Recombinant Human Growth Hormone (rHGH) Engineering Service

Recombinant Human Growth Hormone (rHGH) is a critical biopharmaceutical used to treat growth disorders and various metabolic conditions. Production is challenging: while E. coli provides high expression levels, the product typically suffers from inclusion body formation in E. coli expression , requiring expensive and low-yield refolding. Using eukaryotic systems like yeast can facilitate correct folding and secretion, but the rHGH might still face abnormal glycosylation or degradation issues. The key is establishing a high-yield, high-quality eukaryotic platform that ensures the correct native structure.

CD Biosynsis offers a synthetic biology service focused on high-quality rHGH production. Our core strategy involves optimization of Pichia pastoris secretion expression system , leveraging this host’s ability to correctly fold and secrete complex proteins while minimizing hyperglycosylation problems common in other yeasts. This optimization includes the use of strong, inducible promoters and enhanced signal peptides for high secretion efficiency. Furthermore, we implement modification of protein structural stability through site-directed mutagenesis (if required) or directed evolution to increase resistance to proteolysis and enhance stability during fermentation and purification. This integrated approach aims to deliver high-yield, correctly folded, and clinically active rHGH at a reduced cost.

Pain Points

Achieving high-quality, cost-effective rHGH production faces these key technical challenges:

• Inclusion Body Formation in E. coli: High-level expression in E. coli leads to rapid aggregation of rHGH into inactive inclusion bodies , requiring denaturation and inefficient in vitro refolding steps.
• Abnormal Glycosylation: Although HGH is naturally non-glycosylated, eukaryotic hosts (especially some yeasts) can introduce unwanted N- or O-linked glycosylation , potentially affecting biological activity and increasing immunogenicity.
• Product Degradation: The HGH polypeptide is susceptible to proteolytic cleavage by host proteases in the fermentation broth, reducing the yield of the intact product.
• Low Secretion Efficiency: Achieving high secretion levels in yeast can be difficult due to bottlenecks in the endoplasmic reticulum (ER) quality control and folding machinery.

A high-quality solution must bypass the folding problems of E. coli while ensuring correct processing and high yield in a eukaryotic system.

Solutions

CD Biosynsis utilizes advanced protein and strain engineering to optimize active rHGH production in Pichia pastoris :

Optimization of Pichia pastoris Secretion Expression System

           

We employ high-copy integration of the HGH gene, optimize the alpha-factor signal peptide, and use the powerful AOX 1 promoter for robust, high-density induction.

Modification of Protein Structural Stability

We perform site-directed mutagenesis to remove potential proteolytic cleavage sites and enhance the native disulfide bond formation efficiency, reducing degradation.

Glycosylation Control Engineering

We use specific Pichia strains engineered to humanize glycosylation pathways or knock out key glycosylation genes to ensure a non-glycosylated or clinically appropriate product.

Co-expression of Folding Chaperones

We co-express yeast folding assistants (e.g., Kar2, PDI ) to relieve the load on the ER and promote efficient folding and secretion of rHGH.

This systematic approach is focused on leveraging the benefits of yeast while meticulously controlling the quality and yield of the final secreted rHGH product.

Advantages

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

High Specific Activity and Correct Folding

Secretion in Pichia ensures native folding and disulfide bond formation , leading to a fully active product without in vitro refolding.

Simplified Downstream Purification

Secretion into the medium greatly reduces initial biomass processing costs and simplifies purification steps compared to E. coli inclusion bodies.

Low Immunogenicity Risk

Glycosylation control ensures the product meets clinical standards, minimizing the risk of adverse immune responses . [Image of Cost Reduction Icon]

High Productivity Fermentation

Pichia pastoris is capable of very high cell density fermentation , maximizing volumetric productivity.

Enhanced Stability

Engineered strains and sequences ensure the rHGH is resistant to typical proteolytic degradation during the process.

We provide a specialized platform aimed at maximizing the quality and cost-effectiveness of clinical-grade rHGH biomanufacturing.

Process

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

• Vector Construction and Integration: Design the expression cassette with optimized signal peptide (e.g., alpha-factor) and multi-copy integration sites into the Pichia genome under the AOX 1 promoter.
• Glycosylation Site Analysis and Mutagenesis: Identify and mutate potential N- or O-glycosylation sites if the host strain does not already feature a humanized glycosylation system.
• Proteolytic Site Modification: Perform targeted mutagenesis to eliminate known cleavage sites (e.g., Kex2 sites) while preserving biological activity.
• High-Density Fermentation Optimization: Develop a high cell-density fed-batch fermentation protocol using methanol induction to maximize volumetric yield.
• Quality Control and Bioactivity Assay: Analyze the secreted protein via Western blot, RP-HPLC, CD spectroscopy, and cell proliferation assays to confirm correct size, purity, and activity.
• Result Report Output: Compile a detailed Experimental Report including strain data, fermentation parameters, and final titer/quality metrics , supporting regulatory and scale-up efforts.

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

Explore the potential for a high-quality, cost-effective rHGH supply. CD Biosynsis provides customized strain and process solutions:

• Detailed Secretion Titer and Purity Analysis Report , demonstrating the production efficiency and quality control.
• Consultation on initial capture and polishing chromatography steps optimized for secreted rHGH.
• Experimental reports include complete raw data on volumetric yield (mg/L) and structural homogeneity (e.g., monomer purity), essential for clinical development.