In Part II of our series on “Strategies for Improving Antibody Production in CHO Cells,” we will look at ways to supplement media formulations to improve cell growth, viability and productivity. Earlier this year, I wrote a blog titled “CHO Cells – the Top Expression System for Best Selling Biologic Drugs.” The blog described how CHO cells are the expression system for four out of five of the best selling biologic drugs and as such there is continual effort being made to improve CHO cell biomanufacturing. One area that has undergone many changes in an effort to improve antibody yield is cell culture media.
Cell culture media development and optimization is a complex process. Nearly every CHO cell line that goes on to biomanufacturing will undergo several process development runs aimed at identifying the optimal combination of media ingredients for best results. Often included in the optimization are experiments for culture feeding strategies; metabolic studies to see how the cells are using media nutrients; and an examination of bioreactor conditions to find the perfect conditions for cells. Each of these optimization tools are an important step optimizing cell growth parameters for maximum protein expression for manufacturing. Media supplements are also a very important tool in the optimization process and should be tested as a key media ingredient. For the purpose of this discussion I am going to focus on media supplements, designed to provide a boost to productivity, are quick and easy to use, and are compatible with most media formulations. They can also be added after optimization to increase existing yield and lastly media supplements can give an important boost to yield when you do not have the time or resources to undergo full optimization.
In addition, for this blog I will only focus on animal-free supplements. While early products produced in CHO cells were produced in media that almost always contained animal products, they are undesirable because of the concerns of possible infectious agents. In addition many animal sourced products such as serum are undefined and yield unpredictable results. As a response to concerns from the Food and Drug Administration (FDA), European Medicines Agency (EMA) and other regulatory bodies that discourage the use of animal components there has been a great deal of research conducted around how to remove serum and other animal products from CHO manufacturing. As a result serum-free media has now become the norm. However yield can still suffer along with cell viability and growth now that serum has been removed. The benefits of the supplements discussed below are their ability to maintain important animal-free status while providing some of the key benefits of serum.
One of the newer and most successful categories of supplements are recombinant proteins. The advantages of recombinant proteins are that they are cost effective, consistent, defined and animal-free. While recombinant insulin has long been used in CHO manufacturing, now other recombinant proteins are also available. Two of these proteins – recombinant albumin and recombinant transferrin are key proteins found in serum. Albumin, provides many benefits such as effective energy for cell proliferation as a result of improved lipid and nutrient delivery as well as shear protection, cell membrane stabilization, and removal of toxic substances that may be produced during the biomanufacturing process. Albumin has been shown in studies to increase cell growth and viability as well as antibody productivity. Transferrin is a universal iron carrier designed to deliver the appropriate amount of iron to cells. Due to transferrin receptors on the cells that can up regulate or down regulate based on their iron need, transferrin is 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. Today companies including Sigma, Fisher Scientific, InVitria (CHO Cell Culture), Sheffield Bioscience and Mediatech sell recombinant albumin,and transferrin that can be added to animal-free media and has been shown to improve cell growth, viability and yield of CHO cells while remaining animal-free and defined. In addition, InVitria also manufactures a media supplement designed just for CHO cells called Zap-CHO. Data on their website shows significant improvement when Zap-CHO is added to chemically defined media. Recombinant animal-free insulin is available through Life Technologies and InVitria has animal-free ITSE that contains recombinant insulin, transferrin, selenium and ethanolamine.
In addition to recombinant proteins, plant based hydrolysates are an animal-free supplement that can be added to most media to improve productivity. BD Biosciences has a number of yeast based and soy based hydrolysates for CHO cell culture. Sheffield Bioscience also has yeast based options for CHO cells. Some have been critical of hydrolysates in the past due to product inconsistency and lot-to-lot variation. Interestingly, Sheffield Bioscience has been able to demonstrate that their hydrolysate performance is improved when paired with recombinant albumin (see poster here) that they sell under the name rAlbumin ACF. Unlike recombinant proteins, hydrolysates are not defined, however they are cost effective and lead to improved performance, making up for what may lead to inconsistent manufacturing from batch to batch. Sigma Aldrich offers an purified animal-free hydrolysate called EX-CELL CD Hydrolysate Fusion. BD also produces a purified hydrolysate called BD Recharge. In both of these products they are more defined than traditional approaches to hydrolysates and can be used with recombinant albumin and transferrin to optimize performance.
These are just some examples of quick, easy, and flexible media supplements that are being used to improve CHO production. Has anyone used any of these methods for improving their CHO production? Does anyone have any others to recommend?
|Supplement & Cost||Product||Recombinant||Hydrolysate||Enhanced Growth||Enhanced Productivity||Defined|
|Recombinant Albumin |
Cost per Liter of Media$1-6 per liter
|Sigma recombinant albumin|
|Fisher Scientific rAlbumin|
|Sheffiled Bioscience rAlbumin|
|Mediatech cellgro rhAlbumin|
|Recombinant Transferrin |
Cost per Liter of Media$0.25-$1 per liter
|Sigma recombinant transferrin|
|Fisher Scientific rTransferrin|
|Recombinant Insulin |
Cost per Liter of Media$2 per liter
|Life Technologies AOF Insulin||Yes||No||High||High||Yes|
|InVitria’s Animal-free ITSE|
|Plant-Based Hydrolysates |
Cost per Liter of Media$1-4 per liter
|Sheffield Bioscience Catalog List||No||Yes||Depends on the hydrolysate used, must be screened to find best performance||Depends on the hydrolysate used, must be screened to find best performance||No|
|BD Bioscience Catalog List|
|Purified Plant-Based Hydrolysates |
Cost per Liter of Media$3-10 per liter