Insulin preparation cell disruption

Cell disruption of an insulin preparation by means of High Pressure Homogenization Technology

Human insulin is made by recombinant- DNA technology utilizing S. cerevisiae (baker's yeast) or E. coli as the production organism. It is structurally identical to the insulin produced by the pancreas in the human body.

The production process:

Fermentation & Conditioning

After the fermentation process, the cells are separated by means of a high speed centrifuge. The collected cells are resuspended in buffer to prepare them for the next unit operation. High pressure homogenization is then used for cell disruption.

There are other methods for cells disruption, but there are many advantages of the high pressure homogenization:

• It is a mechanical process that is quick and effective
• It is a continuous process
• The results obtained in laboratory are perfectly reproducible in industrial scale
• You can vary pressure in order to find the right degree of cell rupture
• No need to add expensive enzymes
• No contamination of the product by toxic substances
• The degree of denatured proteins at the end of the process is small

During the homogenization the intracellular material is released generating lots of cell debris. The buffered solution is then cooled down and centrifuged to separate the debris from the intracellular proteins. Because of the size distributions, densities, and shapes of the proteins and cell debris, they will sediment at different rates when centrifuged. The efficiency of the cell disruption can then be evaluated by the content collected after centrifugation.

The proteins are then processed, (washing step, solubilization, clarification, renaturation, etc.) in order to obtain the construction of the peptide chain typical of human insulin.

The homogenization process:

The product before the homogenizer step is kept at low temperature, below 10°C and is homogenized at 1000 bar.

Usually more than one passage is required in order to obtain an efficient level of cell disruption.

The Effects of Homogenization:

Insulin is created as intracellular material by the organisms. High pressure homogenization is a excellent method for production scale cell disruption. Repeated passes through the homogenizer ensure that cells are completely ruptured. Complete disruption of the host cells is important because any undisrupted cells will co-sediment with the intracellular material in the subsequent solid-liquid centrifugal separation. The degree of intracellular soluble protein is often used as a measure of the cell disruption following each discrete passage through the homogenizer.

Homogenizer:

The homogenizer used for the treatment is a machine designed for 1000- 1500 bar with VHP compression block for

• High abrasion
• High viscosity

One homogenizing stage; Knife edge design valve for cell disruption.