Recombinant Human Thrombopoietin (rhTPO) Engineering Service

Recombinant Human Thrombopoietin (rhTPO) is a critical cytokine used in biomedicine to stimulate the production of platelets (thrombopoiesis). It is crucial for treating thrombocytopenia induced by chemotherapy or bone marrow failure. Industrial production in Chinese Hamster Ovary (CHO) cells often suffers from Low expression in CHO cells , resulting in poor product yield and high manufacturing costs. Furthermore, the produced rhTPO can exhibit insufficient biological activity due to sub-optimal post-translational modifications (PTMs) like glycosylation or structural defects, leading to lower therapeutic efficacy.

CD Biosynsis offers an integrated cell line and protein engineering service for high-quality rhTPO production. Our approach addresses both yield and quality issues. The yield problem ( low expression ) is tackled by optimization of fed-batch fermentation process in CHO cells . This includes developing specialized feeding strategies (glucose, amino acids, growth factors) and optimizing physicochemical parameters (pH, DO, temperature shifts) to maximize the viable cell density (VCD) and specific productivity (Qp) of the CHO host. The quality and activity issue ( insufficient biological activity ) is resolved through Modification of receptor binding sites . We employ protein engineering (site-directed mutagenesis or domain shuffling) on the TPO gene to enhance its affinity and specificity for its receptor, Mpl (TPO R). We also engineer the host cell to ensure the rhTPO product receives the appropriate, bio-active N-linked and O-linked glycosylation patterns, which are vital for stability and activity. This combined strategy ensures a high-yield, high-quality rhTPO product ready for clinical application.

Pain Points

Achieving highly active and cost-effective rhTPO production faces these key challenges:

• Low Expression in CHO cells: Suboptimal growth conditions and insufficient nutrient supply in large-scale culture limit CHO cell viability and specific productivity (Qp), leading to low product titer and high cost.
• Insufficient Biological Activity: Improper or incomplete post-translational modifications (glycosylation) and structural instability of the recombinant protein result in poor Mpl receptor binding and diminished efficacy.
• Receptor Binding Competition: The rhTPO is sometimes not able to effectively compete with endogenous TPO for Mpl receptor binding, reducing its therapeutic potential.
• Glycosylation Heterogeneity: The CHO cell system can produce rhTPO with variable glycosylation patterns , which complicates purification and affects product half-life and activity.

A successful solution requires balancing cell growth and productivity with stringent quality control over the final protein structure and activity.

Solutions

CD Biosynsis utilizes advanced cell line and protein engineering to optimize rhTPO production:

Optimization of Fed-Batch Fermentation Process in CHO cells

           

We develop high-density, chemically defined media and use optimized feeding regimens to maximize VCD and Qp, solving the low expression problem.

Modification of Receptor Binding Sites

We use site-directed mutagenesis on the TPO gene to enhance Mpl receptor affinity and improve the stability and biological function of rhTPO.

Glycosylation Pathway Engineering

We engineer CHO glycosylation genes to promote the formation of the specific PTMs required for maximal rhTPO activity and half-life .

Gene Copy Number Amplification

We use DHFR or GS systems to amplify the integrated TPO gene copy number , boosting the expression level in the CHO host.

This integrated approach simultaneously addresses the production (yield) and quality (activity) bottlenecks.

Advantages

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

Significantly Increased Product Titer

Optimized fed-batch process and gene amplification result in $>5$ g/L total protein yield , solving the low expression issue.

High Biological Activity

Modified receptor binding sites and tailored glycosylation ensure the rhTPO has superior Mpl receptor binding and functional efficacy , overcoming insufficient biological activity .

Reduced Cost of Goods Icon

High titer dramatically lowers the cost of production and purification per unit of active dose.

Stable and Scalable CHO Platform Icon

The engineered CHO cell line is robust for industrial bioreactor scale-up while maintaining stable expression.

Consistent Glycosylation Profile

Process and host engineering ensure the rhTPO exhibits a uniform and clinically desirable PTM profile .

We provide a streamlined, high-performance platform for pharmaceutical-grade rhTPO production.

Process

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

• rhTPO Gene Optimization: Codon optimize the TPO gene and introduce mutations to receptor binding sites for enhanced affinity.
• CHO Host Engineering: Integrate and amplify the modified TPO gene into a high-productivity CHO host (e.g., CHO-K1 or CHO-DG44), potentially modifying glycosylation enzymes.
• Fed-Batch Development: Optimize basal media, pH set points, DO, and temperature to achieve maximum VCD and specific productivity.
• Feed Strategy Design: Develop a tailored, chemically defined feeding strategy to prevent nutrient limitation and inhibit byproduct accumulation (e.g., lactate, ammonia).
• Quality Assessment: Analyze rhTPO using SEC, RP-HPLC, MALDI-TOF mass spec to confirm sequence, purity, and PTM profile.
• Functional Assay Validation: Perform in vitro cell-based proliferation assays using Mpl-expressing cells to confirm maximal 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 high-yield, highly active rhTPO supply. CD Biosynsis provides customized cell line and protein engineering solutions:

• Detailed Process Optimization Report , including VCD, Qp, and final titer data from the optimized fed-batch run.
• Consultation on downstream process design to integrate purification steps tailored for the new rhTPO product profile.
• Experimental reports include complete raw data on biological EC}_{50 (Activity) and detailed glycan mapping , essential for regulatory submission.