameter of any successful production system is increased protein expression.

Traditionally, optimal mamma-lian cell growth was achieved by adding animal sera, such as fetal bovine serum (FBS) at a concen-tration of 5–20% to defined basal media. Although sera may provide important growth and regulatory factors, their composition is com-plex and undefined, which can lead to batch-to-batch variability and downstream processing chal-lenges. Furthermore, the potential for contamination by adventitious agents, such as viruses, prions, and bacteria, poses serious bio-safety risks. This has led regula-tory authorities such as the US Food and Drug Administration and European Medicines Agency (EMEA) to issue guidelines that urge biomanufacturers to avoid ingredients of animal or ig in. Regulatory pressures related to safety concerns are therefore driv-ing the biopharmaceutical industry away from the dominance of serum as a media supplement, and toward the use of serum-free, animal-com-ponent free, or even chemically defined media (CDM) for both new and older manufacturing processes.

Serum-Free MediaPlant-derived hydrolysates have been routinely used to reduce or eliminate serum from traditional

basal media formulations, often in combination with a variety of addit ional supplements. These hydrolysates are composed of a mixture of peptides, amino acids, carbohydrates, and lipids, and as a multitude of unidentified compo-nents with indeterminate biologi-cal activity. They are produced by the enzymatic or acidic digestion of a given raw material from var-ious plant sources including, but not limited to soy, wheat, and cot-ton. Some process scientists have been reluctant to use plant-derived protein hydrolysates as medium supplements because of their lack of definition, which impairs their ability to assess the root causes of variability in their production processes. Recent improvements, including novel enzyme digestion techniques, refined processing tech-niques, automation, and formal cleaning validations have resulted in more consistent hydrolysates sold under the trade name of HyPep and UltraPep.1 These improved plant protein hydrolysates are widely accepted as performance-enhanc-ing substitutes for animal-derived media components for a variety of cell lines (e.g., hybridoma, BHK,

CHO, Vero, MDCK).2–4 Several biopharmaceuticals produced using plant-derived protein hydrolysates have reached the market and many more are in various stages of devel-opment.

As an alternative solution to traditional basal media supple-mented w ith an ima l- der ived

serum, high-performing, richly for mu lated CDM have been developed for biopharmaceuti-cal production as stand-alone substrates. The optimized mix-tures of biochemical constitu-ents in CDM have been carefully designed to stimulate cell growth, maintain good cell viability, and promote high protein y ie lds. Although CDM have been used

Figure 1. Cell viability of Chinese hamster ovary cells cultured in chemically defined medium with and without supplementation with HyPep. The plant-derived hydrolysate extended cell viability.

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CDM-C: single chemically defined media

Hydrolysate supplements may provide constituents that are beneficial for performance.

successfully for numerous cell lines—including those that express biopharmaceuticals—their process performance can be limited and their cost is considered to be high.

This article discusses the impact of various supplementation schemes on the performance of cells culti-vated in CDM. It presents examples that use CHO and SP2/0 cell lines grown in 125-mL shake flasks as

models for biopharmaceutical man-ufacturing systems.

Setting Up a Test System The effects of protein hydroly-

sates on the overall performance of a biopharmaceutical produc-tion system can be inf luenced by a number of factors including the specific cell line used, the raw material and process used to manu-

facture the hydrolysate, the hydro-lysate dosage, and the composition of the basal medium. The effect of any supplement on the perfor-mance of a cell culture system is largely dependent on the formula-tion of the basal medium. Because CDM and hydrolysates share a number of common components, the additive effects of these com-ponents may reduce the perfor-mance of a given system as a result of unintentional “overdosing” of certain components. However, hydrolysate supplements also may provide a number of unique con-stituents that are beneficial for performance. Therefore, it is neces-sary to experimentally determine the proper hydrolysate dosage for a given hydrolysate medium combi-nation that will provide the desired optimization effect.

Data for the figures in this article were collected using a sub-clone of CHO-K1 cells engineered to

express secreted embryonic alka-line phosphate (SEAP) by means of a human cy tomega lov i rus (HCMV) promoter, and adapted to grow in suspension in a serum-free medium. Cultures were grown in 125-mL shakeflasks containing a final medium volume of 25 mL and various basal media were sup-plemented with 1 mg/mL G-418. Triplicate cultures were seeded at 3 x 105 cells/mL and incubated at 37 oC, in 5% CO2 at 130 rpm for 12 days. Hydrolysate supplementa-tion was achieved by using 100 g/L

Figure 2. Cell density of CHO cells cultured in chemically defined medium with and without supplementation with HyPep. The plant-derived hydrolysate increased the maximum cell density.

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Figure 3. Secreted embryonic alkaline phosphate production of CHO cells cultured in chemically defined medium with and without supplementation of HyPep. Supplementation with the plant-derived hydrolysate led to a substantial increase in the production of the protein of interest.

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CDM-C: single chemically defined media

Adding HyPep to the media extended cell viability in hydrolysate-supplemented treatments.

stock solutions prepared in each respective basal medium. The data shown are not reflective of all sys-tems, but only depict certain exam-ples from testing conducted using three different commercially avail-able CDM.5,6

Cell Viability and Density During preliminary investigations, a single chemically defined media (CDM-C) diluted to 80% strength with phosphate buffered saline was re-enriched using var ious soy, wheat, and cottonseed hydro-lysates, and the cell viability was examined. It was observed that not all of the hydrolysates tested were able to fully overcome the medium dilution with respect to the overall performance in cell culture (data not shown). Particularly interest-ing results were obtained using the cottonseed-derived hydrolysate, HyPep 7504. This hydrolysate was tested at concentrations of 8 g/L with several commercially avail-able CDM, both at full strength and diluted to 80% concentration with phosphate buffer.5 Adding HyPep to the media extended cell viability in both hydrolysate-supplemented treatments, as shown in Figure 1.

As Figure 2 shows, diluting the CDM resulted in a signif icant reduction in cell density, accompa-nied by a reduction in total SEAP produced (Figure 3). Adding HyPep to the full strength media resulted in a significantly higher maximum cell density as compared to the 100% CDM control. Adding the hydrolysate to the diluted medium more than doubled the amount of total SEAP produced. All hydroly-sate-supplemented samples outper-formed the 100% medium control in growth, viability, and produc-tion of the target protein, demon-strating a significant performance improvement of the cell culture by using this supplement.

Enhanced Cell Performance Attributed to Shift in Metabolism

During the course of these experi-ments, the levels of glucose, lac-tate, glutamine, and glutamate in the culture media were monitored. In a majority of our tests, cultures maintained in CDM were subject to a continuous accumulation of lactate throughout the life of the culture, while those supplemented with the hydrolysate experienced a shift in metabolism with respect to the fate of lactate in the late stages of the production runs. This shift occurred when more abun-

dant carbon sources, such as glu-cose and glutamine, had fallen below certain critical levels or were entirely depleted from the culture medium.6 This may explain, in part, why in most cases hydroly-sate-supplemented cultures exhibit extended g rowth curves and enhanced cell viabilities compared to unsupplemented cultures. In addition, although in this example the shift is seen to a certain degree in the unsupplemented cultures, it is significantly more pronounced in the hydrolysate-supplemented treatments. This ability to effi-

Figure 4. Lactate concentration with and without supplementation with HyPep. Supplementation led to a change of metabolism, resulting in a reduction in the lactate concentration after day 5.

Figure 5. Yield of IgG antibody production. A synergistic effect between a wheat protein hydrolysate (HyPep 4601) and recombinant Albumin (Sheffield rAlbumin ACF) resulted in increased production of the protein of interest.

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CDM-C: single chemically defined media

ciently metabolize lactate correlates well with increased target protein production.

Synergistic EffectsThe performance benef it pro -vided by any medium supplement is subject to its interaction with other medium components pres-ent in the basal formulation, as well as any additional supplements being used. In some instances, a combination of supplements may provide better performance than that observed when supplement-ing with the individual entities. In the course of evaluating the per-formance-enhancing effects of a wheat hydrolysate (HyPep 4601) in SP2/0 hybridoma cells cultivated in a CDM, an interesting result was observed when recombinant human serum albumin (rHSA) was included as an additional supple-ment.7

The data shown in Figure 5 show that supplementation with a combination of the wheat hydro-lysate and rHSA yielded a signifi-cantly higher IgG titer than any other treatment. Although IgG titer increased by more than 30% in the cultures supplemented with rHSA alone, the IgG titer in the cultures supplemented with both HyPep 4601 and rHSA increased to greater than 180% of that of the medium control, despite the wheat hydrolysate’s ineffectiveness as a solo supplement. This shows that cell culture supplements can act synergistically, dramatically

improving the performance of a cell culture system when used in combination.

SummaryThe biopharmaceutical industry faces the challenge of reducing costs while also adopting animal- component–free cell-culture sys-tems. We have observed that although chemically defined cell culture media can be applied to biomanufacturing processes, they are expensive and often do not prov ide opt imal per formance compared to standard media supplemented with sera. This article shows that full strength or diluted CDM supplemented with plant-derived protein hydro-lysates or recombinant proteins such as rHSA provide cost-effec-tive alternatives that can signif-icantly enhance the production titers of the protein of interest. Supplements also can work syner-gistically to further enhance cell-culture performance.

In addition to the beneficial per-formance results shown here for CHO and SP2/0 cell lines, other examples have been reported in the literature for CHO,8,10–14 SP2/0,16 and many other animal host cell lines used in biomanufacturing, including BHK,21,22,24,30,31 VERO, 23,24,26,27,29 HEK,33–35 MRC,32 NS0, CEF, insect lines like Sf9 and High Five,36–40 and plant cells.42–45 These examples show the broad applicability of the approach outlined in this article.

By providing the benefits of enhanced cell density and cell via-bility, underscored by significant target protein production, plant-derived hydrolysates can be used as valuable and practical tools to improve cell culture performance. The inclusion of such supplements has become more and more popu-lar during the development and optimization of upstream pro-cesses. Today, 6 out of 10 biophar-

maceutical manufacturers have indicated they are actively using protein hydrolysate supplements.9 BP

References 1. Babcock JF, Merrill DA, Smith SR.

A novel approach to the production of plant-derived hydrolysates yields medium supplements with enhanced performance in cell culture systems. Poster presentation at 19th Meeting of the European Society of Animal Cell Technology (ESACT), Berlin; 2007.

2. Ganglberger P, Obermüller B, Kainer M, Hinterleitner P, Doblhoff O, Landauer K. Optimization of culture medium with the use of protein hydrolysates. In: Smith P, ed. Cell technology for cell products. Springer Netherlands; 2007. pp. 553–7.

3. Ballez, JS, Mols J, Burteau C, Agathos SN, Schneider YJ. Plant protein hydrolysates support CHO-320 cells proliferation and recombinant IFN-γ production in suspension and inside micro carriers in protein-free media. Cytotechnol. 2004;44(3): 103–14.

4. Merten OW, Kallel H, Manuguerra JC, Tardy-Panit M, Crainic R, Delpeyroux F, et al. The new medium MDSS2N, free of any animal protein supports cell growth and production of various viruses. Cytotechnol. 1999;30(1–3):191.

5. Babcock JF, Antosh A, Hassan T. Partial replacement of chemically defined CHO media with plant-derived protein hydrolysates: Part 1. Poster presentation at 21st Meeting of the European Society of Animal Cell Technology (ESACT). Dublin; 2009.

6. Babcock JF, Antosh A. Partial replacement of chemically defined CHO media with plant-derived protein hydrolysates: Part 2–metabolic effect of hydrolysates. Poster presentation at BioProcess International Conference & Exhibition. Raleigh; 2009.

7. Babcock JF, Antosh A. Performance enhancing synergy between a wheat hydrolysate and recombinant human serum albumin in SP2/0 hybridoma cells. Poster presentation at IBC Antibody Development & Production conference. Carlsbad; 2010.

8. Babcock J, Smith S, Huttinga H, Merrill, D. Enhancing performance in cell culture. Gen Eng News. 2007;27(20):47–8.

9. Sheffield BioScience – company confidential. Internal survey of biopharma producers at IBC Antibody Production Conference. Carlsbad, CA; 2007.

10. Ballez JS, Mols J, Burteau C, Agathos SN, Schneider YJ. Plant protein hydrolysates support CHO-320 cells proliferation and recombinant IFN-γ production in suspension and inside micro carriers in protein-free media. Cytotechnol. 2004;44(3): 103–14. (CHO-K1-origin)

11. Burteau CC, Verhoeye F, Mols JF, Ballez JS, Agathos SN, Schneider YJ.). Fortification of a protein-free cell

Supplements also can work synergistically to further enhance cell culture performance.

culture medium with plant peptones improves cultivation and productivity of an interferon-gamma-producing CHO cell line. In Vitro Cell Dev Biol Animal. 2003;39(7):291–96. (CHO-K1-origin)

12. Kim DY, Lee JC, Chang HN, Oha DJ. Development of serum-free media for a recombinant CHO cell line producing recombinant antibody. Enz Microbiol Technol. 2006;39:426–33. (DG44-origin)

13. Kim SH, Lee GM. Development of serum-free medium supplemented with hydrolysates for the production of therapeutic antibodies in CHO cell cultures using design of experiments. Applied Microbiol Biotechnol. 2009;83(4):639–48. (DG44-origin)

14. Sung YH, Lim SW, Chung JY, Lee GM. Yeast hydrolysate as a low-cost additive to serum-free medium for the production of human thrombopoietin in suspension cultures of Chinese Hamster Ovary cells. Appl Microbiol Biotechnol. 2004;63(5):527–36. (DUKX-B11-origin)

HYBRIDOMA 15. Bonarius HPJ HV, Meesters KPH, de

Gooijer CD, Schmid G, Tramper J. Metabolic flux analysis of hybridoma cells in different culture media using mass balances. Biotechnol Bioeng. 1996;50:299–318. (Murine)

16. Ganglberger P, Obermüller B, Kainer M, Hinterleitner P, Doblhoff O, Landauer K. Optimization of culture medium with the use of protein hydrolysates. In: Smith P, ed. Cell technology for cell products. Springer Netherlands; 2007. pp. 553–7. (SP2/0)

17. Iding K, Büntemeyer H, Gudermann F, Deutschmann SM, Kionka C, Lehmann J. An automatic system for the assessment of complex medium additives under cultivation conditions. Biotechnol Bioeng. 2001;73(6):442–48. (Murine)

18. Jan DC, Jones SJ, Emery AN, Al-Rubeai M. Peptone, a low-cost growth-promoting nutrient for intensive animal cell culture. Cytotechnol. 1994;16(1):17–26. (NS1-derived)

19. Schlaeger EJ. The protein hydrolysate, Primatone RL, is a cost-effective multiple growth promoter of mammalian cell culture in serum-containing and serum-free media and displays anti-apoptosis properties. J Immunol Methods. 1996;194(2):191–99. (Murine-various)

20. Zhang Y, Zhou Y, Yu J. Effects of peptone on hybridoma growth and monoclonal antibody formation. Cytotechnol. 1994;16(3):147–50. (Murine-WuT3)

VACCINE LINES 21. Heidemann R, Zhang C, Qi H, Rule J

L, Rozales C, Park S, et al. The use of peptones as medium additives for the production of a recombinant therapeutic protein in high-density perfusion cultures of mammalian cells. Cytotechnol. 2000;32(2):157–67. (BHK)

22. Keay L. Autoclavable low cost serum free cell culture media. The growth of L-cells and BHK cells on peptones. Biotechnol Bioeng. 1975;17(5):745–64. (L-cells, BHK)

23. Keay L. The growth of L-cells and Vero cells on an autoclavable MEM-peptone medium. Biotechnol Bioeng. 1977;19(3):399–411. (L-cells, Vero)

24. Merten OW, Kallel H, Manuguerra JC, Tardy-Panit M, Crainic R, Delpeyroux F, et al. The new medium MDSS2N, free of any animal protein supports cell growth and production of various viruses. Cytotechnology. 1999;30(1–3):191. (BHK, MDCK, Vero)

25. Mazurkova NA, Kolokol’tsova TD, Nechaeva EA, Shishkina LN, Sergeev AN. The use of components of plant origin in the development of production technology for live cold-adapted cultural influenza vaccine. Bulletin Experimental Biol Med. 2008;146(1):144–47. (MDCK)

26. Mazurkova NA, Troshkova GP, Sumkina TP, Kolokol’tsova TD, Skarnovich MO, Kabanov AS, et al. Comparative analysis of reproduction of influenza virus strains in cell lines perspective for the creation of cultural vaccines grown on nutrient medium on the basis of rice flour protein hydrolysate. Bulletin Experimental Biol and Med. 2008;146(4):547–50. (MDCK, Vero)

27. Mazurkova N, Ryabchikova E, Troshkova G, Getmanova T, Sumkina T, Shishkina L, et al. Morphological and proliferative characteristics of Vero and MDCK cells during culturing in nutrient media on the basis of hydrolysates of plant proteins. Bull Experim Biol Med. 2009;147(4):551–54. (MDCK, Vero)

28. Mochizuki, M. Growth characteristics of canine pathogenic viruses in MDCK cells cultured in RPMI1640 medium without animal protein. Vaccine. 2006;24(11):1744–8. (MDCK)

29. Rourou S, van der Ark A, van der Velden T, Kallel H. Development of an animal-component free medium for Vero cells culture. Biotechnol Progress. 2009;25(6):1752–61. (Vero)

30. Saha SN, Sen AK. Studies on the development of a medium with peptone and casein hydrolysate for the production of foot-and-mouth disease vaccine in BHK-21 cells. Vaccine. 1989;7(4):357–63. (BHK)

31. Saha SN, Sen AK. Partial replacement of serum with peptone and lactalbumin hydrolysate for the production of foot-and-mouth disease vaccine in BHK-21 cells. Acta Virologica. 1989;33(4):338–43. (BHK)

HUMAN CELLS 32. Chun BH, Lee YK, Bang WG, Chung N.

Use of plant protein hydrolysates for varicella virus production in serum-free medium. Biotechnol Letters. 2005;27(4):243. (MRC-5)

33. Han X, Sun L, Fang Q, Li D, Gong X, Wu Y, Yang S, Bing S. Transient expression of osteopontin in HEK 293 cells in serum-free culture. Enz Microbial Technol. 2007;41:133–40. (HEK 293)

34. Pham PL, Perret S, Doan HC, Cass B, St-Laurent G, Kamen A, et al. Large-scale transient transfection of serum-free suspension-growing HEK293 EBNA1

cells: Peptone additives improve cell growth and transfection efficiency. Biotechnol Bioeng. 2003;84(3):33–42. (HEK 293)

35. Pham PL, Perret S, Cass B, Carpentier E, St.-Laurent G, Bisson L, et al. Transient gene expression in HEK293 cells: Peptone addition posttransfection improves recombinant protein synthesis. Biotechnol Bioeng. 2005;90(3):332–44. (HEK 293)

INSECT CELLS 36. Agathos SN. Development of serum-free

media for lepidopteran insect cell lines. Methods Mol Biol. 2007;388:155–85. (Spodoptera frugiperda and Trichoplusia ni)

37. Donaldson MS, Shuler M. Low-cost serum-free medium for the BTI-Tn5B1-4 insect cell line. Biotechnol Progress. 1998;14(4):573–79. (BTI-Tn5B1-4, [High-Five])

38. Ikonomou L, Bastin G, Scheider YJ, Agathos SN. Design of an efficient medium for insect cell growth and recombinant protein production. In Vitro Cell Dev Biol Animal. 2001;37:549–59. (Sf-9 and High-Five)

39. Kwon MS, Dojima T, Park EY. Use of plant-derived protein hydrolysates for enhancing growth of Bombyx mori (silkworm) insect cells in suspension culture. Biotechnol Appl Biochem. 2005;42:1–7. (Bombyx mori)

40. Mendonça RC, DeOliveira EC, Pereira CA, Lebrun I. Effect of bioactive peptides isolated from yeastolate, lactalbumin and N-Z Case in the insect cell growth. Bioprocess Biosys Eng. 2007;30(3):157–64. (Sf-9)

PLANT CELLS 41. Anjum S, Zia M, Chaudhary MF.

Investigations of different strategies for high frequency regeneration of Dendrobium malones ‘Victory’. African J Biotechnol. 2006;5(19):1738–43. (Dendrobium malones)

42. Gamborg OL, LaRue TAG. Ethylene Production by Plant Cell Cultures. The effect of auxins, abscisic acid, and kinetin on ethylene production in suspension cultures of Rose and Ruta cells. Plant Physiology. 1971;48:399–401. (Rosa sp. and Ruta sp.)

43. Gamborg, OL, Miller, RA, Ojima K. Nutrient requirements of suspension cultures of soybean root cells. Experim Cell Res. 1968;50(1):151–58. (Glycine max)

44. Parc G, Rembur J, Rech P, Chriqui D. In vitro culture of tobacco callus on medium containing peptone and phytate leads to growth improvement and higher genetic stability. Plant Cell Reports. 2007;26(2):145–152. (Nicotiana tabacum)

45. Ranch JP, Giles KL. Factors affecting growth and aggregate dissociation in batch suspension cultures of Datura innoxia (Miller). Annals Botany. 1980;46(6):667–83. (Datura innoxia)

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