Partial Replacement of Chemically Defined Media with Plant-Derived Protein Hydrolysates

ABSTRACT

Protein hydrolysates are routinely used as cell culture supplements to enhance the overall performance of many biopharmaceutical production systems. This enhancement is subject to the additive effect of the native hydrolysate components and the supplemented growth or production medium. Therefore, it is necessary to experimentally determine the proper hydrolysate dosage for a given hydrolysate medium combination that provides the desired optimization effect such as better growth promotion, 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 formulated 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 portion of the active ingredients in these rich media.

Sheffield Bio-Science Center for Cell Culture Technology

Optimizing the culture medium is an integral part of upstream process development, and is essential for efficient biopharmaceutical manufacturing. The aim is to design a robust, economical, 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 mammalian cell growth was achieved by adding animal sera, such as fetal bovine serum (FBS) at a concentration of 5–20% to defined basal media. Although sera may provide important growth and regulatory factors, their composition is complex and undefined, which can lead to batch-to-batch variability and downstream processing challenges. Furthermore, the potential for contamination by adventitious agents, such as viruses, prions, and bacteria, poses serious biosafety risks. This has led regulatory 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 origin. Regulatory pressures related to safety concerns are therefore driving the biopharmaceutical industry away from the dominance of serum as a media supplement, and toward the use of serum-free, animal-component free, or even chemically defined media (CDM) for both new and older manufacturing processes.

Serum-Free Media

Plant-derived hydrolysates have been routinely used to reduce or eliminate serum from traditional basal media formulations, often in combination with a variety of additional supplements. These hydrolysates are composed of a mixture of peptides, amino acids, carbohydrates, and lipids, and as a multitude of unidentified components with indeterminate biological activity. They are produced by the enzymatic or acidic digestion of a given raw material from various plant sources including, but not limited to soy, wheat, and cotton. 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 techniques, 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-enhancing 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 development.

As an alternative solution to traditional basal media supplemented with animal-derived serum, high-performing, richly formulated CDM have been developed for biopharmaceutical production as stand-alone substrates. The optimized mixtures of biochemical constituents in CDM have been carefully designed to stimulate cell growth, maintain good cell viability, and promote high protein yields.

Although CDM have been used 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 cultivated in CDM. It presents examples that use CHO and SP2/0 cell lines grown in 125-mL shake flasks as models for biopharmaceutical manufacturing systems.

Setting Up a Test System

The effects of protein hydrolysates on the overall performance of a biopharmaceutical production system can be influenced by a number of factors including the specific cell line used, the raw material and process used to manufacture the hydrolysate, the hydrolysate dosage, and the composition of the basal medium. The effect of any supplement on the performance of a cell culture system is largely dependent on the formulation of the basal medium. Because CDM and hydrolysates share a number of common components, the additive effects of these components may reduce the performance of a given system as a result of unintentional "overdosing" of certain components. However, hydrolysate supplements also may provide a number of unique constituents that are beneficial for performance. Therefore, it is necessary to experimentally determine the proper hydrolysate dosage for a given hydrolysate medium combination 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 alkaline phosphate (SEAP) by means of a human cytomegalovirus (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 supplemented with 1 mg/mL G-418. Triplicate cultures were seeded at 3 x 10⁵ cells/mL and incubated at 37 °C, in 5% CO₂ at 130 rpm for 12 days. Hydrolysate supplementation was achieved by using 100 g/L stock solutions prepared in each respective basal medium. The data shown are not reflective of all systems, but only depict certain examples from testing conducted using three different commercially available 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 various soy, wheat, and cottonseed hydrolysates, 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 interesting results were obtained using the cottonseed-derived hydrolysate, HyPep 7504. This hydrolysate was tested at concentrations of 8 g/L with several commercially available 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.

Figure 1

As Figure 2 shows, diluting the CDM resulted in a significant reduction in cell density, accompanied 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 hydrolysate-supplemented samples outperformed the 100% medium control in growth, viability, and production of the target protein, demonstrating a significant performance improvement of the cell culture by using this supplement.

Figure 2

Enhanced Cell Performance Attributed to Shift in Metabolism

During the course of these experiments, the levels of glucose, lactate, 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 abundant carbon sources, such as glucose 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 hydrolysate-supplemented cultures exhibit extended growth 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 efficiently metabolize lactate correlates well with increased target protein production.

Figure 3

Synergistic Effects

The performance benefit provided by any medium supplement is subject to its interaction with other medium components present 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 supplementing with the individual entities. In the course of evaluating the performance-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 supplement.⁷

Figure 4

The data shown in Figure 5 show that supplementation with a combination of the wheat hydrolysate and rHSA yielded a significantly 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.

Figure 5

Summary

The biopharmaceutical industry faces the challenge of reducing costs while also adopting animal-component–free cell-culture systems. We have observed that although chemically defined cell culture media can be applied to biomanufacturing processes, they are expensive and often do not provide optimal performance compared to standard media supplemented with sera. This article shows that full strength or diluted CDM supplemented with plant-derived protein hydrolysates or recombinant proteins such as rHSA provide cost-effective alternatives that can significantly enhance the production titers of the protein of interest. Supplements also can work synergistically to further enhance cell-culture performance.

In addition to the beneficial performance 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 viability, 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 popular during the development and optimization of upstream processes. Today, 6 out of 10 biopharmaceutical manufacturers have indicated they are actively using protein hydrolysate supplements.⁹

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

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