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Defined supplements that improve growth, productivity and consistency are highly attractive for cell culture optimization projects. And, recombinant proteins are increasingly being used as defined supplements in biopharmaceutical, vaccine and stem cell manufacturing. The use of recombinant proteins is an important step in moving toward defined manufacturing for production cell lines. Recombinant proteins allow for the replacement of proteins that in the past have been only available through animal sourcing or from fractionation of donor blood.This product review will cover six of the most commonly used proteins and their role in manufacturing.
Insulin
Insulin is an essential media component for most cell lines when removing serum from culture. In mammalian cells, insulin regulates metabolism and is responsible for cell signaling that controls glucose and amino acid uptake. Insulin is a key growth factor in cell culture media and has also been shown to promote cell survival and reduce apoptosis.
In the past, insulin has been sourced from animals, primarily pigs. Porcine insulin is the closest to human insulin with only a single amino acid variation, which made it a common choice for cell culture. In the 1980’s recombinant insulin was developed and approved to treat diabetes in humans. As production of recombinant insulin became more widespread, scientists began using the product in cell culture to replace animal-derived insulin.
Due to the length of time that recombinant insulin has been available, its use is well established in cell culture. There are two types of recombinant insulin available: recombinant insulin and animal-free recombinant insulin available. In recombinant insulin that is not animal-free, animal-derived enzymes are used in the manufacturing process, the product is manufactured in facilities where animal-products are used, or manufacturing uses serum during banking. In animal-free recombinant insulin, all products used in the manufacturing process are animal-free and it is manufactured in a dedicated animal-free environment.
Albumin
Albumin is the most abundant protein in serum and has many functions in cell culture. It increases overall cell health and productivity by carrying nutrients (lipids) binding and sequestering toxins and stabilizing hormones and peptides in media. Albumin also binds free radicals that can damage cells and cause apoptosis. As a result of these many benefits, the addition of albumin to serum-free cell culture media often provides an increase in growth and productivity. Albumin is also used in other areas of manufacturing and is often used in formulation to stabilize viral and protein based vaccines.
In many cell lines, scientists have been able to remove serum from media, but have needed to supplement with either bovine derived albumin (BSA) or human blood donor derived serum albumin (pHSA) to maintain healthy cells. Until recently, there were no recombinant albumin options available and this made removing serum from some cell lines nearly impossible without suffering an enormous performance hit. Now that recombinant albumin is available, it is reasonable that adoption of this protein will continue to increase, particularly in applications where completely animal-free manufacturing is required.
Transferrin
Transferrin is also found in serum and in culture is responsible for delivering iron to the cells. Transferrin is a universal iron carrier designed to deliver the appropriate amount of iron to cells in a receptor-mediated transfer of iron from transferrin to the cell. Specific transferrin receptors on the surface of cells up regulate or down regulate iron-bound to transferrin based on their iron need. Transferrin is uniquely able to deliver only the amount of iron needed by the cells. This is important because it eliminates the problem of cells taking in too much iron and causing cell damage, which can happen with other iron sources.
In serum-free systems, transferrin either needs to be replaced by bovine transferrin, human blood donor derived transferrin or other iron sources. Due to the absence of animal-free alternatives, some cell lines were adapted to grow with the use of other iron sources. However, many cell types do not grow as well with iron compounds as they do with transferrin. Now with the availability of recombinant transferrin, there are more animal-free options available for use in media formulation.
Trypsin
Trypsin is a serine protease and its main function is to hydrolyse proteins. For cell culture, it is frequently purified from animal sources, primarily porcine or bovine. It is an important tool used in cell culture to suspend adherent cells either during harvest or moving to a new plate. Trypsin cleaves the proteins that bind the cells to the plate or substrate. These properties make trypsin a key ingredient in vaccine manufacturing, some E. coli based manufacturing (including recombinant insulin production) and for harvesting stem cells.
Any process that uses trypsin to detach cells can’t be called animal-free unless the trypsin is recombinant and animal-free itself. Some sources for recombinant trypsin include the following: Roche carries a recombinant trypsin, Life Technologies carries a trypsin-like protease product called TripLE, and Sigma carries a recombinant bovine trypsin, Trypzean.
Aprotinin
Aprotinin inhibits trypsin and other proteolytic enzymes and prevents protein degradation of cells. The use of aprotinin is an important step in the manufacturing of recombinant proteins produced in E. coli. During cell lysis, enzymes are needed to break the cell walls and release the protein produced in the cells. While these enzymes are necessary to release the recombinant protein from the cells, they can also denature the protein itself and lower overall yield. Aprotinin added at the right stage in the process can inhibit enzyme action and prevent degradation of the recombinant proteins.
Aprotinin is most commonly purified from bovine lung tissue and thus prevents animal-free manufacturing if it is used. There are a few recombinant aprotinin options available; Sigma, Millipore and Cell Sciences all offer a form of recombinant aprotinin.
Lysozyme
Lysozyme is an enzyme that lyses bacterial cell walls. It is an important component of recombinant protein manufacturing in E. coli, as it is often used to extract recombinant proteins or DNA during product harvest. It is preferred over mechanical processes such as sonication and homogenization when shear forces of these processes result in degradation or denaturing of the product. Compared with detergents, Lysozyme does not denature the desired product. Some scientists use Lysozyme in combination with mechanical or detergent-based lysis. In these cases, they reduce potential loss of the target protein by reducing the intensity of sonication or homogenization, or reduce the concentration of detergents used. Recombinant lysozyme is offered by Merck KgA’s EMD Millipore division.
Defined Supplements and Recombinant Advantages
While each protein discussed above provides its own unique function to cell culture, there are some advantages that the recombinant versions offer. When animal-free manufacturing is required, recombinant proteins can offer a viable alternative to animal sourced products. In addition, they can provide more consistency and can also be easier to source with fewer regulatory restrictions than their animal-derived counterparts. While recombinant versions can be more expensive at first glance, it is important to factor in the advantages. After weighing the benefits, recombinant proteins in manufacturing can be quite cost-effective.
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