Recombinant Human Nerve Growth Factor (rhNGF) Engineering Service

Recombinant Human Nerve Growth Factor (rhNGF) is a critical neurotrophic factor used in biomedicine for treating neurological disorders, nerve injuries, and certain eye diseases. Production in Chinese Hamster Ovary (CHO) cells is challenging due to two major bottlenecks: Extremely low expression (rhNGF is naturally expressed at very low levels, making high-titer industrial production difficult) and unstable activity (the protein is a dimer, and its proper folding, disulfide bond formation, and stability are highly sensitive to culture conditions and processing).

CD Biosynsis offers a dedicated cell line engineering service to overcome these rhNGF production challenges. Our strategy for enhancing yield ( extremely low expression ) centers on Gene amplification modification in CHO cells . We utilize robust selection markers (e.g., DHFR or GS) and step-wise selection pressure to integrate and amplify hundreds of copies of the NGF gene into the CHO cell genome, dramatically boosting the transcription rate. To tackle the quality and stability issue ( unstable activity ), we focus on Optimization of signal peptide . NGF requires efficient secretion and processing. We screen or engineer a panel of highly efficient and optimized signal peptides to replace the native NGF signal sequence. An optimized signal peptide ensures the nascent NGF peptide is rapidly and efficiently translocated into the endoplasmic reticulum (ER), where proper folding, disulfide bond formation (essential for the active dimeric structure), and secretion occur. This combined approach targets high production quantity via Gene amplification and high biological quality via Signal peptide optimization .

Pain Points

Achieving stable, high-yield rhNGF production faces these key challenges:

• Extremely Low Expression: The NGF gene is naturally expressed at basal levels, and its expression in heterologous CHO systems often leads to extremely low product titer (typically in the low mg/L range), making large-scale production uneconomical.
• Unstable Activity: NGF must form a correctly folded homodimer stabilized by disulfide bonds. Improper folding or inadequate ER processing results in inactive monomers, misfolded dimers, or aggregation, leading to unstable biological activity .
• Inefficient Secretion: The native NGF signal peptide or the translocation machinery can be rate-limiting, causing intracellular buildup or degradation of the nascent protein.
• Post-translational Processing: NGF is produced as a pre-pro-protein and requires cleavage to yield the mature, active form. Inefficient cleavage reduces the final yield of the usable product.

A successful solution must significantly boost gene transcription while ensuring efficient and correct protein processing and secretion.

Solutions

CD Biosynsis utilizes advanced cell line and protein engineering to optimize rhNGF production in CHO cells:

Gene Amplification Modification in CHO cells

           

We employ the DHFR/MTX or GS/MSX system for chromosomal integration and amplification of the NGF gene, overcoming extremely low expression .

Optimization of Signal Peptide

We replace the native NGF signal peptide with a highly efficient, heterologous signal sequence (e.g., IgG leader) to boost ER translocation and secretion efficiency.

ER Folding and Processing Enhancement

We co-express ER chaperones (e.g., PDI) and proprotein convertases to facilitate correct disulfide bond formation and cleavage of the NGF pre-pro-protein, preventing unstable activity .

Titer and Activity Screening

We use FACS-based and ELISA high-throughput screening to identify clones with the highest gene copy number and specific activity.

This integrated strategy addresses expression quantity, secretion efficiency, and correct folding for high-quality rhNGF production.

Advantages

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

High Product Titer

Effective gene amplification dramatically increases rhNGF expression, transforming extremely low expression into a viable industrial process.

Enhanced Functional Stability

Improved ER folding and processing (facilitated by optimized signal peptide ) ensure the formation of the correct, stable dimer , overcoming unstable activity .

Increased Secretion Efficiency Icon

Optimizing the signal peptide and processing pathways minimizes intracellular degradation and maximizes secreted product.

Reduced Cost of Goods Icon

Higher titer and active fraction lead to lower costs per active dose, making the drug more accessible.

Clinically Relevant Glycosylation Icon

Production in CHO cells ensures appropriate mammalian glycosylation, important for stability and in vivo function.

We provide a reliable, high-yield, and high-activity platform for pharmaceutical rhNGF production.

Process

Our rhNGF engineering service follows a rigorous, multi-stage research workflow:

• Vector Design: Clone the TPO gene with an optimized signal peptide into a DHFR or GS selection vector, and incorporate codon optimization .
• Stable Cell Line Generation: Transfect the vector into a CHO host and isolate high-expressing clones .
• Gene Amplification: Subject the lead clones to step-wise selection pressure (e.g., increasing MTX concentration) to amplify the integrated gene copy number .
• Process Development: Optimize basal media and fed-batch strategy to support high cell density and maximize rhNGF secretion.
• Quality Assessment: Analyze rhNGF using ELISA, SDS-PAGE, HPLC to confirm protein integrity, purity, and dimer formation.
• Functional Assay Validation: Perform in vitro Neurite Outgrowth Assays or receptor binding assays on TrkA expressing cells to confirm stable and high biological activity (EC_50).

Technical communication is maintained throughout the process, focusing on timely feedback regarding yield and product quality attributes.

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

• Detailed Gene Amplification Efficiency Report , showing gene copy number and titer correlation during MTX or MSX selection.
• Consultation on bioreactor process scaling and shear stress mitigation to prevent rhNGF aggregation and preserve activity.
• Experimental reports include complete raw data on biological EC_50 (Activity) and detailed dimer/monomer ratio , essential for stability and clinical validation.