In this podcast, we talked with Dr. Tobias Hertzig, Regulatory Affairs Manager and Dr. Ulrich Tillmann, Global Product Manager, Supplements and Manufacturing Aids both representing Merck KGaA, Darmstadt, Germany. We discussed non-animal origin cell culture media supplements, including regulatory advantages, performance metrics and their use in biologics manufacturing.
I began our discussion by asking Tobias if he could give us a definition of what non-animal origin means and if there was an industry-wide understanding of the term. He explained that he would love to give an industry-wide definition for non-animal origin, but unfortunately there is no industry-wide definition and there is no useful or clear definition provided by the regulators.
Next I asked Tobias if he could talk about the distinctions between primary, secondary and tertiary levels for non-animal origin. He said that these terms are used to distinguish how far away from animal origin the material is. For example, primary describes something that is not directly derived from an animal source. Secondary describes, if you use fermentation as an example, that no animal sourced media ingredients were used in the manufacture. A good example for tertiary would be a recombinant insulin where the material is recombinant, the enzyme used to cleave the protein isn’t of animal origin and the media used to make the enzyme didn’t contain any product of animal origin. It is important to note that these terms aren’t fully defined so end users need to be sure that they understand what the author means by those terms as well.
I then asked Tobias to explain why animal origin is such a big concern in the cell culture realm. He said that the primary concern is that adventitious agents could be present in animal-derived material. However, other concerns could be based on a variety of issues, including: religion, kosher/halal, or lifestyle concerns, like allergies. For adventitious agents, there is concern about zoonotic agents that could cross from animals to humans and cause disease. There are several agents known to cross species, including prions such as TSE and BSE, and also viruses. The cells themselves can be susceptible to viruses and if any viruses are detected, then the manufacturing is not GMP compliant. There are also other concerns about animal-derived materials related to the supply situation including possible import restrictions and shortages.
I asked if he could share the regulatory view on non-animal origin components. He said it is preferred to avoid animal material whenever possible, but due diligence is always required even when non-animal origin is declared. It is important to understand why the material is considered non-animal origin and to work with suppliers to ensure that your understanding of non-animal origin is the same as their definition.
We then switched gears and I asked Ulrich if he could tell listeners about which products in the SAFC® portfolio are of non-animal origin. He explained that the non-animal origin supplements in their portfolio are recombinant proteins that mirror serum sourced versions. For example, instead of Fetal Bovine Serum (FBS), you have recombinant transferrin or albumin and for growth factors you have recombinant insulin or Long® R3. There are also manufacturing aids that are now recombinant, like trypsin for example. The portfolio of recombinant supplements and manufacturing aids is offered under the CellPrime® brand, so when you see that brand you know that it is a recombinant, non-animal origin supplement or manufacturing aid.
For clarification, I asked Ulrich to explain what Long® R3 is. He said Long R3 is a derivative of the IGF-1 growth factor that aids in growth of cells and prevents apoptosis. It also helps with utilization of nutrients in the media. It is similar in action to insulin, but receptors on the cells and their genetic make-up dictate whether they respond better to insulin or IGF-1. The ‘long’ in Long R3 IGF refers to a 13 amino acid N-terminal extension which aids in folding the peptide in E.coli. Glu3 (E) in the human IGF sequence has been replaced by Arg (R). The E to R substitution reduces binding to IGF sequestering proteins thus making the growth factor more available in the media.
I then asked how these supplements are used. Ulrich said that the supplements are being used as raw materials for manufacturing cell culture media. Recombinant insulin or Long R3 are either included in the media during manufacturing or are added by the end user. Media including these supplements are widely used for antibody manufacturing, in cell and gene therapy, and in vaccine and viral therapy media.
I then asked him to talk about mAb (monoclonal antibody) and recombinant protein manufacturing versus regenerative medicine, gene therapy and vaccine manufacturing with respect to supplement use. Ulrich explained that in mAb and recombinant protein manufacturing, companies are trying to reduce extraneous protein load in the media that complicate the purification of the desired product. So in those applications, only low molecular mass recombinant growth factors are used. In cell therapy, you still have many companies using serum components due to a lack of suitable replacements. In cell therapy, you still have many companies using serum components due to a lack of suitable replacements and people are only slowly getting around to using recombinant supplements in manufacturing instead. For instance, if you think about the extension of mesenchymal stem cells or adherent vaccine production cell lines, they are grown on supports from which the cells need to be split. In order to keep risk low, recombinant trypsin should be used instead of bovine or porcine based trypsin.
Next Ulrich described how just because something says ‘plant-derived’ on the label, it doesn’t necessarily mean that it is non-animal origin. It is important to closely look at their manufacturing processes. Where the plants are grown and under what kind of controlled conditions. If it isn’t tightly controlled, contamination can happen such as by animal manure.
We then talked about if there is a difference in performance between non-animal origin and animal origin supplements. Ulrich said that he doesn’t think that there is a difference in performance but with serum you have other issues, such as possible TSE/BSE contamination, introduction of proteins that you don’t want in your process, particularly as you move to purify the desired product. With differences between cell lines, it is important to titrate NAO supplements into cell cuture media in order to achieve the right concentration. MS supplies supplement sample pack sizes to those who make their own media and also offers custom media formulations (cell culture media containing supplements) to help companies get the best performance from their cell line.
I then asked Ulrich about the challenges in developing non-animal origin supplements. He said that you must be sure that you have complete visibility on the manufacturing process. When you rely on suppliers for supplements and manufacturing aids, you must audit the suppliers and all the details of the materials they use and their manufacturing facility. You must assess their level of non-animal origin production. Products within the SAFC® portfolio use the definition that there can be no animal components in the raw materials used to formulate the supplement/manufacturing aids growth media; no animal origin material in their manufacturing process or in the facility; no shared equipment and no contact with animal origin used elsewhere in the manufacturing plant.
I closed our talk by asking if either Tobias or Ulrich had anything to add for listeners. Tobias said to be sure to “keep your eyes open”. A non-animal origin declaration doesn’t mean that you don’t need to thoroughly understand the process of vendors and always apply the precautionary principle. Ulrich said there are so many definitions of non-animal origin, it is important to understand what a supplier means by their definition. Ask the supplier, conduct a thorough audit, and ensure that their definition and process matches with what you had in mind.
This post is sponsored by the SAFC® portfolio brand of MilliporeSigma