GMP (Good Manufacturing Practice) growth factors and cytokines designed for therapeutic manufacturing are a critical component in defined medias. To date, the Cell Therapy industry has accepted the term GMP for this reagent class despite the fact that there is no direct oversight by regulatory authorities. These proteins are intended to be used during further manufacturing and do not come in direct contact with the patient. In fact, Cell Therapy manufacturers need to take steps to ensure that reagents used for further manufacturing are removed before the cells can be used in the clinic. In addition, FDA-regulated, clinical grade proteins that can be directly used as therapeutics may also be described as GMP, leading to confusion of the term “GMP” within different contexts.
We recently published a blog, “GMP Proteins for Cell Therapy Manufacturing: Top 6 Things to Know,” that discusses what you should look for in a supplier. It was a popular piece with readers, so we asked the author, Tim Manning, if he would host an Ask the Expert session on the topic. Tim was kind enough to agree to answer questions from readers this week on the topic of GMP protein manufacturing.
Tim Manning is the Product Manager for Proteins at R&D Systems, a Bio-Techne brand. He has an extensive knowledge of protein production and manufacturing, quality control, and GMP guidelines. Before joining Bio-Techne, Tim received his PhD in from the University of Alabama at Birmingham and was a Postdoctoral Fellow at the University of California, San Diego.
If possible, you would prefer to use cytokines and growth factors manufactured following guidelines set by the United States Pharmacopeia (USP) and/or the European Pharmacopeia (Ph. Eur.). However, this is not always possible, and reagents made for research use only (RUO) may be the only material available. If this is the case, you may be able to still use these reagents in your process. First, you should have confidence in the ability of the supplier to be able to provide consistent material that will meet your needs. Also, involve regulatory authorities as early as possible to ensure that the use of the reagent is acceptable and won’t result in a stumbling block to approval. Lastly, your supplier may have the ability to start manufacturing an RUO protein using USP or Ph. Eur. guidelines. At R&D Systems, we’ve worked closely with biotech and pharmaceutical companies on many of these types of projects.
How much detail can you share about your manufacturing protocols to make sure they meet the needs of our application?
Some information can be provided via questionnaires submitted to a supplier and answered by their Quality Assurance (QA) personnel. In fact, this is often a good way to begin to ensure basic requirements can be met. Additional information is typically shared during an on-site visit or audit of the facility. Even then, there may be proprietary information that a supplier does not wish to provide. In this is the case, a Drug Master Files (DMF) can be submitted by the supplier to regulatory authorities in the market of interest. This document contains extensive detail about the manufacturing process, including proprietary information. This allows the supplier to protect their intellectual property, while providing a mechanism for regulatory authorities to access the information, and for a customer to reference the DMF in a regulatory submission in the market of interest.
I would suggest moving to GMP as early as possible, potentially even during R&D work. This is especially the case if you are using cytokines or growth factors from a supplier that doesn’t also offer GMP for further manufacturing. Proteins are made in biological systems and are, therefore, susceptible to variability. Growth factors from different suppliers can appear identical on a Certificate of Analysis (C of A), yet still exhibit differences in folding, glycosylation, or impurities that can impact activity in your system. The earlier you incorporate the appropriate materials into your process, the easier and less expensive it is to make changes if they become necessary. Often cost is the main reason for delay. In order to keep costs down, work with your supplier to develop supply agreements that take into account bulk purchases, or bundling of other reagents and instrumentation.
Yes. Each cytokine is tested for performance using cell-based bioassays that include proliferation, induced cytokine secretion, cytotoxicity, and others. Before a cytokine is released to the public, a bioactivity specification is established based on the consistent performance of a minimum of three bulk lots. After that, each new bulk lot must meet this defined activity. Lyophilized vials are also tested for bioactivity following each new bottling run and must meet the specification. In addition, controlled stability testing is done to ensure that the cytokine retains its specified activity during storage. Details regarding activity testing for a given cytokine are regularly shared during audits of our facilities.
The main differences come down to extensive documentation, training, oversight, and added testing. GMP proteins include Quality Assurance reviews on all GMP batch and bottling records before release. Three validation lots are made before the product is released to ensure our ability to produce consistent lots. Validated equipment and methods are used throughout production. Our documentation extends to employee training, material traceability, equipment logs, and lot specific certificates of analysis. Each batch is tested for mycoplasma, host cell protein, and N-terminal sequencing of the first 10 amino acids. In addition, individual GMP bottlings also have additional bioactivity and bioburden testing. This is all done to ensure the production of safe and consistent proteins.
I work in an academic lab and we have talked about using higher quality proteins in our research just to avoid lot variability, but it would involve a cost increase. Are there any other benefits to using GMP proteins in a research setting?
As you indicate, one of the main reasons for shifting to GMP is to ensure consistency. This comes from all of the extra analytical testing, documentation, and oversite of the manufacturing process. Protein inconsistencies can lead to negative results and missed opportunities, false positives that lead you down an incorrect path, or results that simply can’t be repeated. All are incredibly frustrating to the researcher and, in the long run, can be more expensive than spending a bit more up front. However, cost is always a factor and must be considered. One suggestion I can make, if you haven’t already, is to develop a relationship with your vendor’s sales representative. At R&D Systems, our reps do an excellent job at finding ways for researchers to save. You might find that shifting to GMP is not as expensive as you think.
I seem to recall seeing something about the variation of animal sourced growth factors and cytokines having unexpected impacts on various cell types. Is this something you have experience with?
The vast majority of discussions we have about animal-free vs animal-sourced growth factors involve the risk of introducing pathogens. However, there are other points of variability that could be introduced by using animal-derived materials. “Animal-sourced” might mean that the natural growth factor is purified from a preparation like plasma. It may also refer to recombinant proteins produced in mammalian cell lines, or that the manufacturing process uses animal-derived components like serum. Natural proteins typically have more inherent variability than recombinant proteins, which are manufactured from scratch in a more controlled environment. For recombinant proteins, host cell protein is a potential contaminant that could affect your cells. Typically, host cell proteins would be found at very low, or undetectable, concentrations and the risk is small. However, if your cell type is exquisitely sensitive, it could have an impact. Lastly, growth factors produced in eukaryotic cells may be glycosylated, a post-translational modification that can affect activity. Consistent manufacturing conditions should lead to consistent glycan patterning, although anytime you rely on biology there is an inherent risk of variability.
Do you have any recommendations for transitioning from research use products to cGMP produced products in terms of culture?
It’s easiest to transition if you can get GMP from the same vendor that supplies your research use only (RUO) protein. Although the manufacturing process and analytical testing is more stringent, there may be very little difference between the proteins in the vials. It is perfectly valid to ask if the manufacturing steps and materials that are used are essentially the same. If a GMP version isn’t available, some vendors will take on a custom project to manufacture your protein using GMP guidelines. Even though the transition should be seamless in these instances, conducting confirmatory testing by repeating experiments should be done. Have a good understanding of what your needs will be if your therapy is successful. The further you are into clinical trials, the more difficult and expensive it is to change. A simple first step is to submit a questionnaire to the supplier’s Quality Department to ensure that they can meet your minimum requirements. Typically there would also be an on-site audit where you could see the facility for yourself and get more detailed information about manufacturing processes. Speak with your colleagues and look for supplier that has experience. The areas of Cell Therapy and regenerative medicine are so new and evolving, an experienced supplier can add value to the relationship.
We have been having some trouble with mycoplasma in our lab, we feel we are doing all the right things for good lab practices, is there any chance this be coming from our raw materials?
As you know, mycoplasma can be very hard to deal with. They are resistant to some antibiotics, may be small enough to slip through typical lab filters, and they can linger on surfaces and equipment. Some potential sources of introduction are shared cell lines between labs, contaminated reagents such as serum or natural proteins, or they can even be transmitted by lab personnel. Therefore, it is possible that raw materials could be a source if the supplier isn’t diligent. At R&D Systems, we ensure that any serum used in our manufacturing process is tested free from mycoplasma. In addition, mycoplasma testing is part of our routine process for cell banks and bulk lots used for GMP manufacturing.
I am interested in your recommendations for setting up secondary suppliers. What if the GMP version is hard to source? Is it ok to have a research use only as a secondary or tertiary?
It is OK to use research use only (RUO) reagents, even as a primary source when GMP for manufacturing isn’t available. If this is the case, you should still be asking the same questions of the supplier that you would if they were manufacturing using GMP for Cell Therapy. You still have the same concerns about lot-to-lot consistency, scalability, and supply chain. At minimum, a vendor should have an ISO 9001:2008/2015-certified quality management system. This means the supplier has been independently audited, and should have a document and record management system that includes manufacturing records, a supplier qualification monitoring program, and more. R&D Systems’ RUO reagents are manufactured under this system. Also, early communication with regulatory authorities can also ensure there are no issues using the reagent. Lastly, a supplier with the capability can initiate a custom project to supply your reagent under GMP for manufacturing guidelines.
What kind of support is available for FDA filings. We are beginning to think about moving to GMP what should we expect from suppliers?
Some basic things to expect include a lot-specific Certificate of Analysis that will detail the identity of the protein and analytical testing that has been done to ensure the material is meeting specifications. A Certificate of Origin may be obtained that provides traceability of the materials used in manufacturing of the protein. This would be key document for those wanting to establish they are working with animal-free raw materials. Drug Master Files (DMFs) may also be submitted to regulatory authorities by the supplier. DMFs provide details about manufacturing process and can be referenced during filing. They also provide a mechanism for submitting proprietary information. A supplier should be very open to audits of their facilities where you can review batch records that provide critical information about lot-to-lot consistency. Lastly, look for someone with experience. Chances are they’ve run across most questions, and they can be an asset as you move through the process.
Unless requested differently, our GMP proteins are lyophilized in glass vials, which is typically very stable. We have environmentally controlled stability chambers that are in continuous use to ensure that there is a documented understanding of protein stability during storage. Although GMP proteins as not labeled as sterile, microbial testing is done on all bulk product and on each bottled lot of GMP proteins. In addition endotoxin testing is performed. Lot specific sterility testing as per USP or Ph. Eur. can be done upon request.
If by system, you mean cell culture vessels, this is highly dependent on the type of cell lines used and the protein being expressed. Currently these include a combination of glass, stainless steel, CellSTACK, and WAVE systems.
If you mean the source, this also very dependent on the protein. In most cases we use an E.coli expression system, the most widely used cell type for animal-free processes. However, in some cases proteins require folding or glycosylation that is not conducive to the use of E. coli. Here we typically use an Sf9 insect cell line that has been certified animal-free. We do also have some examples where we use CHO or NSO lines to ensure we have the best combination of consistency and activity. These are still made using GMP guidelines for Cell Therapy manufacturing, but are not considered animal-free.
How do you confirm the identity of your cytokines and what tests do you run for potential contaminants?
Our identity testing is performed by SDS-PAGE for molecular weight and analysis by densitometry to verify purity. We also use mass spectrometry and N-terminal sequencing of the first 10 amino acids. Endotoxin, bioburden, host cell protein, and mycoplasma testing is also done. Additional testing may be performed depending upon the protein expressed and the cell line utilized to manufacture the product. Requests for additional analytical testing are regularly addressed for customers.