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

Jun 02, 2010


Sheffield Bio-Science Center for Cell Culture Technology
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.

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.