Defined Supplements – The Role of Recombinant Proteins in Biopharmaceutical Manufacturing

By on August 28, 2012

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, animal-free manufacturing for all production cell lines. Animal-components in manufacturing are problematic due to the inherent inconsistency of products derived from animals. Primary challenges include sourcing difficulties, high batch-to-batch variation, and safety concerns involving infectious agents. 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 the six most commonly used proteins, their role in manufacturing, and a table of suppliers that offer these products in recombinant form. I hope this is helpful to anyone looking to either boost existing serum-free media or formulate an animal-free production platform.

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. Most media contains recombinant insulin, but there is a difference between recombinant insulin and animal-free recombinant insulin. Some versions of recombinant insulin still use animal-derived enzymes in the manufacturing process, are manufactured in facilities where animal-products are used, or use serum during banking. A common use of animal derived trypsin is in the manufacture of recombinant insulin, which makes the recombinant insulin a potential source of contamination. Therefore, truly animal-free recombinant insulin uses no animal-derived products in manufacturing (such as trypsin) and ensures an animal-free manufacturing facility. There are now sources of animal-free trypsin from Roche and others, which can replace animal-derived trypsin (see below).

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 cell lines where serum is still being used or where blood proteins are added. The use of recombinant albumin can help transition cell lines from serum to serum-free and it can also serve as an animal-free replacement for either pHSA or BSA in culture.

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. However, bovine transferrin is up to 100 fold less potent than human transferrin for the culture of murine and human cells. The use of bovine transferrin requires higher levels of transferrin, which increases the cost of serum-free medium. Due to the absence of animal-free alternatives, some cell lines were adapted to grow with the use of iron chelates. However, many cell types do not grow as well with iron-chelate compounds as they do with transferrin. In the past, the past the lack of available animal-free transferrin, prevented some cell types from reaching full growth potential in serum-free media.

With the increasing availability of recombinant transferrin, productive animal-free cell culture media is possible for more cell lines, especially when combined with other recombinant proteins like insulin and albumin. In addition there is evidence to suggest that CHO cells could benefit from the use of transferrin over other iron sources. Please see our blog “Transferrin – Nature’s Solution for Iron Replacement in Serum Free Culture,” for more information about the advantages of transferrin in cell culture.

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. The challenge is that there aren’t many available sources for recombinant trypsin at the moment. Roche carries a recombinant trypsin. Life Technologies carries a trypsin-like protease product called TripLE, manufactured by Novozymes. Sigma carries a recombinant bovine trypsin, Trypzean, manufactured in corn and available through the Sigma product website.

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, please see below.

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.

The first use of Lysozyme relied on Hen Egg White (HEW), or Chicken lysozyme. However, it is notoriously variable in its activity ranging from less than 10,000 activity units per mg to 50,000 units per mg. Recently, Recombinant Lysozyme was developed that is 5 times more active than HEW/Chicken lysozyme and completely animal component free. This product boasts consistency and high activity, so it is quickly being adopted by the industry. It is offered by Merck KgA’s EMD Millipore division. The link to this product is provided in the table below.

Defined Supplements and Recombinant Advantages

While each protein discussed above provides its own unique function to cell culture, there are some universal advantages that the recombinant versions offer. Recombinant proteins deliver a better safety profile and truly animal-free manufacturing. They are also more consistent and easier to source with fewer regulatory restrictions when compared with their animal-derived counterparts. In addition recombinant proteins are purer than animal derived proteins and sometimes require less product to do the same job. While recombinant versions can be more expensive at first glance, it is important to factor in the considerable advantages. After weighing the benefits, recombinant proteins in manufacturing are quite cost-effective.

Recombinant Proteins
Product Company Animal-free or Animal Products used in manufacture
Recombinant Insulin Life Technologies AOF recombinant insulin (manufactured by Biocon) Animal-free
Sheffield rInsulin ACF Animal-free
Sigma recombinant insulin Animal Product Used
FEF Chemicals recombinant insulin (manufactured by Novo Nordisk)
Animal-free Grade Animal-free
Standard Grade Animal Product Used
Recombinant Albumin InVitria Cellastim Animal-free
Sigma recombinant albumin Animal-free
Fisher Scientific recombinant albumin Animal-free
Sheffiled Bioscience rAlbumin ACF Animal-free
Mediatech cellgro rhAlbumin Animal-free
Recombinant Transferrin InVitria Optiferrin Animal-free
Sigma recombinant transferrin Animal-free
Fisher Scientific recombinant transferrin Animal-free
Recombinant Trypsin Roche Recombinant Trypsin Animal-free
Sigma Trypzean Animal-free
Life Technologies TripLE (Trypsin Replacement) Animal-free
Novozymes rTrypsin (Trypsin Replacement) Animal-free
Recombinant Aprotinin Sigma recombinant bovine aprotinin Animal-free
Millipore recombinant bovine aprotinin Animal-free
Cell Sciences recombinant bovine aprotinin Animal-free
Recombinant Lysozyme Millipore CellPrime recombinant human lysozyme Animal-free