All Qkine proteins are animal origin-free, but why is this so important? And why aren’t all recombinant proteins produced in animal free expression systems?
Why produce growth factors in animal cells?
Firstly, it can be necessary. Eukaryotic (animal) cells have all the machinery needed to properly fold eukaryotic proteins. Some proteins are also post-translationally modified by phosphorylation, glycosylation or lipidation, and these modifications can be required for full bioactivity [1]. This means that producing certain eukaryotic proteins in a bioactive form in prokaryotic (bacterial) cells can be difficult or impossible, depending on protein folding or modifications needed.
Unique protein engineering technology has allowed Qkine to overcome some of these challenges, particularly with the TGF-β family, with a sequence of buffer-controlled folding steps allowing us to create the only commercially available animal origin-free bioactive TGF-β1.
Some growth factors are difficult to express in E. coli rather than mammalian cells, but there are many advantages to animal origin-free growth factors:
- Protein purity and identity – prevents cross contamination with endogenous growth factors and other proteins
- Sterility – free of animal pathogens and mycoplasma
- Scalability and cost
- Ethical considerations
- Reduced therapeutic risk and regulatory compliance
What does animal origin-free mean?
There are a few different classifications for ‘animal-free’ recombinant proteins; these include animal derived component free/ADCF and animal origin-free/AOF. Qkine proteins are animal origin-free meaning all raw materials and consumables are completely animal origin free.
Animal derived component free (ADCF):
- Raw materials used directly in the product or as a starting material (e.g. E. coli growth media) are not derived from animal or human tissue, cells or body fluids
- Raw materials used directly in the product or as a starting material do not contain animal or human tissue, cells, or body fluids at any stage of their manufacture process
On top of this animal origin-free (AOF) also means:
- Consumables used within the production process (e.g. filters) are not derived from animal or human tissue, cells or body fluids and no animal or human tissue, cells or body fluids are used at any stage of their manufacture process.
Read more about our animal origin-free manufacturing
What are the issues with human and mammalian cell-derived recombinant proteins?
Protein purity and identity
Producing growth factors in a cell culture environment, with animal cell components and serum present increases the risk of contamination with aggregates and animal components. During purification there is a risk of isolating other similar growth factors along with the desired growth factors. For example, animal-derived recombinant GDF-15 was shown to be cross-contaminated with TGF-β1. As GDF-15 works through different signaling pathways the subsequent stimulation of the SMAD pathways by TGF-β1 caused confusion during the study, wasting research time, resources and funding [2]. See our article on GDF-15 for more on this.
Animal origin-free manufacture cannot always guarantee purity. Cross contamination with other bioactive proteins can also come from poor manufacturing practices, such as re-using purification columns, labware and poor quality control [3]
Qkine GDF-15 QC for SMAD2/3 (activin A/TGF-β) bioactivity. Bioactivity was determined using a SMAD2/3 luciferase reporter assay in HEK293T cells. Cells were treated (in triplicate) with a serial dilution of GDF-15 in the presence or absence of 10 µM of the activin A inhibitor follistatin. In this lot, contaminating activity was seen (green line), which was inhibited by follistatin (black line), confirming that contaminating activin A is causing this activity. Data from Qk017 lot #010 (not for sale)
We have seen evidence at Qkine of how easily cross-contamination can occur if standard laboratory practices are followed. In our R&D laboratory, the team were refining processes for GDF-15 manufacture. Despite thorough decontamination and extensive cleaning of a purification column that had previously been used for activin A, a related protein, when our scientists purified GDF-15 on this column we saw clear evidence of contamination of GDF-15 with activin A in a bioactivity assay. Activin A activates the SMAD 2/3 pathway with femtomolar EC50, but GDF-15 is an unusual TGF-β super family member and has been proven not to activate this pathway.
Luckily our extensive quality testing caught this issue and the protein was never cleared for sale. As cross contamination in commercial sources of GDF-15 is such a common problem Qkine has built a bioassay into our standard lot-specific quality control procedure for this protein to ensure there is no activation of SMAD2/3 in our preparations.
For more on this read our blog on growth factor cross-contamination
Sterility
Human- and animal-derived materials have a risk of introducing adventitious agents, including viruses, parasites, bacteria, mycoplasma and agent(s) responsible for transmissible spongiform encephalopathies (TSEs). Ensuring sterility of recombinant proteins which are going to be used in cell culture media is vitally important for both research use and cell and gene therapy (CGT) use [4]. In research, introduction of mycoplasma or other infectious agents can negatively affect experimental data and introduce variability. In cell and gene therapy preparation introduction of adventitious agents can pose a direct risk to human health.
Animal origin-free expression systems have a lower risk of the introduction of animal pathogens, Qkine also perform sterility and mycoplasma testing on every lot of recombinant protein to ensure reproducibility in research cell culture and safety for cell therapy grade ex vivo applications.
But what about endotoxins?
One concern with bacterially expressed proteins is the potential for endotoxin contamination. Endotoxins can stimulate inflammatory signaling pathways and influence cell responses and proliferation, leading to experimental variability. They can persist without live bacteria being present and can also collect on plasticware and labware even after sterilization. Because of the concern of endotoxin contamination in bacterial expression systems products are generally tested during quality control (QC) processes. However, even when bacterial expression systems are not used endotoxin contamination can come from general contamination within the lab environment so can be present in recombinant proteins no matter the expression system.
Qkine test every lot to ensure undetectable endotoxin levels in their E. coli expressed proteins.
Mammalian expressed does not mean endotoxin free, during testing Qkine have found that mammalian-expressed recombinant proteins can have high endotoxin levels and testing may not be a routine or reported part of QC for these proteins.
Scalability and cost
Mammalian cell culture is costly, requiring expensive cell culture media and equipment. This limits the reliability and scalability of the production of recombinant proteins in mammalian cells [5]. In contrast production of recombinant proteins in bacteria is relatively cheap, not requiring expensive media or equipment. Bacterial doubling times are also faster and easier to control [5]. These factors mean increasing production scale is more reliable and less costly, securing the supply chain and allowing for bulk production.
Ethical considerations
Mammalian cell culture for recombinant protein involves the use of large volumes of bovine serum; as previously discussed this comes with the risk of introduction of adventitious agents but it also has potential ethical implications [6].
Reduced therapeutic risk and regulatory compliance
FDA guidance includes recommendations when developing CGT products and tissue-engineered medical products (TEMPs) that are manufactured using human- and animal-derived materials. Their considerations include donor screening and testing, adventitious agent testing and screening, risk assessment, and materials management [7].
Although only published as guidance, the FDA recommend the use of materials that are free of human- or animal-derived proteins (e.g., tissue culture media free of human or animal-derived materials, recombinant proteins), because they may have fewer safety risks and may be less variable in their composition, thus avoiding donor-to-donor variability, and avoiding the variability in how such proteins affect cellular or tissue growth and properties.
So why and why not animal origin-free?
- Currently some bioactive proteins can only be produced in mammalian cells due to post-translational modifications needed for their function.
- It is always best to use animal origin-free proteins where possible to ensure purity, reproducibility and for translational considerations.
- Qkine's unique protein engineering has created uniquely bioactive animal origin-free proteins such as DKK-1, EPO and TGF-β family proteins.
Qkine growth factors are manufactured to the highest quality standards and are purification tag free, carrier protein free and free from animal-derived contaminants, delivering low endotoxicity and high purity. All our proteins conform to our Nine-point Qkine Quality Commitment.
At Qkine, we are committed to raising the standards of growth factors, cytokines and related proteins to better support long-term and complex cell cultures. We are a science-led team, please reach out with any questions or requests to support@qkine.com.