About 60−70% of all recombinant protein pharmaceuticals are produced in mammalian cells like CHO (Chinese Hamster Ovary cells), NS0 (mouse myeloma), BHK (Baby Hamster Kidney). We currently work in the following areas related to cell culture bioprocess engineering
Glycosylation modulation
Glycans attached to the protein are synthesized by a series of sequential steps within the ER and Golgi. Unlike DNA and protein sequences which are hard-coded in the genome, glycans attached to the protein are heterogeneous resulting in a complex glycan profile on the protein comprising different glycan molecules in varying proportions. The glycan profile of recombinant proteins expressed in animal cells depends on the clone and process conditions, and can affect the activity of the therapeutic protein e.g. increased high mannose glycoforms on the protein can result in faster clearance. Biosimilar protein therapeutics present a unique scientific challenge in the need to replicate the innovator drugs glycan profile. Our group is working on understanding factors that affect glycosylation which can then make available design parameters to engineer desired glycan profiles on a recombinant glycoprotein.
Novel platforms for mimicking fed batch mode with pH control in small scale culture platforms like shake flasks
During cell line, process and/or media development, several clones/conditions are tested (typically in shake flasks in batch mode) for their growth and productivity characteristics and the best is taken forward for testing under production conditions. Production processes are rarely carried out under batch mode. This could hence lead to suboptimal selection during the initial stages.
We are developing slow release polymer hydrogel systems for nutrient feeding and pH management in mammalian cell cultures in order to mimic fed batch conditions in shake flasks. This will enable initial screening under conditions which are more representative of production conditions.
Similar hydrogel platforms are also being developed for microbial cultures.
Transient expression of recombinant proteins
When small amounts of glycoproteins are required rapidly, as is the case for pre-clinical evaluation of drug candidates, the protein can be expressed transiently in a few days. Furthermore, for some therapeutic proteins a small quantity may be sufficient to meet the desired needs. This is likely to be the requirement if the concept of individualized medicine becomes a reality. Transient expression involves transfecting the plasmid into the host cells and culturing the transfected cells expressing the recombinant protein for a few days until the plasmid is lost. As is obvious, the process involves a new transfection for every batch of protein made. This can cause variability in protein quantity and quality due to changes in parameters like the state of cells at transfection and transfection efficiency. The reproducibility (or lack thereof) of the transiently expressed product has not yet been comprehensively evaluated.
If consistency in protein quality and quantity is achieved and demonstrated, it is envisaged that in addition to rapid production of small amounts of therapeutic proteins, transient expression can also provide a platform to provide larger amount of proteins rapidly: for example for rapid production of vaccine antigen in response to a pandemic, while longer term solutions are established . For this to be economical, plasmid requirements need to be significantly reduced. Strategies to achieve this reduction are being explored.
Cell engineering for more efficient metabolism
We are using evolution-based strategies to better understand metabolic plasticity in animal cells which can then be used to design cells with more efficient metabolism.
For instance, we have reported that adaptation of CHO cells to limiting inorganic phosphate concentrations over a period of more than 2 months results in selection of a population also showed better growth characteristics compared to control in batch culture replete with Pi (higher peak density and integral viable cell density), accompanied by a lower specific oxygen uptake rate and cytochrome oxidase activity towards the end of exponential phase. Surprisingly, the adapted cells were also able to survive better upon glutamine withdrawal while growing to a lower peak density under glucose limitation. This suggests long term Pi limitation may lead to selection for an altered metabolism with higher dependence on glucose availability for biomass assimilation compared to control. Metabolomics analysis of U-13C glucose fed cultures indicate that adapted cells have a higher pyruvate carboxylase flux.