Insulin and Insulin Purification
The discovery of insulin has been many years in the past, experiencing a transition from nothing to something, from animal insulin to human insulin and then to insulin analogs.
There are three phases of insulin analogs, and insulin specifications have evolved from the initial single short-acting insulin to the current rapid-acting, short-acting, intermediate-acting, long-acting, ultra-long-acting, and premixed insulins available in various dosage forms.
Comparing Three Generations of Insulin
|By source||Animal insulin||Human insulin||Human insulin analogs|
|Availability||First generation||Second generation||Third generation|
|Production time||1923||1982||Early twenty-first century|
|Extraction from animal pancreas||Genetic engineering technology||Genetic engineering technology|
|Vintage||Making medicine available to diabetics; inexpensive||The same molecular structure as human insulin does not cause allergic reactions, high safety, and efficacy||More in line with the human physiological insulin secretion curve, more increased safety, and effectiveness compared to second-generation insulin, reducing the risk of hypoglycemia; flexible injection time|
|Drawbacks||Higher side effects; the presence of heterologous allergic reactions||It cannot completely simulate the human insulin secretion metabolic curve, and it is difficult to accurately adjust blood glucose and risk of hypoglycemia; injection time requirements are strict||The relatively complex and expensive synthesis process|
|representative product||Neutral Insulin Injection, Zinc Arginine Insulin Injection||Zinc Arginate Recombinant Human Insulin Injection, Recombinant||Glycine Insulin, Menthol Insulin|
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.
The production process of insulin analog API
Insulin analog API can be produced by E. coli or yeast, mainly through strain construction, fermentation, purification, enzymatic digestion, modification, purification, lyophilization, and other steps. Except for the modification step, the rest of the process is roughly the same as that of human insulin. Modification refers to the addition of amino acids or acylated side chains to the insulin peptide chain after enzymatic cleavage, and if the added amino acids have protecting groups, they must be deprotected. For example, in the manufacture of insulin API, threonine ester is attached at position B30 and the protecting group is removed after reverse phase purification.
Factors influencing the production process of insulin
Fermentation of API is the most central part of the insulin production process, and the current market application of human insulin and insulin analogs are produced by both E. coli fermentation and yeast fermentation.
(1) API Fermentation Cycle and Volume
Insulin production uses an E. coli fermentation cycle of about 2-3 weeks and a yeast fermentation cycle of about 1 week.
Too long a period will lead to an increase in impurities, so the fermentation time must be strictly controlled; too large a fermentation volume will reduce the expression rate per unit of fermentation broth, increase the process impurities, reduce the product yield, and increase the production cost.
(2) Reproducibility and Enzyme Cutting Efficiency
After fermentation and expression of insulinogen inclusion bodies in E. coli, the inclusion bodies need to be denatured to form correctly folded insulinogen, whereas the process of producing insulin and its analogs by yeast secretion expression system is relatively simple and does not require complex denaturation technology. In addition, the fermentation of insulin forms a single chain in vivo, which needs to be enzymatically cleaved by instrumental enzymes in vitro and then form a disulfide bond to become a double chain, and some insulins need to be enzymatically added to certain amino acids side chains, and so on.
2. Separation and Purification Link
Insulin API needs to be separated and purified after fermentation, of which the most important is reverse-phase purification chromatography. In order to achieve good results in reversed-phase purification, the flow rate of chromatography solution is theoretically very low, but considering the process time and process efficiency, some companies basically have the flow rate of chromatography in downstream high-pressure purification at more than 12 columns/hour, and some companies may actually have 1-3 columns/hour, and there are also some companies improve the efficiency of single chromatography by increasing the diameter of chromatography columns.
Theoretically, the larger the diameter of a chromatography column, the higher the purification efficiency, but the main bottleneck is the flow rate of the pump, if the pump flow rate and pressure can not meet the process requirements, will limit the expansion of production scale; the larger the diameter of a chromatography column, the higher the process parameters of high-pressure pump, resulting in pressure and flow rate to meet the needs of a larger scale.
(1) Aseptic production of preparations and fillings
Insulin is a sterile preparation, and the preparation process is mainly divided into preparation, preparation, and filling, insulin preparation process is relatively simple compared to raw materials, the most important thing is the aseptic guarantee, that is, preparation and filling must be carried out under aseptic conditions, which requires training and confirmation of personnel dressing, sterilization/disinfection of equipment, sterilization/determination, and filtration of materials, periodic validation of the aseptic process, the establishment of A/B clean environment and daily monitoring. B clean environments are established and monitored daily.
(2) Preparation Volume and Filling Rate
In the production process of insulin preparation, the main key processes that limit insulin production efficiency or yield improvement are preparation volume and filling speed. The preparation volume of insulin preparation will limit the batch size of the product, and the filling speed of insulin preparation will limit the production efficiency, but in general, both of them have a general influence on the insulin production capacity.
(3) Accuracy of formulation dosing
At present, enterprises usually use automated program control feeding, with personnel operating part of the double-check and on-site supervision to ensure the accuracy of formulation feeding, so the degree of impact on insulin production capacity is small.