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CHO Cell Culture – Ten Tips from the Experts on Expression, Media, and Bioprocessing
It has been a year since Cell Culture Dish launched our Ask the Expert feature and in that time we have amassed a great deal of valuable information on a number of important topics. For today, I have compiled a list of ten tips on CHO cell culture from Experts through our Ask the Expert sessions and guest blogs and provided links to the source material. I hope you find these tips useful in your culture.
Successful gene cloning and clone selection are key elements of creating and selecting a CHO cell line for biomanufacturing. Traditional cloning methods involve many steps from sequence to clone selection and offer little control over the final gene. Since the process is imprecise, often the resulting gene is of poor quality, has mutations, or produces no gene at all.
Today’s de novo gene synthesis technologies offer easy, fast, and affordable access to nearly any desired gene sequence. A viable alternative to cumbersome cloning and mutagenesis steps carried out in individual labs, gene synthesis offers researchers maximum design flexibility and makes it possible to receive a 100% correct clone in around 5 business days. In addition, this technology makes every sequence available to scientists and offers the ability to utilize the redundancy of the genetic code to optimize protein coding genes and open reading frames of choice for maximum protein expression. Gene optimization and synthesis services offer an attractive solution for cloning needs, thus enabling researchers to simplify their workflow, free internal lab resources for value-added work, and to gain speed in research and development by increasing the success rate based of gene expression projects.
When evaluating the success of a gene synthesis program, expression must be the key parameter for success. Quality of the algorithm must be tested and verified by confirming that the optimized gene candidates were able to express and that expression levels were increased when compared with their natural counterparts. In addition, gene optimization is not only a tool to make expression success rates more predictable and improve expression yields in general, but also optimized genes represent a powerful tool in functional genomics as the sequence-modified counterpart can be used for the rescue of an siRNA-mediated knockdown.
Cell Culture Media
There are several ways of utilizing cell metabolism characteristics for medium optimization. Firstly, the metabolism rate of key medium components such as amino acids and vitamins can easily be determined by spent media analysis. The metabolic profiles can then be used for design of stoichiometrically balanced medium formulations. Since metabolic characteristics are dynamic, utilization of metabolism rates measured from both the cell growth phase and stationary production phase are best for stoichiometric balancing of basal and feed media. Secondly, cellular metabolism is an interrelated network and metabolism characteristics can guide the focus of medium optimization. One example is within the glycolysis pathway and TCA cycle. For a cell line that tends towards lactate production, medium optimization can address this issue by switching the metabolic flux towards the TCA cycle. Finally, cell metabolism characteristics can also impact the balance between biomass formation and protein production. For a cell line with high growth rate but low productivity, limiting the metabolism rates through medium optimization can force the cells work on protein production thereby improving product yield.
The concentration of glutamine and/or asparagine in your cell culture media should be assessed and limited since both amino acids can be deaminated leading to ammonia as the byproduct. During late stationary phase of mammalian cell culture, cells may utilize alanine to generate pyruvate and this process also produces ammonia. Supplementation of pyruvate into the culture is a possible solution to alleviating ammonia accumulation. Additionally, the issue can potentially be addressed from a process perspective by implementing a temperature shift scheme or adjusting the harvest time.
Nutrient depletion causes cell apoptosis which can impact productivity levels and/or product quality therefore it is important to supplement media with depleted components. In order to achieve the best performance, a metabolically balanced feed formulation and an optimal feeding schedule should be designed and applied.
There are approaches to improve media solubility, such as alternating pH (either acidic or basic) and temperature (mostly warm),that can accelerate/facilitate solubilizing media compounds. However, they don’t necessarily improve solubility (maximum concentration in a saturated solution) issues. Therefore, it is important to also evaluate the composition of your media formulation to assess whether there are components that are at the limit of solubility. Additionally, chelation can be considered for stabilizing the solution of a difficult component. One can also consider exchanging a less soluble component for a biologically equivalent compound with higher solubility to overcome solubility limitations.
The reason 1X liquid media from a manufacturer is often better then medium made in your lab from a dry powder media (DPM) is the difference in the quality of water. The better commercial media companies monitor endotoxin and heavy metal contaminants in their water and have processes in place to control them. Water grows bacteria rapidly and unless you control growth you will have endotoxin produced by the gram-negative organisms. Many systems have a bacterial filter at the end of the line and the pressure of the water passing through will rupture the gram-negative bacteria and release endotoxin. It is a good practice to change this filter once a month. It is also true that the use of ceramic grinding stones produces a lot of heat. This is why the most heat-labile components are added last during a DPM run, so that they are exposed to the heat the least amount of time. Like with a liquid medium, the timing of addition of the media components is a science in itself. If made correctly and you have high quality water, DPM should be fine as the components in a standard formulation are usually there in excess. There are ways to make a DPM which allows for the fine powder to be sprayed through an orifice so rapidly as to result in the components seeing minimal heat (jet milling). Some manufacturers have switched to this method while others still use ball milling or use one or the other dependent on the size of the run.
You need to be aware that different nutrients are used at different rates and what is overly utilized is cell specific. With CHO cells glutamic acid, aspartic acid and cystene are self limiting and need to be part of a perfusion medium. Glutamine or glutamine dipeptide needs to be added at about 0.5 mM in the medium and with CHO cells the glucose concentration should be monitored and adjusted daily to 1.0 G/L.
Deciding which strategy to follow for media development is complex and also very important. Cell culture media is an important factor in determining product yield, product quality, and cost of manufacturing. Careful analysis of company goals, timelines, budgets, and competencies are necessary in selecting the right strategy.
Key Factors to Consider
- Stage of product development
- Media Formulation Complexity
Media Development Options
- Off the shelf media
- Partner to develop a custom media formulation
- Develop media in-house
- Hybrid – develop media in-house, but employ a media partner to optimize for specific cell lines
One of the first steps is to establish a reliable scale-down model in the bench-top reactors. We have used the XDR-10 in the scale-down mode to design and predict what we will see in the large scale systems. The control methods and hardware are critical. Special attention should be given to the dissolved gasses, both pO2 and pCO2, as these parameters will affect the amount of base required as well. You should examine a sparge porosity you are likely use at large scale. This way you can get a relative understanding of the volume to volume gas requirements. This will also provide process information on the amount of foam that may be generated. Further, this will give an early look at the potential impact of interfacial shear resulting from the sparging. The agitation rate (rpm) should also be closely considered. Often times stress on the cells/culture can alter metabolite profile.
One of the key components for single-use system manufacturing is the system’s ability to control the critical process parameters such as dissolved oxygen (D.O.), pH, temperature and agitation within your desired target range. Then, what are the appropriate or optimal control strategies that will achieve these critical process parameters. Dissolved oxygen for example: select a gas sparge element with a porosity that will provide an adequate kLa, without generating an excess amount of foam or negative levels of interfacial shear. This can be achieved through an appropriate sparge pore size, along with appropriate agitation rate (rpm). Additionally, a controller, with the capability for gas cascading (i.e. air/oxygen blending), look-up tables and PID tuning can benefit this process greatly.