Brief description of technology processes for recombinant insulin production

Brief description of process stages

1. Preparation of solutions and growth medium.

A processing machine operator is taking necessary reagents from intermediate depot and makes accurate weighted amounts in weighting area according to instructions. Weighted amounts are transferred to growth media preparation and handling area, a section for solutions and growth medium preparation. Growth medium is sterilized in technological vessel by autoclaving, with that it is aseptically transferred into inoculation and basic vessel via tubing line. Flasks with growth medium for inoculum cultivation are separately sterilized in autoclave. After sterilization cooled flasks with growth medium are transferred through pass-through window into primary inoculation area (alternatively flasks for inoculums growth may be sterilized in pass-through autoclave connecting media preparator and primary inoculation area). Solutions are transferred from technological vessels to bioreactors in fermentation area via tubing lines during cultivation process. Additionally to fermenters ammonia lines should be conducted from ammonia storage area for its usage during cultivation process.

1. Inoculation transfer.

Microbiologist takes inoculation vessel from working bank storage area and transfers it into primary inoculation area through pass-through window.

A microbiologist enters into primary inoculation area through personnel sluice and performs the inoculation of flasks, the flasks are placed into thermoshaker for 12-16 hours, and they are then transferred through pass-through window into fermentation area.

1. Preparation of inoculation vessel and fermenter (bioreactor) for inoculation.

Processing operator(s) enter into fermentation areas through personnel sluice. Processing operator delivers needed amount of growth medium to bioreactor or inoculation vessel via sterile tubing lines. Necessary solutions are supplied into portable vessels via the lines by processing operator, and necessary amounts of solutions are added into inoculation vessel and bioreactor through sterile filter. Validated parameters are adjusted (рН, temperature).

1. Inoculation of producer-strain culture inside inoculation vessel.

Inoculation flasks are taken from pass-through window between fermentation area and primary inoculation area, then inoculation of inoculum bioreactor is conducted. Fermenter mixer and control equipment are switched on; sterile compressed air is delivered (at the rate of 2 fermentation volume per minute). In the course of cultivation in inoculation vessel appropriate solutions are entered through sterilizing filter, and necessary pH levels are maintained by adding the ammonia solution. At appropriate intervals samples for analysis are taken from inoculation vessels.

1. Inoculation and cultivation of growth in working fermenter.

The content of inoculation vessel is transferred into working fermenter via sterile tubing line. Fermenter mixer and control equipment are switched on; sterile compressed air is delivered (at the rate of 2 fermentation volume per minute). In the course of cultivation in inoculation vessel appropriate solutions are entered through sterilizing filter, and necessary pH levels are maintained by adding the ammonia solution. At appropriate intervals samples for analysis are taken from inoculation vessels.

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1. Bacterial mass collection.

Upon completion of cultivation process through sterile pipeline the content of fermenter through heat exchanger (cooling down to +10/+15⁰С) is delivered into separation and inclusion bodies cleaning area to self-discharge separator. Bacterial mass concentrate as a suspension is collected into refrigerated technological vessel (+10/+15⁰С). Waste solution from growth medium after separation is transferred via tubing line into vessel in sterilization area and further on it is transferred into pasteurizer for disinfection. After pasteurizer through heat exchanger cooled solution is drained into sewage system

1. Destruction of bacterial cells.

Obtained bacterial mass suspension is dissolved with cooled water until specified volume is reached, solutions applied are added as required, temperature of +10/+15⁰С is adjusted; the bacterial mass suspension is supplied to disintegrator to disintegration area. Destruction of bacterial mass is followed by additional heating of suspension by 20-25⁰С. After disintegrator the suspension collected in vessel is transferred through cooling heat exchanger via tubing line into separation and washing area for washing of inclusion bodies.

1. Washing of inclusion bodies

Suspension collected in reservoir and obtained after destruction is refrigerated the temperature range of +10/+15⁰С is transferred to self-discharge separator. After separation suspension of inclusion bodies is collected in technological vessel, cooled water until specified volume is reached; appropriate solutions are added as required. Suspension is kept and mixed in technological vessel during one hour; thereafter it is again delivered to separator. There are 4 washing cycles. Precipitate of washed inclusion bodies after separation is collected into technological vessel via tubing line in sterilization area and is further transferred to pasteurizer for sterilization.

1. Dissolution of inclusion bodies

Suspension of washed inclusion bodies is transferred via tubing line into technological vessel in the area of dissolution of inclusion bodies. Appropriate components and solutions for dissolution are applied. Dissolution process is accompanied by solution refrigerating, wherefore controlled heating of solution is required (20-25⁰С). Inclusion bodies dissolution lasts for 3 hours, thereafter it is transferred to drum separator for clarification. Clarified solution is transferred via tubing line to technological vessel for refolding area.

1. Refolding.

Weigh components of buffer solution for refolding are prepared in weighting area adjacent to refolding area. Weigh reactors are entered into technological vessels for refolding, filled with required volume of water cooled down to +10⁰С.

Needed amount of clarified protein solution is taken from technological vessel and transferred into technological vessel with refolding buffer. During 6 hours protein is incubated and mixed in refolding buffer under temperature control.

1. Precipitation of hybrid protein.

After refolding finalization required solution for protein precipitation is entered, and pH level is adjusted. Mixing is stopped, and protein is left for settling for 2-3 hours.

After settling of refolded hybrid protein the certain amount of supernatant is decanted with pump, and sediment of supernatant residues is transferred through self-discharge separator into hybrid protein collection area.

Suspension of amorphous precipitate of refolded hybrid protein is fed into portable vessel and transferred to separation area.

12. Fermentative conversion 1 (tryptic cleavage)

Protein suspension obtained at the previous stage is dissolved with purified in reactors water with jackets and stirrer and mixed during 20-30 minutes until protein is dissolved, рН level is adjusted to 2,5 – 3 by adding 2 М of hydrochloric acid. In analytical laboratory total protein concentration of solution is identified by spectrophotometer, as well as refolded hybrid protein concentration (HELC) and Zn⁺² concentrations (ААS).

Trilon B and glycine are added to hybrid protein solution, рН 11.3 – 11.52 is adjusted by М sodium hydroxide solution.

Obtained hybrid protein solution is refrigerated up to 10 – 12°C by supplying cold water into vessel jacket.

Thereafter trypsin is added to hybrid protein solution (at the rate of 0.125 g of trypsin for 1 kg of total protein). Tryptic cleavage is carried out under the temperature of 10 – 12°C during 16 – 18 hours until maximum concentration of Arg-(В31)-insulin has been reached. Arg-(В31)-insulin concentration control is carried out during every 2 hours.

Tryptic cleavage is stopped by adding 2 М of hypochlorite solution to adjust рН 2.5 – 3.0.

2-propanol is added from measurer to Arg-(В31)-insulin solution and mixed until concentration of 20 %
(volume percent) is reached, then mixing is stopped, and solution is stored during 10-12 hours under the temperature of 10 – 12 ºС for ballast protein precipitation.