Advances in Animal-Free Manufacturing of Biopharmaceuticals

Published on: 
BioPharm International, BioPharm International-05-02-2007, Volume 2007 Supplement, Issue 3

In addition to existing guidance, in January 2007 the FDA announced further proposals to prohibit the use of certain bovine materials as ingredients in some medical products or as elements of product manufacturing.

The US Food and Drug Administration (FDA) and the Department of Agriculture have erected a series of barriers to protect humans from exposure to the fatal agent linked to bovine spongiform encephalopathy (BSE), and measures announced by the FDA in January 2007 build on those barriers. With its heavy reliance on mammalian cell-based expression systems, and with the problem of serum-free versus protein-free versus productivity, the biopharmaceutical industry continues to need chemically undefined or defined supplements of animal and human origin. To move toward avoidance of animal- or human-derived materials used during biopharmaceutical manufacturing, the industry is increasingly looking at alternative raw materials and is investigating the benefits of non-mammalian expression systems that offer simpler and chemically defined growth conditions. New recombinant raw ingredients are becoming more readily available and have been developed specifically for use in large-scale mammalian cell culture. Animal-free microbial expression systems have made such products possible, and these may themselves offer solutions to some regulatory and productivity issues the biopharmaceutical industry faces.


Biotherapeutics—including recombinant proteins, monoclonal antibodies, and nucleic-acid-based drugs—represent the largest growth sector in the pharmaceutical industry, with a market size estimated at $33 billion and projected to reach $70 billion by the end of the decade. Of the 31 therapeutic proteins approved since 2003, 17 are manufactured using mammalian cell lines.1 Mammalian cell culture is the method of choice for manufacturing large complex proteins, especially those requiring posttranslational modifications, such as glycosylation.

Traditionally, a range of animal-derived protein products—including fetal calf serum, peptones derived from acid or enzyme, hydrolysates of casein, gelatin, meat, egg, lactalbumin, and bovine serum albumin (BSA) or human serum albumin (HSA)—have been used for mammalian cell culture media for research and commercial-scale manufacturing of biopharmaceuticals. These ingredients, together with supplements such as human holo transferrin and insulin-like growth factors (IGF), are added to promote cell growth, survival, and optimized productivity.


Due to the threat of contamination from bovine materials infected with BSE and the threat of transmission of variant Creutzfeldt-Jakob disease (vCJD) from HSA, regulatory authorities worldwide have issued guidelines that govern the use of animal-derived materials in pharmaceutical products. For example, both the European Medicines Agency (EMEA)2 and the Center for Biologics Evaluation and Research (CBER) of the US Food and Drug Administration (FDA)3 have issued guidelines for the controlled use of animal-derived materials for pharmaceuticals from sources with a risk of BSE or vCJD infection.

In addition to existing guidance, in January 2007 the FDA announced further proposals to prohibit the use of certain bovine materials as ingredients in some medical products or as elements of product manufacturing. These proposals are the latest in a series of BSE safeguards that would bar material that harbors the highest concentrations of this fatal agent in infected cattle.

To ensure that companies comply with these prohibitions, the FDA proposes that records be kept to demonstrate that any bovine material used as an ingredient in these medical products or as part of their manufacturing process should meet FDA requirements. This proposal is in addition to measures that companies must already take to reduce risks from serum-derived products, including:

  • evidence of select sourcing of animals

  • testing for the presence of adventitious agents

  • inactivation or removal treatments for contaminating agents.

Current efforts in the US to minimize the risk of BSE infection from pharmaceuticals are in line with those in place in the EU since 2003.4 These US regulations require manufacturers of medicinal products to undertake and document extensive risk assessments on all animal-derived materials used in any aspect of pharmaceutical production, including in the preparation of active substances, excipients, adjuvants, and raw starting and packaging materials. Those assessments should be based on:

  • source animals and their origin

  • the nature of animal-derived material used and cross-contamination procedures

  • systems in place for ensuring product consistency and traceability.

Today the use of animal-derived materials in pharmaceutical production places an increasingly onerous burden on product manufacturers to comply with established requirements.



With the construction of further barriers to the use of animal-derived materials, there are growing needs and trends in the industry to move toward the use of serum-free, chemically defined, and most recently, protein-free media formulations for use in mammalian cell culture. Using chemically defined media components not only reduces the risk of introducing adventitious agents, but it also assists both upstream and downstream process optimization.

While major advances have been made in this area, there are still drawbacks to the use of these products, especially for industrial-scale culture.5,6,7 Transfer to serum-free or protein-free media can be very stressful for cells, and it typically requires a period of adaptation or "weaning." Successful adaptation can be time-consuming and cell-line-dependent, and a lack of protein components can lead to a less robust and less reproducible process.

It is recognized that the addition of specific growth factors and proteins (which are naturally found in serum) can enhance cell viability and productivity. Serum-free media, which is still used extensively in research laboratories, can contain proteins that may or may not be animal- or human-derived. Similarly, to enrich protein-free media, hydrolysates may be added that are not always non-animal in origin.

Recently, dedicated research and development into the production of defined, recombinant alternatives to three critical components of serum (insulin, transferrin, and albumin) have led to a range of animal-free recombinant protein ingredients that are designed and manufactured specifically to optimize mammalian cell growth and productivity at the industrial scale. With these ingredients, researchers should now be able to achieve the performance associated with protein-supplemented media while avoiding the associated risks of traditional animal-derived supplements.

Studies with recombinant ingredients have shown equivalent or improved performance in a wide range of cell types up to commercial scale.8,9,10 Manufactured to recognized quality standards and offering continuity of supply, these ingredients provide the confidence in critical raw material quality that is required of products used in long-term biopharmaceutical manufacturing.

Recombinant Insulin-Like Growth Factor

Growth factors are essential for long-term growth and proliferation of cell lines in serum-free media formulations. Recombinant therapeutic insulin has been used as a growth factor in serum-free cell culture. However, its primary use for the treatment of diabetes has led to supply and availability issues for cell culture users.

Recombinant insulin-like growth factors are now available as a dedicated raw material, manufactured exclusively for the industrial cell culture market. Studies using this mitogen as a supplement in cell culture have shown that it results in performance that is equivalent to or better than recombinant insulin in numerous cell types, including: Chinese hamster ovary (CHO), baby hamster kidney (BHK), human embryo kidney-293 (HEK293), Vero, PER.C6, Madin-Darby canine kidney (MDCK), and fibroblasts (data not shown).

LONGR3 IGF-I is a recombinant insulin-like growth factor (IGF-I) analogue manufactured in E. coli Studies comparing the effectiveness of LONGR3 IGF-I with insulin for sustaining cell growth and viability in serum-free CHO cell cultures have found that recombinant LONGR3 IGF-I protein is better able to sustain viability under production conditions than is insulin.8 Insulin is mostly used at supra-physiological concentrations, and its ability to promote growth and survival is attributable to its activation of the IGF-I receptor at these high concentrations. LONGR3 IGF-I is also more potent than insulin at activating the IGF-I receptor, even at 200-fold lower concentrations in both CHO and HEK293 cells (Figure 1).11

Figure 1. Combined results from subculture experiments showing the percentage cell growth with DeltaFerrin-supplemented serum-free media relative to growth obtained with holo human transferrin and other commercially available chemical chelators (ferrous ammonium sulphate, ferrous sulphate, and Invitrogen B), over five subcultures in Vero-PP, Madin-Darby canine kidney (MDCK), and baby hamster kidney (BHK)-21-PP1-C16

Recombinant Transferrin

Animal- and human-derived transferrins have traditionally been used as cell culture supplements to facilitate optimal iron metabolism. Studies in CHO cells have found that a combination of both IGF-I and transferrin is necessary to maintain viability and proliferation upon the withdrawal of serum from the culture media.12

In an effort to move away from animal-derived transferrins, investigators look to using combinations of chemical-based iron chelators. However, a recent study questioned the ability of the chelators to consistently support a wide range of cell lines in serum-free media. Additionally, in comparing various commercially available iron chelators (ferrous ammonium sulphate, ferrous sulphate, and Invitrogen B) against a plasma-derived recombinant human transferrin (DeltaFerrin), the authors found that the recombinant human transferrin (DeltaFerrin) was the only alternative growth factor that showed almost equal potency to native human holo transferrin over five subcultures in Vero-PP, MDCK, and BHK-21-PP1-C16 (Figure 2).9

Figure 2. Combined results from subculture experiments showing the percentage cell growth with DeltaFerrin-supplemented serum-free media relative to growth obtained with holo human transferrin and other commercially available chemical chelators (ferrous ammonium sulphate, ferrous sulphate, and Invitrogen B), over five subcultures in Vero-PP, Madin-Darby canine kidney (MDCK), and baby hamster kidney (BHK)-21-PP1-C16

The use of chemical chelators for industrial-scale manufacturing may also be limited by the chelators' unpredictability in controlling and managing the redox cycle and cell oxidation processes. An animal-free recombinant human holo transferrin, specifically designed and manufactured for industrial cell culture applications, provides the benefits of natural plasma-derived transferrin, with the defined, regulatory-friendly aspects of chemical alternatives.

Recombinant Human Albumin

Albumin is a 66-kDa plasma protein with a variety of physiological and stabilizing functions in vivo.13,14 In cell culture, albumin (usually in the form of BSA or HSA) is known to serve several important functions, including acting as a carrier protein and protecting cells from mechanical stress, and it is also thought to be a major serum survival factor, functioning as an inhibitor of apoptosis.15

Several recombinant human albumin (rHA) products are now commercially available and are offered as alternatives to HSA for a range of biopharmaceutical applications. rHA expressed from S. cerevisiae has been shown to exhibit similar safety, tolerability, and pharmacokinetic/pharmacodynamic profiles to native human albumin,10 and it has shown equivalent capacity to protect immunological, biological, and biochemical cell function.15

rHA from other yeast hosts, such as Aspergillus oryzae (for example, rProbuminAF), is being developed specifically for industrial cell culture use. Used as an ingredient in mammalian cell culture, such recombinant albumins can deliver quality, regulatory benefits, and performance benefits for both research and commercial-scale applications.


New animal-free cell culture ingredients derived from microbial expression systems (such as those described above) offer a valuable solution to the quality and performance limitations of supplement regimes currently used with serum-free and protein-free media formulations for industrial-scale mammalian cell culture. Such ingredients, designed and manufactured exclusively to support biopharmaceutical manufacturing, bypass many regulatory hurdles and offer a secure, consistent supply for commercial-scale production.


Mammalian expression systems historically have been characterized as slow and expensive vehicles for the large-scale manufacturing of therapeutic proteins. They are, however, essential for the production of therapeutics that require complex post-translational modifications (PTMs), particularly glycosylation. O-linked and N-linked glycosylation patterns can influence protein stability, ligand binding, immunogenicity, and serum half-life, and they are significant in the context of efficacy of a wide range of biopharmaceuticals.1

On the other hand, microbial systems as models for the animal-free production of recombinant proteins on an industrial scale have much to offer the biopharmaceutical industry.

Unlike mammalian-based technology, microbial systems are typically grown in simple, chemically defined media. And, in the case of the proprietary E. coli and S. cerevisiae systems (which enabled the development of the recombinant ingredients described above) the entire production process is free from the use of animal- or human-derived materials.

With faster growth rates, high yields, and well understood genetics, microbial systems remain the obvious choice for expression of non-glycosylated peptides and proteins. Of the 31 therapeutic proteins approved since 2003, nine are produced in E. coli.15 However, in spite of this prokaryote lacking the ability to perform eukaryote -specific PTMs, it is widely used for the production of insulin, growth hormones, and growth factors. Yeast-based systems confer the advantages of high expression levels, easy molecular manipulation, and low costs of goods; in addition, as eukaryotes, yeast can perform PTMs, such as proteolytic processing, folding, disulphide bond formation, and glycosylation.

Since the 1980s, yeasts have been used for the large-scale production of recombinant proteins of human, animal, and plant origin. The brewer's yeast S. cerevisiae is an extremely well characterized eukaryotic system with "generally regarded as safe" (GRAS) status, and it was widely used for early protein production, including the first commercialized recombinant vaccine.16

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.


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., SMTE@novozymes.comMAREE DEVINE is a commercial operations manager at Novozymes Delta, Ltd., Nottingham, UK, +44 (0) 115.955.3355,


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