JPS6322161B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing a lyophilizate, and more particularly, to a process for producing a lyophilizate, in which an aqueous solution, suspension or emulsion containing viruses, bacteria, blood cells, serum or enzymes is prepared in liquid nitrogen. The present invention relates to a method for producing a freeze-dried product, which comprises drying a granular frozen product formed by dropping it into a low-temperature liquid under reduced pressure at a temperature of 0° C. or lower.

The method of the present invention will be explained in detail using an aqueous suspension of rubella virus as an example.

Rubella virus was purified from the filtrate obtained by culturing rubella virus (hereinafter referred to as purified virus), bovine serum albumin was added as an active protective agent and excipient, and hemagglutination activity was determined using barbital buffer. A purified virus aqueous suspension was prepared to have 4 units.

When 25 microliters of the purified virus aqueous suspension is dropped into liquid nitrogen in a suitable container using a microdropper, granular frozen matter is instantly formed. To prevent frozen substances from joining together, the liquid nitrogen container is vibrated from the outside using a rotator or the like.

This frozen product was scooped up from the liquid nitrogen, placed in a freeze dryer with a shelf temperature of -40°C, and dried under reduced pressure for 16 hours to obtain a purified rubella virus granular freeze-dried product. The lyophilized granular frozen product obtained in this way instantly freezes on a large surface area and dries in that shape, so the hemagglutinating activity of purified rubella virus does not decrease, and it can be restored. Excellent solubility over time.

When the purified rubella virus granular lyophilized product was reconstituted using 25 microliters of distilled water,
It was found that the hemagglutinating activity of 4 units was maintained as before lyophilization.

The present invention can be used not only for rubella virus but also for producing freeze-dried products from aqueous solutions, aqueous suspensions, or aqueous emulsions containing bacteria, blood cells, serum, or enzymes.

Furthermore, the unit of distribution is not limited to the hemagglutinating activity unit as described above, but can be obtained as a desired lyophilized product in units such as weight, activity, or number of cells per dose.

Freezing methods are not limited to the liquid nitrogen mentioned above.
Dry ice, acetone, fluorocarbon, etc. can also be used.

Examples of the present invention are shown below.

Example 1 After washing sheep red blood cells, they were fixed with a physiological saline solution containing formaldehyde, and then 10-20 ppm
of tannic acid as a coupling agent.

2.5 ml of sheep red blood cells (carrier) prepared as above
% w/v in phosphate buffered saline;
1 volume of this and 1 volume of human immunoglobulin (30mg/dl)
Sheep red blood cells were incubated for 1 hour in a constant temperature bath at 37°C, centrifuged, and washed twice.
% w/v as a freeze-drying protectant in phosphate buffered saline to which amino acids and bovine serum albumin were added, stirred to make it homogeneous, and then suspended in liquid nitrogen using a microdropper. twenty five
Microliters were added dropwise. At this time, vibrations are applied to the liquid nitrogen bath from the outside. The dropped droplets instantly froze in a minimum volume form in a liquid nitrogen bath. This frozen substance is scooped up from the liquid nitrogen bath with a wire mesh, placed in a vial, placed in a vacuum freeze dryer with a shelf temperature of -40°C, and freeze-dried under reduced pressure for 12 hours to obtain the desired human immunoglobulin. Sensitized red blood cell freeze-dried product was obtained.

Example 2 Avian and mammalian red blood cells were washed and fixed using a physiological saline solution containing 2% glutaraldehyde, and the remaining glutaraldehyde was washed away by centrifugation to deactivate the membrane components of the red blood cells. To prevent this, amino acids, sugar, and bovine serum albumin are used as protective agents, and red blood cells are contained at 10% w/v.
to obtain a suspension of red blood cells. Fifty microliters of this was added dropwise into dry ice/acetone using a micropipette or dripping device. The frozen solids were scooped up using a gold wire in the same manner as in Example 1, placed in a freeze dryer with a shelf temperature of -40°C, and freeze-dried under reduced pressure for 16 hours to obtain the desired freeze-dried red blood cells. Ta.

Example 3 Protein was precipitated from the streptococcus culture filtrate by the ammonium sulfate method, and streptokinase was further obtained from this protein portion by column chromatography. 10 microliters of an aqueous solution adjusted to have a streptokinase activity of 10,000 U/ml was dropped into liquid nitrogen, and the formed frozen solids were placed in a freeze dryer with a shelf temperature of -40°C as in Example 1.
Freeze-drying was carried out under reduced pressure for 16 hours to obtain a freeze-dried product having 100 U streptokinase activity per drop.

Example 4 10 pieces/50ml of A using Coulter counter
A suspension of group hemolytic streptococci was prepared. While homogeneously dispersing the mixture using a mixer, 50 microliters of the mixture was dropped into dry ice and acetone to obtain frozen fine particles. This is taken out of the refrigerant using a wire mesh, distributed one by one into cooled containers, and freeze-dried under reduced pressure for 10 hours using a freeze dryer with a shelf temperature of -30°C or lower to achieve the desired freeze-drying. I got something.

By the above method, a lyophilized group A hemolytic streptococcus containing 10 bacteria in 50 microliters was obtained.

Claims (1)

[Claims] 1. A method for producing a lyophilized product in which a granular frozen material is dried under reduced pressure at a temperature of 0° C. or lower, wherein the granular frozen material is an aqueous solution, aqueous suspension, or an aqueous solution containing a virus, bacteria, blood cells, serum, or enzyme. A method for producing the freeze-dried product, characterized in that the emulsion is formed by dropping the emulsion into a low-temperature liquid such as liquid nitrogen.