Optimizing the Purification Process for Recombinant Human Insulin

In recent years, diabetes patient growth has driven insulin demand, but affordable insulin is in short supply. Efficient, economical insulin production is crucial. Mainly produced via Escherichia coli (E. coli) and yeast due to rapid growth and low cost, E. coli faces complex downstream processing.

Overview of Downstream Processing:

Purifying recombinant human insulin from E. coli inclusion bodies involves multiple steps, including recovery, washing, solubilization and oxidation, cleavage, buffer exchange, chromatographic purification, precipitation, renaturation, enzymatic cleavage, and formulation.

Yaohai Bio-Pharma boasts extensive experience in recombinant insulin production and purification, coupled with a team of experts, ensuring that your insulin production is completed with high efficiency. Leveraging our experience from hundreds of projects, Yaohai kindly summarizes how to optimize the Downstream Purification Process for Recombinant Human Insulin.

Inclusion Body Recovery and Washing:

Cells are disrupted using mechanical methods (such as ultrasonication, high-pressure homogenization) or lysozyme treatment, followed by washing buffers (containing urea, Triton X-100, etc.) to remove impurities. During washing, parameters need to be optimized to ensure protein quality while controlling costs.

Inclusion Body Solubilization and Oxidation:

High-concentration denaturants (such as guanidine hydrochloride and urea) are used to solubilize inclusion bodies, with dithiothreitol or β-mercaptoethanol added to prevent the formation of incorrect disulfide bonds. pH and temperature significantly impact the solubilization and oxidation reactions.

Cleavage and Buffer Exchange:

Cyanogen bromide cleavage is used to remove the N-terminal initiating amino acid, but it poses toxicity and low specificity issues. Through dialysis, ultrafiltration, or size exclusion chromatography, Buffer exchange removes harmful reagents in preparation for subsequent operations.

Chromatographic Purification and Precipitation:

Multiple chromatographic techniques are employed to purify insulin, including affinity chromatography, ion exchange chromatography, reversed-phase chromatography, hydrophobic interaction chromatography, and size exclusion chromatography. Precipitation methods, such as pH precipitation and zinc crystallization, are used to remove impurities and concentrate proteins.

Renaturation:

The solubilized protein is diluted in a renaturation buffer to promote the correct formation of disulfide bonds and protein folding. Protein-folding liquid chromatography and dialysis are also methods used for renaturation.

Enzymatic Cleavage and Formulation:

Trypsin and carboxypeptidase B are used to cleave the C-peptide, forming the active insulin heterodimer. Finally, crystallization and lyophilization are employed to remove residual salts and buffers, preparing a stable formulation. Specific additives (such as zinc and protamine) are added to meet different clinical needs.

Conclusion:

Downstream processing of insulin from E. coli is complex. Future focus should be on optimizing these steps for efficient, economical production to meet demand and reduce costs.

Yaohai Bio-Pharma is also actively seeking institutional or individual global partners and offers the most competitive compensation in the industry. If you have any questions, please feel free to contact us: [email protected]