Wayne K. Way, Ph.D., Head of Protein Preparation and Reagents, MilliporeSigma, Bellefonte, PA, an affiliate of Merck, KGaA Darmstadt, Germany
Eleni Sommerkamp, B.S, Product Manager- Protein Prep & Reagents, Extraction, MilliporeSigma, St. Louis, MO, an affiliate of Merck, KGaA Darmstadt, Germany
In the biopharmaceutical industry, quality is pivotal. For a final product to meet quality standards, quality must be built into the production process. Biopharmaceuticals, however, are made from biologically sourced materials, which are inherently variable. This is why choosing the right materials from the beginning is so important. Qualified vendors, robust production processes, reliable equipment, consistent quality control testing, and reproducible product performance – all these boxes have to be ticked. There are specific considerations and obstacles when developing a biopharmaceutical product as it must meet strict quality control specifications. The goal is to develop a product that has a good yield each time it is produced and that meets certain specifications regarding concentration, performance, microbiological safety, as well as other criteria. To reach these goals, understanding the production process and addressing appropriate critical points is a good place to start.
The protein extraction process
To produce biopharmaceuticals, a variety of methods and materials can be used, with a common approach being to start with recombinantly expressing a protein in a host cell system. Once the cells are grown, the protein is extracted. To do this, the cells are lysed for the protein to be released. To protect the protein of interest, protease and phosphatase inhibitors are then added to the extraction solution. Because DNA and RNA are released alongside the protein during cell lysis, nucleases must be added to degrade these nucleic acids into smaller molecules. Once this step is completed, the extracted solution can be further purified, concentrated, and tested.
Maximizing protein yield
During protein extraction, there are two points at which yield can be addressed: cell lysis and protein protection. Various methods exist to lyse cells, and the choice should be based on the types of cells used for expression and the properties of the protein being extracted. Examples of cell lysis methods include chemical or detergent treatment such as BugBuster® Protein Extraction Reagent, single enzymatic digestion using lysis enzymes like lysozyme, or a mixture of enzymes like MetaPolyzyme. Effective options also include mechanical methods like bead-beating, sonication, and even freeze-thaw treatments. It is also typical to use a combination of the mentioned methods. The goal is to extract the highest amount of protein possible under conditions that minimize damage.
Once the protein is extracted, it is exposed to proteases and phosphatases that have also been released from the cell. These enzymes will degrade and modify proteins, so to protect them protease and phosphatase inhibitors are added to the solution. This is not a one-size-fits-all step, and there are options for which inhibitors should be added to the process. There are commercially available inhibitor cocktails specific for proteases, phosphatase, or a combination of both. The ReadyShield® products, for example, are non- freezing solution cocktails that are tailored to the sample type, while SIGMAFASTTM inhibitor tablets include versions with and without EDTA. If this critical point is not considered, the final yield and final performance of the product will be greatly impacted.
Removing contaminant nucleic acids
During extraction, the nucleic acids DNA and RNA are also released from the cell, causing the solution to become viscous and making it more difficult to work with. Additionally, in biopharmaceutical production, DNA and RNA contamination must be removed to meet final product specifications. For these reasons, nucleases are added to the extraction solution. Some, such as the Benzonase® nucleases, are active under extraction conditions and will degrade both RNA and DNA at efficient rates. These are ideal reagents to use for efficient nucleic acid degradation during protein extraction.
Avoiding downstream interference
When using reagents during the extraction process, the final product specifications and the eventual product use must always be considered. There are reagents that can interfere with final testing or may contaminate the final product. Therefore, it is necessary to use reagents that can be removed once they have served their purpose so they will not interfere with the intended use of the final product or will not cause interference with downstream testing. For example, while it is important for a nuclease to degrade DNA and RNA during the production process, these enzymes should not be active in the final product. Nucleases can be inactivated by high temperatures and extreme conditions, and Benzonase® nuclease can also be removed by chromatography.
Changes, opportunities & outlook
The biopharmaceutical market is growing as new innovations are utilized and commercialized. Government interest and investment in this area of life science is causing a spike in research opportunities, the development of new technologies, and future advancements in this promising field. FDA and quality regulations must also be considered to ensure that a quality product is developed. Giving consideration to the critical points during protein extraction can improve costs, yield, efficiency, and develop a consistent and robust process to produce recombinant biopharmaceuticals.
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