Recombinant Coagulation Factor VIII (rFVIII) Engineering Service

Recombinant Coagulation Factor VIII (rFVIII) is a life-saving therapeutic protein essential for treating Hemophilia A. The supply chain faces instability due to the insufficient supply of plasma sources , which also carries inherent safety risks. Furthermore, manufacturing rFVIII in industrial host cells like Chinese Hamster Ovary (CHO) cells is hampered by the low expression yield , driven by the large size and complex structure of the protein.

CD Biosynsis offers a specialized mammalian cell engineering service to enhance rFVIII production. Our core strategy involves the optimization of the gene amplification system in CHO cells , utilizing robust selection markers and gene circuit tuning to maximize the copy number of the FVIII gene. This is combined with the engineering of signal peptides and promoters to enhance transcription initiation and improve the efficiency of protein secretion. This integrated approach aims to establish a high-yield, stable, and cost-effective rFVIII manufacturing platform.

Pain Points

The successful and economical large-scale production of rFVIII is challenged by:

• Supply Limitations and Safety: Dependence on plasma sources poses risks of pathogen transmission and is inherently unstable, driving the need for reliable recombinant production.
• Low CHO Expression Yield: FVIII is a massive protein (2,351 amino acids), making its stable expression and secretion in CHO cells intrinsically difficult, resulting in low volumetric productivity .
• Gene Amplification Instability: Achieving and maintaining high FVIII gene copy numbers using traditional selection systems can be prone to genetic drift and instability during prolonged culture.
• Secretion and Degradation: A significant portion of synthesized FVIII is often retained in the ER (endoplasmic reticulum) , incorrectly folded, or degraded before successful secretion.

A cost-effective solution must enhance CHO cell capacity for both gene expression and protein handling.

Solutions

CD Biosynsis utilizes advanced cell line and expression system engineering to address rFVIII challenges:

Optimization of the Gene Amplification System in CHO Cells

           

We employ dihydrofolate reductase (DHFR) or Glutamine Synthetase (GS) systems with optimized selection pressures to achieve high and stable FVIII gene copy numbers.

Engineering of Signal Peptides and Promoters

We screen and engineer optimized promoters for high transcription and refine the FVIII signal peptide to enhance efficient translocation into the ER and subsequent secretion.

ER Folding and Secretion Pathway Tuning

We overexpress key ER chaperones (e.g., BiP) and folding assistants to mitigate stress and increase the rate of correctly folded, secretion-ready FVIII.

Intron Optimization for Expression

We investigate the strategic placement and optimization of introns within the FVIII gene construct to potentially enhance transcription, mRNA stability, and overall translation.

This systematic approach is focused on overcoming the intrinsic difficulties associated with expressing large, complex proteins like FVIII in CHO cells.

Advantages

Our rFVIII engineering service is dedicated to pursuing the following production goals:

Stable, High-Titer Expression

Optimized gene amplification aims to produce a CHO cell line with consistently high and stable FVIII gene copy numbers for sustained production.

Enhanced Secretion Efficiency

Signal peptide and ER folding optimization are focused on maximizing the fraction of FVIII that is correctly processed and secreted, increasing volumetric yield.

Reduced ER Stress

Tuning ER folding capacity is intended to improve cell viability and specific productivity by mitigating stress responses during high expression.

Increased Product Safety

Recombinant production completely bypasses the risk of human pathogen contamination associated with plasma-derived products.

Highly Scalable CHO Platform

CHO cells are the regulatory standard for biologics, ensuring that the engineered platform is readily scalable for clinical and commercial supply.

We provide a specialized platform aimed at maximizing the yield and quality of rFVIII biomanufacturing.

Process

Our rFVIII CHO strain engineering service follows a rigorous, multi-stage research workflow:

• Vector Construction and Optimization: Design the FVIII expression vector with optimized promoter/enhancer elements and the chosen selection marker (DHFR/GS).
• Signal Peptide and Gene Tuning: Screen and integrate the most efficient signal peptide sequence and optimize FVIII gene sequence elements (e.g., codon usage, intron placement).
• Gene Amplification and Clone Selection: Transfect CHO cells and subject them to step-wise DHFR/GS selection pressure to isolate high gene copy number clones .
• ER Pathway Co-expression: Integrate and optimize the expression of ER chaperones or other folding factors to enhance the correct processing of the FVIII protein.
• Bioreactor Performance Validation: Test the final engineered CHO clone in fed-batch bioreactors to assess volumetric productivity, specific productivity, and product quality .
• Result Report Output: Compile a comprehensive Experimental Report detailing vector maps, gene copy number analysis, cell line stability data, and final titer/activity assessment , supporting regulatory submission.

Technical communication is maintained throughout the process, focusing on timely feedback regarding gene copy number stability and protein quality.

Explore the potential for a stable, high-yield rFVIII supply. CD Biosynsis provides customized cell line engineering solutions:

• Detailed Gene Copy Number and Titer Analysis Report , illustrating the success of the amplification and expression strategy.
• Consultation on bioreactor culture strategies optimized for FVIII production and cell viability.
• Experimental reports include complete raw data on protein activity and stability , essential for clinical and commercial development.