Advances in Animal-Free Manufacturing of Biopharmaceuticals - New recombinant raw ingredients developed for use in large-scale mammalian cell culture are becoming readily available. - BioPharm

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Advances in Animal-Free Manufacturing of Biopharmaceuticals
New recombinant raw ingredients developed for use in large-scale mammalian cell culture are becoming readily available.


BioPharm International


Despite its commercial pedigree, S. cerevisiae has recently suffered from a negative image in the industry due to a number of perceived disadvantages in comparison to methylotrophic yeasts (e.g., Pichia pastoris). These perceived disadvantages include mitotic instability of recombinant strains, undesirable over-glycosylation, and difficulties in adapting production strains to industrial scale.17 Pichia systems offer strong promoters, stable integration of expression plasmids, and high-density fermentation. However, they also have a number of limitations at large scale, including the use of hazardous chemicals (e.g., methanol induction), lack of moderately expressed promoters, and few selectable markers.18

Advances in the molecular biology and process development of S. cerevisiae have overcome many of the issues outlined above, and have produced an effective microbial expression technology for the animal-free production of certain classes of biotherapeutics.19 Extensive strain development has created a proprietary S. cerevisiae-based system, delivering a highly competitive cost of goods, typical of a microbial fermentation-based process.

S. cerevisiae strains now exist that typically grow to high cell densities in short fermentation cycles in animal-component–free chemically defined media. Various desirable traits—including genetic stability, high copy number expression plasmids, protease deficient mutants, and strains deficient in the enzymes involved in O-linked glycosylation—have been engineered.20 The systems are optimized for the production of recombinant proteins where glycosylation does not naturally occur or can be designed out without impacting product efficacy. This yeast-based system also overcomes many problems associated with prokaryotic alternatives, such as improper disulfide bond assignment, risks of protease degradation, and inclusion body formation.

The constitutive expression system has not given rise to any problems with post-translational modifications, such as proteolysis or other forms of degradation that would make a separate induction phase advantageous. To produce recombinant proteins in high yields and of optimum quality, S. cerevisiae has been subjected to a series of genetic manipulations. The success of the design of the disintegration vector, designed as whole 2-μm vectors in an otherwise plasmid-free background, dispels the common view that yeast episomal plasmids are unstable for industrial use, as no plasmid loss occurs during the production process in semi-continuous operation over a time scale of months.

Commercial-scale manufacturing of Recombumin—the first recombinant human albumin approved by both the EMEA and the FDA for use in the production of human therapeutics—has successfully validated the disintegration vector system and the automatic procedures developed in the laboratory for the control of the fed batch process function at plant scale.

CONCLUSION

Microbially derived recombinant ingredients are entering the market. These directly address the dilemma of optimizing mammalian cell productivity and performance while avoiding the regulatory and quality risks associated with animal- and human-derived supplementation. Designed for industrial cell culture, recombinant alternatives to albumin, transferrin, and insulin, in particular, offer the biopharmaceutical industry alternative tools with which to develop a fully defined, animal-free, industrial cell culture process that is regulatory compliant and delivers optimal cell productivity.

Furthermore, advances made in the molecular engineering and process development of microbial expression technologies (such as S. cerevisiae) have already delivered a completely animal-free expression system for the industrial-scale production of a wide range of therapeutically relevant recombinant proteins, including antibody fragments, protease inhibitors, enzymes, transport proteins, cytokines, anti-angiogenic polypeptides, anti-inflammatory polypeptides, and growth hormones.

Such products and technologies enable the biopharmaceutical industry to continue to ask questions of its current methods and processes today, in the hope of finding new and innovative solutions which will optimize its product pipeline tomorrow.

SARA MORTELLARO is a marketing manager at Novozymes GroPep, Ltd.,
MAREE DEVINE is a commercial operations manager at Novozymes Delta, Ltd., Nottingham, UK, +44 (0) 115.955.3355,

REFERENCES

1. Walsh G. Biopharmaceutical benchmarks. Nature Biotech 2006;24:796.


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