Designing proteins for gene therapy
Watch this on-demand webinar to learn about Abcam’s manufacturing workflow for bioactive protein production
Dr Deborah Moore-Lai, Director of Protein Development at Abcam
Recombinant proteins require strict quality control, as quality issues affect the reliability of the intended downstream application. Cell and gene therapy research in particular demands superior protein quality that adheres to strict quality control and full validation.
In this SelectScience on demand® webinar, Dr Deborah Moore-Lai, Director of Protein Development at Abcam, presents a new line of high-end bioactive proteins. During the session, Moore-Lai details Abcam’s workflow, as well as the company’s mission to help scientists achieve their goals in cell and gene therapy research and other areas of research. research.
Read on for highlights from the live question-and-answer session or sign up to watch the webinar anytime that’s convenient for you.
Q: Tell us more about your custom protein production capabilities: how long does custom work take?
LMD: The process begins with the initial submission, where we’ll look at the streak you wish to have voiced. From there, we’ll determine if there are any potential challenges with the streak. We then do a small-scale pilot study to determine the expression levels. From there we will determine the scale and amount of supernatant we would need to generate large-scale protein production. We can do this with or without markers, and in a variety of different expression systems, whether mammalian or bacterial. After doing the pilot study, we are better able to estimate the total duration of the schedule. Between 8 and 12 weeks is typical.
Q: What additional proteins and / or expression systems will Abcam bring to the catalog this year?
LMD: We currently have mammalian and bacterial systems. For 2021, we are introducing the expression of insects and potentially yeasts.
Q: What is the benefit of mammalian marker-free proteins?
LMD: The advantages are that the proteins produced by mammals have the optimal post-translational modifications. They are also folded correctly as they are expressed and then secreted into the medium. Our mammalian proteins are typically produced using HEK293 cells, sometimes CHO, depending on the cell type with the highest expression. In terms of tag-free, it’s as close to native as it gets. In the workflow, we express the proteins with tags and cleave them during the purification process. The final tag is just a single glycine on the N-terminus of the protein.
In addition, the reproducibility of the proteins in this product line is very high. Whether you buy a batch of protein today or two years from now, performance will still be due to rigorous quality control.
Q: What is the benefit of the additional QC tests Abcam performs on proteins?
LMD: One of the first processes in QC is to validate the size of the protein. After that, we examine the purity of the proteins using reverse phase HPLC. The protein must be 95% pure or more, which means that there are no fragments or aggregation in this protein sample. We then perform an intact mass analysis where we calculate whether the mass is within 10 Daltons of the theoretical mass of the protein. If it is beyond 10 Daltons, we are failing the protein and it does not upgrade to premium bioactive grade status.
We also make sure that the ultra-low endotoxin levels are below 5 EU per milligram of protein, which is almost sterile. Finally, we perform a functional test to determine the potency of the protein; these assays include assays for proliferation, adhesion, chemotaxis and cytotoxicity; these are supplemented to ensure that the protein is functionally active.
Q: What specific aspects of the protein manufacturing process are you most proud to overcome to produce this line?
LMD: One of the hardest parts of setting up the workflow and what we’re most proud of is the cleansing. It is often a three to four step purification process to separate the tags and then to repurify the protein to separate the cleaved protein from the tags. At this point, if a protein is still not pure enough, we subject it to gel filtration or ion exchange to further separate the specific protein from any contaminants. During this process, the purity is tested at each step to ensure that the final purity is above 95%.
Q: What makes Abcam proteins different from other proteins on the market?
LMD: This range of premium proteins is differentiated by the fact that they are exclusively derived from mammals and without labels. Many of our competitors will offer active proteins from bacterial systems. With this, proteins are often not folded correctly and have to be refolded after being purified. In addition, they often do not include any relevant post-translational modification.
Learn more about Abcam’s premium bioactive protein range >>
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