Development of efficient bioprocesses for the PEGylation of therapeutic proteins

6th Call
Developed by Pramod Wangikar
Department: Chemical Engineering

Problem statement: One of the common problems faced by cancer patients undergoing chemotherapy is
febrile neutropenia. Neutropenia refers to a condition where the patient has too few neutrophils, a type of
white blood cell. As a result, the patient becomes very prone to infections and has frequent fever
occurrences (febrile neutropenia). Recombinant protein therapeutics are one of the ways of treating this
condition. Granulocyte colony-stimulating factor (G-CSF, filgrastim) is a blood growth factor used in this
treatment. This growth factor helps the bone marrow of the patient to produce more neutrophils. G-CSF is
administered in a PEGylated form, adjunct to standard-dose chemotherapy. The PEGylation process
significantly improves the pharmacokinetic properties i.e. the movement of the drug within the body and
retention of G-CSF without compromising its performance. Our industrial partner, Epygen Biotech (Navi
Mumbai) has developed a production process for G-CSF. However, it is required to optimize and scale up the
PEGylation and purification process in order to improve the protein yield and reduce the cost of a final
therapeutic product. The proposed project is therefore aimed at bioprocess optimization for the PEGylation
of recombinant GCSF.

Project details: Recombinant protein therapeutics are commonly used and often require conjugations
to improve stability. This in turn reduces the yield and eventually increases the cost of production. This
project aims at bioprocess optimization for one of such proteins, G-CSF, and the PEGylation of recombinant
G-CSF. Our aim is to construct a recombinant E. coli producing higher G-CSF. The crucial step would be to
develop a bioprocess, that yields more recombinant proteins which would require reactor studies
as well as process modeling. After purifying recombinant GCSF, site-specific PEGylation reaction conditions
and catalyst optimization to achieve maximum conjugation in a shorter time duration will also be done. The
conjugated protein will be purified and checked for quality with other commercially available market players.
Activity assays and pharmacokinetic profiles will also be part of the processing. The major steps will include
genetic Engineering of E. coli for GCSF production, large-scale production of G-CSF, standardizing PEGylation
reaction and scaling up, in vitro assays, and pharmacokinetics with available commercial therapeutic protein.
We have already developed a process for high cell density of recombinant E.coli using a fed-batch by following
cues obtained by process modeling. We have already filed a patent for the same. Further, we have optimized
the process for recombinant G-CSF-producing bacterial cell growth in bioreactors for obtaining higher protein

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