New recombinant protein ingredients offer a sustainable solution to the regulatory and productivity issues currently facing the growing biopharmaceutical industry argue Maree Devine and Sarah Mortellaro of Novozymes
A wide range of animal-derived protein products, such as foetal calf serum, hydrolysates of casein, gelatine, egg/lactalbumin and bovine/ human serum albumin, have traditionally been used for the manufacture of biopharmaceuticals. However, the theoretical risk of the transmission of adventitious agents (such as variant CJD and HIV) via the continued use of animal and human-derived products remains a major concern for the regulatory authorities.
To this end, supporting guidance from the EMEA and FDA for the manufacture of biopharmaceuticals and medical devices, strongly encourages the use of "animal-free" components. There are various ranges of recombinant protein ingredients currently being manufactured without the use of animal-derived components, and specifically developed for the industry to satisfy these guidelines.
Recently, dedicated research and development of recombinant alternatives to three critical components of serum - albumin, transferrin and insulin - has created a range of defined, animal-free, recombinant protein ingredients designed to optimise mammalian cell growth and productivity at industrial scale. Studies with these recombinant ingredients, outlined below, have demonstrated equivalent or improved performance in a range of cell types up to commercial scale.
It is widely accepted that optimal cell culture performance is achieved using protein supplemented media; however, animal and human sources of proteins are not consistent or sustainable options. The ingredients described here enable process development scientists to achieve the performance of protein-supplemented media while avoiding the risks and inconveniences associated with animal- and human-derived sources.
There has recently been strong regulatory direction towards "Quality by Design", whereby the reliance on final product testing is reduced by identifying and removing variables, throughout the manufacturing process, which can affect product quality.
The importance of consistent biological raw materials is a big part of this initiative. Manufactured to recognised quality standards and offering continuity of supply as well as batch-to-batch consistency, the recombinant ingredients developed by Novozymes and described below offer the confidence in critical raw material quality that is required of products used in long-term biopharmaceutical manufacture.
Recombinant insulin has been used as a growth factor in serum-free cell culture to enable long-term growth and proliferation. But its primary use as a therapeutic for the treatment of diabetes has led to supply and availability issues for industrial cell culture users.
LONG R3IGF-I is an analogue of human insulin-like growth factor I (IGF-I) manufactured in E.coli, specifically for industrial cell culture. Studies comparing its effectiveness with insulin for sustaining cell growth and viability in serum-free cell culture have found that LONG R3IGF-I was better able to sustain viability under production conditions than insulin.1 Both LONG R3IGF-I and insulin elicit their response via the IGF-I receptor to promote growth and survival. Studies have shown that LONG R3IGF-I is more potent than insulin at activating the IGF-I receptor, even at 200-fold lower concentrations in both CHO2 (Fig. 1) and HEK293 cells (data not shown).3
Further studies using LONG R3IGF-I, as a supplement in cell culture, have shown that it results in equivalent or better performance compared to recombinant insulin in numerous cell types including CHO, BHK, HEK293, Vero, PER.C6, MDCK and fibroblasts (data not shown).
Animal- and human-derived transferrins have traditionally been used as cell culture supplements to facilitate optimal iron metabolism. An animal-free recombinant human transferrin analogue, specifically designed and manufactured for industrial cell culture applications, provides the benefits of plasma-derived transferrin with the defined, regulatory-friendly aspects of chemical alternatives.
CellPrime rTransferrin (referred to as rTransferrin) is a recombinant human holo transferrin that facilitates iron transportation in mammalian cells. Recent independent studies have shown that rTransferrin supports the growth of transferrin-dependant MDCK, BHK and Vero cells over a sustained period in a manner indistinguishable from human holo transferrin, and was the only alternative that consistently supported cell growth over multiple passages4. Furthermore, rTransferrin has been shown to be an equally potent growth factor in cell- culture media as serum transferrin from current commercially derived sources.5
Studies in CHO cells have highlighted a significant growth enhancement effect when LONG R3IGF-I and rTransferrin were added in combination to serum-free media (Fig. 2). Viable cell densities were higher over the 14-day cell culture period when the two components were added together than when either component was added alone.
In cell culture, albumin (usually in the form of BSA or HSA) is known to serve several important functions, such as acting as a carrier protein and protecting cells from mechanical stress such as shear force. It is also thought to be a major serum survival factor, functioning as an inhibitor of apoptosis.6 Several recombinant human albumins (rHAs) are now commercially available and are offered as alternatives to Human Serum Albumin (HSA) for a range of biopharmaceutical applications.
HSA has a wide range of beneficial in vivo functions and physical properties that have been exploited for use in biopharmaceutical applications, particularly as an excipient.
Its amphiphilic properties make it suitable as an additive to inhibit adsorption of the active protein to the container, via competitive adsorption mechanisms, while its surface-active character enables it to fulfil the role of a surfactant, thereby preventing protein aggregation. In some instances, HSA stabilises the conformational structure of the active molecule to maintain its bio-activity throughout product shelf-life. HSA also has a high glass transition temperature, which, in combination with its amphiphilic nature, makes it an ideal vehicle for cryoprotection.
Safety concerns provide the strongest motivations to develop rHA as a suitable alternative to HSA, for use as an excipient in biotherapeutics. As well as avoiding any known or future adventitious agents, the likely advantages of using rHA over HSA include increased batch-to-batch consistency, which, for industrial applications, could mean the difference between performing several timely and costly batch verifications per year or not, and breaking a heavy reliance on an increasingly unpredictable supply chain.
Recombumin is an rHA made by Novozymes at its cGMP-compliant facility in Nottingham, UK. It is the only commercially available rHA approved by the FDA and EMEA for use in the manufacture of biotherapeutics; it is used in the production of childhood vaccines for measles, mumps and rubella M-M-R II (Merck & Co.) and M-M-RVAXPRO (Sanofi Pasteur MSD) and is supported by a Type V Biologics Master File (BMF) with the FDA.
A Phase I study has been conducted comparing the safety, tolerability, pharmacokinetics and pharmaco-dynamics of rHA (Recombumin) with HSA.7 Two double-blind, randomised trials were performed in healthy volunteers using intravenous (IV) and intramuscular (IM) administration. No serious or potentially allergic events were noted with either product in the IV study. Furthermore, there was no immunological response to either product, and dose level did not influence the study outcomes. The study concluded that rHA (Recombumin) and HSA exhibited similar safety, tolerability, and pharmacokinetic/pharmaco-dynamic profiles, with no evidence of any immunological response.
High quality recombinant protein ingredients, offer the industry the performance benefits of native proteins. In addition, such ingredients, designed and manufactured exclusively to support biopharma manufacture, ease the regulatory hurdles associated with animal derived equivalents and offer a secure, consistent supply for commercial-scale production. Defined recombinant ingredients developed exclusively for biopharmaceutical manufacturing are critical tools for improving product quality and compliance and will support the increasing growth of the industry by offering a safe and sustainable route for the development of tomorrow's biopharmaceuticals.