Insulin and Insulin Purification

Insulin API production process

1. Animal insulin API production process

Animal insulin API production process is roughly the use of acidic ethanol from the animal pancreatic fluid extracted from the impurity-containing insulin, adjusting different isoelectric points to remove the heterogeneous proteins, extracted by low-temperature evaporation to remove ethanol, separated from the oil and fat, with sodium chloride salt precipitation to obtain the crude insulin product. The crude product in acetone solution to adjust the isoelectric point, further removal of heterogeneous proteins, adding zinc salt in citric acid buffer, recrystallization is obtained after the boutique. Because animal insulin is generally extracted directly from the pancreatic tissue of animals to obtain insulin, and the human body itself produces insulin in the structural differences, the body will produce an immune response after use, resulting in a decline in the effect. Therefore, although animal insulin is cheap, it has been eliminated from the market.

2. Human insulin API production process

There are three kinds of production processes for human insulin API, namely, the recombinant production process of E. coli based on the expression of A and B chains respectively, the fermentation production process of E. coli, and the fermentation production process of yeast. These three methods use DNA recombination technology to insert the human insulin expression gene into a plasmid and introduce it into the host cell so that the host cell can express human insulin-related products. After the expression product is enzymatically cleaved and denatured, it finally forms human insulin with natural conformation, which greatly reduces immunogenicity, and its local and systemic allergic reactions and other adverse reactions are also significantly reduced.

(1) Recombinant production method in E. coli based on the separate expression of A chain and B chain.

This process first constructs an insulin A chain and B chain E. coli expression system, respectively, and uses E. coli to produce insulin, respectively.

The A and B peptide chains are artificially linked by disulfide bonds in vitro to form active human insulin. This process is complicated and the efficiency of joining A and B chains in vitro is low, so it is basically no longer used.

(2) Fermentation production process using E. coli bacteria.

This process uses E. coli to express the inclusion bodies of insulinogenic and refold the inclusion bodies to form the correctly folded insulinogenic, which is then enzymatically cleaved and isolated for purification to finally obtain insulin. The whole process takes about 2-3 weeks, among which the re-sexing takes longer, and each production step requires industrial water or water for injection, which requires enough time for the materials and raw materials to flow through the special material pipeline.

The insulinogen expressed by E. coli is wrapped in inclusion bodies, and the inclusion bodies cannot be secreted from the body, it is necessary to break the bacteria and dissolve the inclusion bodies to collect the insulinogen, which needs to be separated several times. In the isolation process, it is easy to mix more impurities, so it needs to be purified several times, which requires high purification technology, and the insulinogenic expressed by E. coli is inactive and needs to be processed in vitro for rejuvenation.

(3) Fermentation production process using yeast.

There are two types of yeast secretory expression systems, Saccharomyces cerevisiae and Picrosporum. Saccharomyces cerevisiae secretes and expresses insulin precursors in the pancreatic egg.

In the presence of leucase and excess threonine ester, human insulin ester was generated by enzymatic transpeptidation, then deesterified and purified with trifluoroacetic acid to finally obtain recombinant insulin. In the construction of Saccharomyces cerevisiae, by removing Thr at position 30 of the B chain in the cDNA molecule, the C peptide connecting the A and B chains will be shortened, and then it will be connected with the leader peptide factor, which can effectively increase the expression of single-chain insulin, and then recombinant human insulin can be obtained after enzymatic transpeptidation and reverse-phase purification.
The process of producing human insulin API by yeast fermentation can be summarized into 2 steps, i.e., fermentation to produce human insulin precursor and purification and post-processing of human insulin precursor. The whole process took about 1 week, with the fermentation and purification sessions being more time-consuming.

Compared with the advantages of easy handling, low price, and high expression capacity that E. coli fermentation has, yeast fermentation, although the insulin precursor is easily digested when it is expressed, the process is simpler because its insulin replication process has been completed in yeast fermentation and culture, and it can be enzymatically cleaved after clarification without the need of complex denaturation technology. At the same time, because the insulin precursor can be secreted directly into the body without breaking down the bacteria, fewer impurities are introduced. In addition, Saccharomyces cerevisiae can be produced in a continuous process, with one-third to one-quarter of the volume of fermentation broth harvested from a batch each day, allowing it to go directly to the next process, and production can continue for two to three months once started, an advantage not possible with the E. coli fermentation production process.