Recombinant Protein Customization Service

Background

Recombinant proteins are produced by genetically engineered expression systems to obtain desired target proteins. Compared to proteins isolated from natural sources, recombinant proteins offer high activity, high purity, batch-to-batch consistency, stringent quality control, and can be optimized for specific characteristics such as solubility, stability, and formulation. Types of recombinant proteins include biopharmaceuticals, industrial enzymes, diagnostic proteins, vaccines, food additives, cosmetic proteins, peptides, biosensors, and biocatalysts.

Application Fields of Recombinant Proteins

Recombinant Protein Expression Systems

Recombinant protein expression systems are generally categorized into prokaryotic expression, eukaryotic expression, insect cell expression, and mammalian cell expression.

Expression Systems	E. coli Expression	Yeast Expression	Insect Cell Expression	Mammalian Cell Expression
Typical Hosts	BL21 / Rosetta / Origami	Pichia pastoris Saccharomyces cerevisiae	SF9 Cell/Hi-Five Cell	CHO Cell/293 Cell
Advantages	1. Genetically stable, fast cultivation cycle, and low cost. 2. High protein yield and simple separation and purification. 3. Commercialized vectors and strains are available.	1. Fast cultivation cycle, relatively low cost, and genetically stable. 2. Supports high-density cell cultures. 3. Proteins are easily secreted into the culture medium, simplifying purification.	1. Proper protein folding, disulfide bond pairing, and post-translational modifications. 2. High expression levels, capable of accommodating large molecular insertions. 3. Can express multiple fragments simultaneously.	1. High expression yield after system optimization. 2. Post-translational modifications. 3. Activity is closer to that of natural proteins.
Disadvantages	1. No post-translational modifications, difficult to form correct disulfide bond pairing. 2. Expressed proteins are often insoluble, prone to forming inclusion bodies, with low expression levels. 3. Tends to produce endotoxins and contains botulinum and toxic proteins.	1. Long fermentation cycle. 2. Different types of glycosylation modifications. 3. High polysaccharide concentration in the culture supernatant, complicating downstream purification.	1. Protein expression is regulated by viral promoters. 2. Viral infections lead to cell death. 3. High cultivation cost and long fermentation cycles. 4. Lower expression levels and high technical requirements for operation.	1. High cultivation cost and long fermentation cycles. 2. Lower expression levels and high technical requirements for operation. 3. Susceptible to viral and mycoplasma contamination.
Expression Levels	300~10000 mg/L	300~10000 mg/L	1~500 mg/L	0.2~10000 mg/L
Protein Glycosylation Levels	NONE	LOW	MEDIUM	HIGH
Production Expression Cycle	2-4 DAYS	5-7 DAYS	6-8 DAYS	7-20 DAYS

CMO Service Platform Overview

Hzymes has many years of experience specializing in the development of biological raw materials and comprehensive technical solutions. We offer extensive capabilities in custom development and large-scale manufacturing of recombinant proteins, including GMP-grade production. Our fermentation platforms range from 5L, 50L, 500L, 5T, 10T, 30T, 60T to 100T, with matched chromatography systems of various capacities, and freeze-drying platforms from 0.2㎡, 0.5㎡, 2㎡, 10㎡ to 20㎡.

We maintain a robust quality management system, strictly control the manufacturing processes, and implement rigorous release standards for intermediate and final products to ensure batch consistency. This fully supports production needs from R&D to pilot and industrial-scale manufacturing.

1. Fermentation Process

Fermentation production begins with small-scale fermentation using shake flasks to determine the optimal growth conditions of microorganisms before scale-up in bioreactors. In high-density fermentation systems, culture methods and conditions (e.g., batch fermentation, fed-batch, or continuous fermentation) are optimized to significantly increase cell density, ensuring a balance between cost and efficiency. Key parameters monitored include pH, temperature, dissolved oxygen, CO₂ concentration, aeration, agitation, and the resulting shear forces.
Different fermentation equipment imposes different constraints on parameter control:

• Shake flasks are typically used for strain revival and cultivation;

• 5L fermenters are used to test initial fermentation parameters;

• 50L fermenters confirm conditions before industrial-scale production.

Metrics	Conventional Cultivation	High-Density Fermentation (E. coli Expression)	High-Density Fermentation (Yeast Expression)
OD₆₀₀	7~20	60~200	300~600
Wet Weight (g/L)	10~20	80~260	300~500
Protein Expression Level (mg/L)	5~500	300~15000	3000~10000
Production Volume	100~400L	100~400L	100~400L
Cycle Duration	2-4 DAYS	3-7 DAYS	7-9 DAYS

Hzymes' 500L fermenter platform is used for process development and scale-up.

Hzymes offers: fermentation platforms of various scales for process development and scale-up production, with optimized key parameters to ensure stable and reproducible fermentation processes.

Hzymes' Project A (E. coli expression) and Project B (yeast expression) both achieved high-density fermentation using the 500L platform.

Control Parameters & Test Indicators	HZYMES High-Density E. coli Fermentation Medium	HZYMES High-Density Yeast Fermentation Medium
Culture Medium	HZYMES High-Density E. coli Fermentation Medium	HZYMES High-Density Yeast Fermentation Medium
Culture Temperature	Online Real-Time Control
Fermentation pH	Cascade Acid-Base Feed, Constant Fermentation pH
Dissolved Oxygen	Cascade Aeration and Agitation, Maintaining Controllable Range
Feed	HZYMES High-Density E. coli Fermentation Feed Medium	HZYMES High-Density Yeast Fermentation Feed Medium
Fermentation Duration	4d	7d
Fermentation Volume	400L	350L
Final OD600	160	435
Biomass Yield	95kg	129.2kg
Biomass Wet Weight	238g/L	369.1g/L
Protein Expression Level	1000mg/L	5460mg/L

2. Purification Process

• Affinity Chromatography (AC):
Nickel affinity chromatography is commonly used for purifying His-tagged proteins. This method often achieves over 85% purity in a single step and is widely used for purifying antigens and enzymes for biochemical diagnostics.

• Size Exclusion Chromatography (SEC):
This technique separates molecules based on size: large molecules elute faster as they do not enter resin pores, while smaller molecules enter the pores and elute later. The method is simple and only requires one buffer, but has low sample capacity and requires high-end equipment, which may increase costs.

• Ion Exchange Chromatography (IEX):
This method utilizes reversible interactions between charged molecules and oppositely charged groups on the resin. By adjusting buffer pH or ionic strength, target molecules can be bound and eluted selectively. IEX is widely used in the purification of proteins and nucleic acids.

• Hydrophobic Interaction Chromatography (HIC):
HIC separates proteins based on hydrophobicity differences. Proteins interact with hydrophobic resins to varying degrees and are eluted sequentially by gradually reducing salt concentration in the buffer, enabling effective separation and purification.

Scale	Development-Level Small-Scale Testing	Hundred-Gram Scale-Up	Kilogram-Scale Production
Chromatography Column Specifications	16-100 mm	150-450 mm	800 mm
Protein Yield	1-60 g	100-800 g	1000-2000 g
Protein Purity	≥95%
Microbial Load	<1 cfu/g
Bacterial Endotoxin Content	<10 EU/mg
Cost Reduction	/	60-100% compared with the trial	120-200% compared with the trial

Hzymes offers: purification platforms of various scales to rapidly transfer and scale purification processes, producing high-activity, high-quality proteins.

Project C (E. coli): A custom purification process developed by Hzymes' platform achieved 99.9% purity, endotoxin level <10 EU/mg, expression yield of 20 mg/g wet cell, and batch yields exceeding hundreds of grams.

Items	Quality Standards	Test Results
Appearance	colorless and clear liquid	colorless and clear liquid
Clarity	complies with regulations	complies with regulations
pH Value	7.25 ± 0.25	7.32
Protein Concentration	36-40 mg/mL	39.52 mg/mL
Protein Purity	≥ 97%	99.9%
Residual E. coli Host Proteins	≤ 10 ng/mg	≤ 10 ng/mg
Residual E. coli Host DNA	≤ 1 ng/mg	≤ 1 ng/mg
Bacterial Endotoxin Content	≤ 10 EU/mg	≤ 10 EU/mg

3. Lyophilization Process

Recombinant protein liquid formulations require cold-chain transportation and are prone to structural instability in solution, leading to aggregation or denaturation. Increasingly, products are being developed with lyophilization processes to enhance stability and reduce transportation costs.

Hzymes offers: various-scale lyophilization platforms and extensive lyophilized formulation development experience. Automated loading/unloading systems are employed during freeze-drying to ensure aseptic processing.

CMO Service Workflow

1. Customized Service Workflow

2. Standard Service Workflow

Service Features

Overview of Biopharmaceutical Products