Principles Of Biopharmaceutical Engineering: Using A CHO Cell Line In Suspension Culture To Produce Therapeutic Protein
CHO cells are the most profoundly used to produce Recombinant therapeutic proteins and monoclonal antibodies in the Biopharmaceutical Industry since a long time now. CHO cells provide many advantages to have earned that kind of prominence in the market,It acts as the perfect ‘host’ for the proteins because; performing genetic engineering and incorporating genes that encode our protein of interest is convenient to carry out in the CHO cell machinery.
CHO cells provide the post-translation modifications and produce glycoproteins that are required to makes the protein of interest compatible and bioactive in humans. CHO cell line shows remarkable adaptability to various kinds of culture conditions, gets easily habituated to grow in serum free media which also makes them the most preferred cell line to produce recombinant protein. Since it has been two decade since the CHO cell lines have been established as host cells successfully, validation of a new plant requiring CHO cells cultivation probably is easier to obtain from regulators like the FDA. CHO cells have gene amplification systems like DHFR mediated or glutamine synthetase mediated that overcomes the problem of low specific productivity. Thus using CHO lines for the production of therapeutic protein implies to be a secured decision.
With the advancement on the cell culture technology, the production ability has risen way higher than what we were getting years ago. According to a study we can produce 10g/l of therapeutic protein from CHO cells. This was achieved because of progression in the optimization of the culturing technique, media development and high yielding stable producer cells, improvement in the culture to produce higher titers lowers our requirements of huge equipment and manufacturing cost also meets the market demand faster.
Upstream Production
In this report we are discussing about what Bioreactor technology is better to choose for the upstream production of therapeutic protein from CHO cells. Upstream is the first process where biomolecules are grown. The upstream process starts when Cells are taken out of the working banks and thawed. These are cells that prepared after transfecting them with the gene of our interest. Cells are made into working banks, Propagation of working bank cells and generating starter cultures, culture is subcultured until it reaches a volume enough to inoculate a seed reactor.
From the seed reactor, the harvest is transferred to bigger production scale bioreactor (batch, fed-batch) CHO cells are majorly grown in the fed batch cell culture process because it maintains high volumetric productivity and with low operational complexity.
The basic requirements of cells while upstreaming is to create an environment in the bioreactor with proper growth media, oxygen supply, chilled water supply to maintain an optimum temperature, CO2 supply, sparger is used to maintain CO2 in the system and remove the extra build up of the air. Impeller is required in the vessel to break the bubbles and increase the mass transferred from liquid to gas and maintains the pH as the rate of transfer affects the CO2 in the media.
Harvest from this is transferred to downstream for further processing. Sterility is the most basic need to be maintained while working with Biopharmaceuticals at every step but in upstream, contamination specially from other other cells is to be taken care of.
Decision
So for the production facility to be designed to manufacture 1000 kg of therapeutic protein by using CHO in the suspension; we assume our titer to reach 10g/L, thus, a 100KL of suspension is required to for fermentation.
To produce 100KL liter of culture we can choose to either go for the traditional hard-piped stainless steel bioreactors or we could use single use bioreactors. I would suggest using single use bioreactor of 2000L size as they are available in the market upto that size currently. Running 25 batches in two single use bags simultaneously for an year will give me 100KL of culture, (2*2000*25) 100KL of cell culture will be sufficient to provide 1000 kg of yield assuming the titer is 10g/L.
Well it depends on the product and time that we have to manufacture the product. If in case our required therapeutic protein is a biosimilar and has to reach market before the competition rises, it might be a good idea to install two single use bioreactors simultaneously.
Discussion
To justify my choice I would like to discuss about the factors that make single use more convenient and suitable for our requirements.
The pharmaceutical industry has incorporated the use of disposable plastic material since 1980’s with disposable bags and filters, even while cell culturing or in the initial phases of the Biopharmaceutical production, use of disposable plastic vials or T flasks has been considered safe and a good practice since it avoids cross contamination and its sterility is trusted. The use of disposables may sound like new trend but is being used in the industry since last 30 years, started with small scale filter capsules to large volume parenterals for serum and culture media as well as buffers and by mid 1990’s we had large scale single use processing bags. By 2000s there were stirred tank-liner bioreactors and disposable rocking bag bioreactors. Today the single use technology include material from depth filters, sterile disconnectors and single use tangential flows filtration systems and membrane chromatography units etc etc. the single use market has grown and has created products suitable for all scales and applications, from upstream to fill finish. (
Single use bags for upstream production of therapeutic proteins are manufactured in a wide range to replace stainless steel and glass vessels. The bag systems are pre assembled, sterile and pyrogen free. The many benefits of using single use bioreactors start from:-Capital Investment: When comparing the capital investment, it is not rocket science to figure out that plastic is cheaper than stainless steel but there are other factors that reduce the capital investment when using single use. Sterility and assembly if the single use bags are outsourced as they are delivered in those conditions. The pipelines of water and steam required for steam in place (SIP) and clean in place (CIP) are not required in single use and the designs are much smaller so it saves on area and cost, reduced requirements for huge media and buffer holding vessels, facility’s layout and automation strategy changes if we decide to use single use systems. Clean room requirements, floor to floor heights, HVAC designs. The contained areas will no longer be required as we use the tube fusing systems and aseptic connections can be made from bag to bag and there is no need of making them under LAF cabinets. Thus, building requirement reduces and also plant can be made multiple product plant that will increase the manufacturing capacity that will ultimately improve the revenues. Because of less construction requirements, the time of construction of the facility also reduces.
Maintenance cost
Labour: a study by on cost of goods (CoG ) analysis by Rentschler shows 63% less cost in case of using single use bioreactors (rogge) while using single use, there are less things to do manually in the process thus the cost if labour is reduced. Quality checks and (QA/QC/RA) costs are also cut down. Maintenance engineers are also less often required and need not be hired but called only in need.
Utilities: Consumables for sure are increased in case of using the disposable bioreactors but when thinking about the utilities.
Power, water, purified air. So much of energy used in purifying water for cleaning and making water for injection is saved that eventually saves money. Less of CIP media/chemicals and fewer spare parts include in the cost reduction. Since there is less complex equipment involved, there is also lower effort of preventive maintenance. Process cost: one of the highest cost and time consuming work in the bio manufacturing is transferring of sterile material i. e liquids, gases and reagents from one part to another through pipes that also require CIP, SIP and validation and adds so much more to the cost. Turn over time: on the same lines of cost, turn over time makes a lot of difference. Turn over time is time required to prepare the equipment after a batch for another one. Because of high levels of cleaning, sterilizing and revalidating required in the stainless steel bioreactors, and then to perform quality checks on them. Whereas there are no CIP of unit operation systems in single use. It indirectly saves money for the company because more batches and more product can be produced in less time. Also as mentioned earlier when making biosimilars or a product for which the market is competitive, losing on time can really make a difference to your revenues.
Safety and validation requirements: Since closed systems are easier to generate in case of single use bioreactors, the risk of contamination is less as there is no need for an air vent filter. And the key feature for a disposable bioreactor is that it is a pre sterile cultivation container that is formed by FDA approved plastics and is only made for single use, thus it reduces chances of bio burden.
Validation requirements: IQ/OQ/PQ time required for single use facilities in their commissioning phase of a new facility is less compared to the time required for set up of traditional equipment.
Cross contamination: facilities can be multi product as there is reduced risk of cross contamination. Since the last few years trend shows that validation failures were mostly dues to risk if cross contamination so single use disposable bags save from that hustle and chances of loss.
Future trends and flexibility of single use
With all the growing need of requirements of highly productive cell lines, we ll be able to get more titer, that will in turn require less amount of cell culture and smaller equipment. A fixed at place, highly complicated. Expensive stainless steel bioreactor doesn’t provide anything when it is not in use. In case of single use bioreactors; there is flexibility and move to smaller units whenever required. With the coming up concept of personalized medicines where T cells are taken from an individual and after modifications in the genome are made to grow in bioreactors. In such cases, huge investment done on the steel bioreactors will be all wasted. Thus, if I am part of a new building facility, I would suggest a technology that can be competent to future trends in the industry.
