Microbial systems
Despite the increase in protein drugs derived in non-microbial systems, microbial systems still remain convenient and powerful tools to produce recombinant proteins. However, their efficacy is evident only after countering intrinsic challenges such as:

• Lack of or unconventional Post-Translational Modifications (PTMs);
• Proteolytic instability;
• Poor solubility;
• Activation of cell stress responses.

Microbial set of cellular systems, Escherichia coli and Saccharomyces cerevisiae, still remain robust choices for protein drug production. At Inbiopro, we have demonstrated viable process and product optimization techniques to ensure maximized gains through these systems.

Escherichia coli

The enterobacterium E. coli spurred the historical development of microbial physiology and molecular genetics; it still remains the first-choice for the production of recombinant proteins. With a steady generation of information and molecular tools (such as engineered phages, plasmids, and gene expression cassettes) broadening its scope, it is widely used for primary cloning, genetic modification, and small-scale production for research.

However, recombinant proteins obtained in E. coli lack the PTMs present in most eukaryotic proteins. Glycosylation is the most common PTM but many others, such as disulfide bond formation, phosphorylation, and proteolytic processing may be essential for biological activity. PTMs play a crucial role in protein folding, processing, stability, final biological activity, tissue targeting, serum half-life and immunogenicity of the protein; therefore, a PTM deficient version might be insoluble, unstable, or inactive. Furthermore, through genetic engineering of the underlying DNA, the amino acid sequence of the protein can be changed to alter its ADME (absorption, distribution, metabolism, and excretion) properties. All these affect the quality of proteins produced and limit large-scale use in factories for producing recombinant pharmaceuticals.

Saccharomyces cerevisiae

Protein production in yeast is often undertaken when:

• The target protein is not produced in a soluble form in the prokaryotic system;
• A specific PTM essential for its biological activity cannot be produced artificially on the purified product.

Yeasts offer many advantages that bacterial systems do:

• Cost-effectiveness, faster processes, and technical feasibility;
• High-density cell cultures that can also be reached in bioreactors.

Moreover, mutant strains producing high amounts of heterologous protein are already available.

Though yeasts offer many PTMs such as O-linked glycosylation, phosphorylation, acetylation and acylation, the N-linked glycosylation patterns differ from higher eukaryotes. The sugar side chains of high mannose content affect the serum half-life and immunogenicity of the final product. The production of recombinant proteins also triggers conformational stress responses and produced proteins sometimes fail to reach their native conformation. With recent advances in technologies like Phybrid™ we can hope for more enabling processes and success in newer modified peptide molecules.

The challenges:

Many bacterially expressed proteins need to be folded correctly into their 3-dimensional structure as in that of humans. Many processes involve using toxic chemicals to solubilize and refold them. At Inbiopro, we are committed to using non-hazardous chemicals for protein refolding, and thus avoid or minimize the chances of side effects.

Aggregates and related impurities are formed during the course of folding, due to de-amidation and oxidations; we remove them with utmost care before formulating the product. Apart from that:

• Our resin library is continuously updated with valuable resins. We proactively use latest resins to benefit from better selectivity and resolution. This helps remove the contaminants to a greater extent.
• We follow a Quality by Design (QBD) based platform for designing process parameters and combining effective process and facility validations. We are committed to delivering batch-to-batch reproducible material quality and evaluate different formulations of the product to conform to international standards.