Following cases of mycoplasma contamination in the 1970s, endotoxin concerns in the 1980s, and bovine spongiform encephalopathy (BSE) scares in the 1990s, the biopharmaceutical industry continues to push for thorough and transparent information about the origin of raw materials used in the manufacture of therapeutics. In particular, companies often ask cell culture media suppliers for documentation of the animal-origin free (AOF) status of media and reagent components. Many biotherapeutics manufacturers have begun applying varying definitions and standards of AOF, however, depending on the regulatory authorities they work with and their own level of concern; many companies also have begun to demand detailed information about the complete supply chain of components used in media and reagents. The consequences of these in-depth investigations can be significant in terms of the cost and availability of materials. To help navigate what is possible and practical, this article reviews the levels of inquiry required for selecting AOF cell culture components; how to qualify and validate the claims; and finally, ways to help balance risks with patient safety without affecting product availability.
In addition, media suppliers are now being asked to provide cell culture media in which all the components of the cell culture media meet the standard of secondary level AOF. This term has no universal definition, but for the purposes of this article, secondary level AOF will be used to refer to media components that have been manufactured without the use of products derived directly from animals. For example, if a protein component has been manufactured using recombinant E. coli grown in a culture containing no animal-based peptones, the component is classified as secondary level AOF.
As suppliers and manufacturers have gained a better understanding of the media manufacturing process and how to remove AO components, the desire has emerged to go even further. The most challenging standard of AOF is the tertiary level, which requires media manufacturers to ensure that none of the components used to manufacture the raw materials are derived from an animal. In practice, this would mean that the protein in the example above would only be considered tertiary AOF if the AOF peptone were derived from a recombinant process in which none of the ingredients in the broth used to make the peptone were directly derived from an animal.
It is good that the precise AOF status of media components be understood and documented, and that there be a high level of transparency regarding manufacturing change for raw materials. It should be noted, however, there have been no indications that AO materials at the tertiary level constitute any safety hazard in cell culture–based pharmaceutical products. To provide anything above the secondary level of AOF requires a significant audit trail including reviews of bill of materials and various manufacturing steps, which are currently not specifically required by any regulatory agency. Currently, tertiary level AOF components are not being actively demanded by many manufacturers of biotherapeutics mainly because of how difficult it is to establish, control, monitor, and maintain supplies of such products.
What Levels of AOF are Achievable? A Practical Example
Insulin is widely used in mammalian cell culture to delay apoptosis and thereby increase productivity. In the past 20 years, because methods of producing recombinant human insulin have come to the forefront, there has been a shift away from using bovine- or porcine-derived insulin.4 Recombinant insulin is generally a primary level AOF component. However, as worries about TSE and other animal-origin contaminants have increased, many biopharmaceutical companies have begun to ask media suppliers to consider producing secondary level AOF insulin, even though the therapeutic standard insulin that patients receive as an injectable remains primary level AOF.
Currently, one major insulin manufacturer is using a recombinant form of non-animal derived trypsin to produce a secondary level AOF insulin for bioprocess use. This secondary level AOF version is more expensive and less readily available than primary level AOF insulin. The India-based pharmaceutical company Biocon is developing a secondary level AOF insulin. Once it is available, biotherapeutics manufacturers will have another source for secondary AOF level insulin.
If all the media components and enzymes used in processing insulin were derived from fermentations that use AOF components, this would produce tertiary level AOF insulin. In the future, insulin manufacturers may consider this option as a way to gain a competitive advantage. The perceived risk of contamination is still very low, however, so one might question whether continuing down this path to greater and greater levels of AOF status will provide sufficient benefits to justify the effort.