In a previous Cell Culture Dish blog, the history and importance of hybridoma technology was discussed. While there is no question that this technology led to incredible monoclonal antibody treatments and a real breakthrough in the advancement of medicine, it fell short when it came to manufacturing. Hybridoma cell lines are sufficient for producing small amounts of antibody, but when these drugs move from research to commercial applications, much larger amounts are required. This meant that a new host cell line would need to be chosen to take the drug into large-scale commercial manufacturing. When evaluating possible host cell lines for these drugs, several factors need to be considered. Primarily the technology has to work, i.e. it has to be able to correctly produce folded proteins that have the proper posttranslational modification. They also have to easily be genetically modified and have the capacity for high expression levels. Lastly the cell lines have to be safe and not susceptible to adventitious agents. Through years of using CHO cells in the lab for many types of studies, it was confirmed that they met many of these requirements.
CHO cells had been used for many years in laboratory work beginning in 1919 for typing pneumococci. Their use declined after domestication problems led to hereditary diseases due to imbreeding. Then in 1957, Dr. Theodore T. Puck from the University of Colorado’s Department of Medicine first established ovary cells in culture plates and found that they had good viability and grew rapidly. In light of this new research, CHO cells began being used in many types of lab studies, which ultimately led to their selection as a host cell line for recombinant proteins. According to Jayapal, Wlaschin, Yap, and Hu (2007) nearly 70% of all recombinant protein therapeutic productions is done in CHO and sales for biologics produced in CHO is over $30 billion worldwide.
The first product produced in CHO cells, approved in 1987, was a recombinant therapeutic protein manufactured by Genentech called Activase. Activase is a tissue plasminogen activator for treatment of acute myocardial infarction. Early biopharmaceutical manufacturing using CHO cells was much different than production today. The first manufacturing runs were conducted as batches and ran for 7 days with yields around 100 mg/liter and the media in which the cells grew contained animal products, such as fetal bovine or fetal calf serum. Then bioreactors began to be used and run times moved to 10-14 days with media and nutrients being replenished during the run. Now commercial biopharmaceutical manufacturing utilizes bioreactors 10,000 – 25,000 liters and larger in size. Media and nutrients for the cells are monitored continuously as well as oxygen levels, CO2 levels, and waste product. This constant information about cell health has led to more efficient and productive culture with average production yields from 1-6 grams/liter.
The manufacturing process has undergone continual improvements since commercial production began. In addition to the equipment used, cell line development and media formulation has become critical to successful, high yield manufacturing. Current biomanufacturing in CHO cells includes cell line development and screening to select the CHO cell line, which has a combination of the highest cell viability, growth, expression level, and product quality. Once the best cell line is selected, the medium in which the CHO cells are grown is optimized through testing of various media, supplements, and other ingredients. Finally, the entire process is scaled up for large-scale bioreactor production.
In addition to the above culture improvements, culture media has also been made safer. Early products produced in CHO cells were produced in media that almost always contained animal products. which are undesirable because they are undefined and and yield unpredictable result due to high batch-to-batch variation. The Food and Drug Administration (FDA) and other regulatory bodies discourage use of these animal components because of the serious safety concerns involving the risk of viral and prion (the infectious causative agent associated with Mad Cow Disease) contamination. As a response to these concerns, in the early 1990’s there was a great deal of research being conducted around how to remove serum and other animal products from CHO manufacturing, but yield often suffered. Scientists began to look for animal-free supplements that could be used to improve yield. Some supplements existed, such as hydrolysates, but often these supplements were undefined and exhibited lot-to-lot variability making manufacturing more challenging. The latest breakthrough has been cell culture that is completely animal-free and defined, which has paved the way for a new class of recombinant supplements that meet both criteria.
For further reading see:
- Jayapal K. P., Wlaschin K. F., Yap M. G. S., & Hu W-S. (2007). Recombinant protein therapeutics from CHO cells — 20 years and counting. Chem. Eng. Prog., 103(7), 40–47.