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Recombinant Protein

Growth Factor, Insulin, etc

Recombinant protein therapies – including major biologics like monoclonal antibodies – are produced by inserting target DNA into bacterial or mammalian host cells, expressing the encoded proteins, and purifying them from cellular systems. Widely used as host, Escherichia coli (E. coli) plays a key role in recombinant protein production. There have been various approved therapeutic proteins produced in E. coli, contributing to the low cost. Despite these advantages, the production process is complex and requires strict control over key indicators such as host residuals and process impurities. BRC Bio offers quality testing for E. coli and plasmid products, meeting the requirement of gene therapy, gene-modified cell therapy, vaccine, nuclear acid drug etc.

Biosafety Strategy for Cell and Gene Therapy

Recombinant Protein

Genetic Stability Studies

The genetic stability and clonality of CHO producer cell lines are closely related to the quality consistency of monoclonal antibody products. BRC Biotech offers comprehensive solutions for cell line development (CLD), clonality, and genetic stability of producer cell line, ensuring stable and consistent product quality.

Regulatory for Genetic Stability Testing

Cell substrate used in biologics production should be evaluated the genetic stability in both master cell bank (MCB) and end of production cell (EOPC) as described in regulatory guidelines, monitoring its genetic locus, coding sequence of the expression cassette or the RNA transcript, the copy number of the transgene. As well, restriction enzyme mapping is suggested to demonstrate integrity and integration pattern of transgene.

Refer to ICHO5D

The genetic stability is recommended in WHO by comparing the profiles in both MCB/WCB and ECB/EOPC. The aspects such as the copy number of GOI (gene of interest), the location of GOI, and the sequences using sanger sequencing should be included.

Refer to WHO TRS 978 / Annex3

Traditional Genetic Stability Testing Methods

Study Item	BRC Method
Purity	GOI and Flanking Sequencing RT-qPCR
Genomic Structure	Restriction enzyme analysis by Southern Blot
Insert Gene Copy Number	qPCR
Transcript Size	Northern blot
Cell Line Identity	CO1
Karyotype	Karyology

GMP-compliant testing system

Full-validated methodology

Completed project management

Meet the quality requirements of biopharmaceuticals

Comply with the international regulations and guidance

Professional regulatory technical support

Genetic Profiling Analysis based on Next Generation Sequencing Technology (NGS)

Recombinant-constructed Chinese hamster ovary cells (CHO) are widely used in the recombinant proteins and therapeutic antibodies production. The construction techniques might lead to off-target effects potentially, However, almost all construction techniques can potentially lead to off-target effects, multiple integration sites, and structural variations in the backbone sequence.

According to ICH 05B and WHO guidelines, it is required to compare the genetic stability between master cell banks (MCBs) and end-of-production cell lines (EOPCs), including the accuracy of coding DNA sequences, insertion site structure stability, and insertion copy number, etc.

Compared with traditional Southern Blotting, FISH and amplicon sequencing, TLA (Targeted Locus Amplification) next generation sequencing technology will bring a new solution for genetic characterization analysis.

BRC is the unique authorized domestic partner of Cergentis 1. Cergentis proprietary TLA-based assays offer clear advantages over standard approaches, most of which are unable to resolve all essential genetic characteristics in one experiment, e.g. as integration mutagenesis or sequence information. Briefly, genomic DNA is crosslinked, fragmented and circular DNA fragments are generated. The locus of interest is amplified and sequenced with NGS technology, and the sequence data are subsequently analyzed.

1： Cergentis is now BRC biotech is Cergentis is Now Solvias NL

Characterization		TLA+NGS	WGS	Amplicon/Capture based	qPCR/ddPCR	Southern Blot	FISH
Integration site		✓	—	—	×	x	✓
Structural variation		✓	×	—	×	×	×
Convergent integration		✓	×	×	×	×	×
Integrate a vector or GOl	SNVs	✓	—	✓	×	×	×
	Indels	✓	—	—	×	×	×
	Rearrangement	✓	×	×	×	×	×
	Tandem	✓	✓	×	×	×	×
Copy number		—	✓	—	✓	×	×

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