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.
Concern about the risk of animal-derived transmissible spongiform encephalopathies (TSE) has long been a driver for biopharmaceutical
companies to eliminate animal origin (AO) materials from their cell culture processes.1 This trend has prompted research by manufacturers of cell culture media into producing media that can maintain productivity
without using animal-derived serum, proteins, or lipids.2,3 To reduce the risk of known contaminants such as BSE and yet unknown agents, biopharmaceutical companies are pushing cell
culture manufacturers to take increased levels of care in choosing raw materials for their products.
The term animal-origin free (AOF) generally is used by cell culture component suppliers to refer to any components not directly derived from eukaryotic
animals (excluding higher plants, fungi, protozoa, and algae). This level of animal-origin free is known as primary level AOF. Currently, there is no standard industry definition that addresses the status of the lower life forms listed in the animal
kingdom (e.g., sponges, worms, insects), although some biopharmaceutical companies define an animal source as any multicellular
form with specialized eukaryotic cells that have their own means of locomotion. This raises the question of what the definition
of animal should be in this case.
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.
In addition, requiring tertiary level AOF media severely restricts the possible sources. As shown in Figure 1, one media supplier
has made an effort over the past decade to qualify secondary AOF components, and in doing so, has made progress in achieving
a higher level of AOF status for many components. Clearly, the higher biopharmaceutical manufacturers raise the bar for AOF
status, the fewer components will qualify for use in their media.
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.
Cell culture media suppliers have responded by ensuring that the cell lines they use to produce insulin are cultured in AOF
media. However, one of the greatest stumbling blocks to producing secondary level AOF insulin is at the step where pro-insulin
is cleaved to produce active insulin (Figure 2). One of the enzymes used in the cleavage is trypsin, which is traditionally
derived from bovine or porcine sources.
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.