Strain Stability for Recombinant Protein Production

1.?????Why is it important to study strain stability for recombinant protein（rProtein）production?

Escherichia coli (E. coli) is the most used genetic engineering bacterium due to its mature gene cloning & expression system, rapid reproduction, and easily controllable metabolism. E. coli exhibits strong tolerance and applicability to the insertion of foreign genes, which is advantageous for gene manipulation and promotes the expression of target proteins.

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2.?????How to construct strains for rProtein production?

The production of human recombinant DNA protein biologics involves the preparation of engineered cells, fermentation or cell culture, target protein extraction & purification, and formulation. The construction process usually involves several steps, including: vector construction, transfection, pool screening, clone screening, the establishment of RCB (Research Cell Bank)/PCB (Primary Cell Bank)/MCB (Master Cell Bank)/WCB (Working Cell Bank) and strain stability studies. Quality control（QC）of raw materials used in cell culture is essential to ensure compliance with established quality standards for their intended use.

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3.?????What are the factors affecting strain stability?

The stability of plasmid DNA (pDNA) is important for achieving expected experimental results in RCB construction, which is the foundation for subsequent MCB and WCB construction. If the recombinant pDNA maintains its original structure and replication characteristics during the growth of the host strain, it is considered to be stable. pDNA stability is influenced by many factors, including host strain, copy number of plasmids, size of the target gene fragment, fermentation culture method, and induction expression method. The choice of culture medium for the recombinant strains also has a certain impact on plasmid genetic stability.

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4.?????What are the conditions for examining strain stability?

Passage stability: To ensure stable expression and prevent negative effects such as cell mutation or loss of expression ability, it is important to limit the number of passages or cell population doublings during the production of expression vector bacteria or cells. When determining the maximum number of passages allowed, consider the cell phenotype, genotype, molecular integrity, and expression consistency, as well as the host cells or vector's consistency at the end of the production process. The range of passages involved in these factors should meet or exceed the specified maximum limited passage number of the cells.

Storage stability: To reduce genetic variation in bacterial strains, a dormant state is induced by creating conditions that suppress metabolic activity and growth. Basic measures for preserving bacterial strains include using a nutrient-poor environment, low temperature, dryness, and vacuum sealing, based on the physiological and biochemical characteristics of the microorganisms.

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Yaohai Bio-Pharma is China's first and largest biologics CRDMO (Contract Research, Development and Manufacturing Organization) focusing on microbial expression systems. It provides customized end-to-end solutions from DNA design and synthesis, microbial strain engineering and construction to drug substance manufacturing in GMP or non-GMP level and products fill & finish across diversified modalities, such as recombinant proteins, peptides and polypeptides, enzymes, single-domain antibodies (sdAbs), plasmid DNA and mRNA, glyco-polymers, virus-like particles (VLPs), to meet global customers’ clinical and commercial needs in biological drugs, biosimilars, vaccines and diagnostics for human and veterinary use.

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