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Human cytomegalovirus immunoglobulin for intravenous injection and preparation method thereof

A technology of human immunoglobulin and cytomegalovirus, which is applied in the field of human immunoglobulin and its preparation, can solve the problems of low ethanol precipitation purification efficiency, high hardware and operating costs, and low process yield, so as to reduce energy consumption and labor intensity, improve product purity, improve the effect of purity and yield

Active Publication Date: 2014-04-09
SHENZHEN WEIGUANG BIOLOGICAL PROD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Long-term practice has shown that the low-temperature ethanol method has the following defects: First, ethanol is a protein denaturant, which can easily cause IgG structure changes and denature inactivation during the separation process, resulting in low titer recovery and may also lead to new The formation of antigenic determinants; secondly, compared with column chromatography technology, the ethanol precipitation purification efficiency is low, and it is usually necessary to reduce the protein recovery rate to meet the purity requirements of the product, so the yield of the process is low and the product purity is not high; thirdly, Because the separation process usually needs to be carried out under low temperature conditions, there are still defects such as high hardware and operating costs, and high labor intensity.

Method used

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  • Human cytomegalovirus immunoglobulin for intravenous injection and preparation method thereof
  • Human cytomegalovirus immunoglobulin for intravenous injection and preparation method thereof
  • Human cytomegalovirus immunoglobulin for intravenous injection and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Example 1, such as figure 1 as shown,

[0051] (1) Take 20 portions of human plasma whose anti-CMV high titer was determined by enzyme-linked immunosorbent assay, thaw the slurry at 25°C, and the volume after mixing is 11540ml.

[0052] (2) Add 2310ml of normal saline to adjust the plasma protein content to 49.53mg / ml, add glacial acetic acid to adjust the pH to 6.18, add 3926ml of absolute ethanol to adjust the ethanol concentration of the suspension to 22%, adjust the reaction temperature to -5.0°C, and stir the reaction After 4 hours, the reaction was completed and centrifuged to obtain FI+II+III precipitates.

[0053] (3) FI+II+III precipitate was dissolved in 11500 ml of sodium acetate buffer solution with a pH of 4.93 and a concentration of 50 mmol / L, stirred at 4° C. for 12 hours, and centrifuged to separate the supernatant.

[0054] (4) Adjust the pH of the supernatant to 4.57 with 4 mol / L acetic acid, add octanoic acid at a concentration of 60 mmol / L, stir an...

Embodiment 2

[0066] (1) Take 20 portions of human plasma whose anti-CMV high titer was determined by enzyme-linked immunosorbent assay, thaw the slurry at 30°C, and the volume after mixing is 11410ml.

[0067] (2) Add 2280ml of physiological saline to adjust the plasma protein content to 50.30mg / ml, add glacial acetic acid to adjust the pH to 6.48, add 4603ml of absolute ethanol to adjust the ethanol concentration of the suspension to 25%, adjust the reaction temperature to -4.5°C, and stir for 6 Hours, the reaction was completed and centrifuged to obtain FI+II+III precipitates.

[0068] (3) FI+II+III precipitate was dissolved in 10500ml of sodium acetate buffer solution with a pH of 5.16 and a concentration of 50mmol / L, stirred at 2.2°C for 12 hours, and the supernatant was separated by centrifugation.

[0069] (4) Adjust the pH of the supernatant to 5.07 with 4 mol / L acetic acid, add octanoic acid at a concentration of 30 mmol / L, stir at 25° C. for 2.5 hours, and centrifuge the supernata...

Embodiment 3

[0082] (1) Take 20 portions of human plasma whose anti-CMV high titer was determined by enzyme-linked immunosorbent assay, and thaw the plasma at 15°C, and the volume after mixing is 11570ml.

[0083] (2) Add 2310ml of normal saline to adjust the plasma protein content to 49.69mg / ml, add glacial acetic acid to adjust the pH to 6.32, add 4603ml of absolute ethanol to adjust the ethanol concentration of the suspension to 20%, adjust the reaction temperature to -4.8°C, and stir for 6 hours After the reaction is completed, centrifuge to obtain FI+II+III precipitate.

[0084] (3) The FI+II+III precipitate was dissolved in 12500 ml of sodium acetate buffer solution with a pH of 5.02 and a concentration of 80 mmol / L, stirred at 6.0° C. for 14 hours, and the supernatant was separated by centrifugation.

[0085] (4) Adjust the pH of the supernatant to 5.50 with 1 mol / L sodium hydroxide, add octanoic acid at a concentration of 40 mmol / L, stir at 23° C. for 1 hour, and centrifuge the sup...

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Abstract

The invention discloses a human cytomegalovirus immunoglobulin for intravenous injection and a preparation method thereof, and aims to improve the purity, yield and safety of the product. In the invention, the specific activity of the human cytomegalovirus immunoglobulin for intravenous injection is not less than 2.5 PEI-U / mg, the anti-CMV titer is not less than 100 PEI-U / ml, the purity is greater than 98.2%, and the protein content is 51-55 mg / ml. Caprylic acid precipitation and anion exchange chromatography are used instead of the partial ethanol precipitation step in the traditional low-temperature ethanol method, thereby keeping IgG in the supernate all the time so as to keep the IgG activity; and processes of caprylic acid virus inactivation and nano film virus removal are used, thereby effectively ensuring the safety of the product. Researches show that the preparation method disclosed by the invention improves the purity, yield and safety of the product, saves the energy and reduces the production cost.

Description

technical field [0001] The invention relates to a human immunoglobulin and a preparation method thereof, in particular to an intravenous injection of cytomegalovirus human immunoglobulin and a preparation method thereof. Background technique [0002] Cytomegalovirus CMV (Cytomegalovirus) is a DNA virus of the genus β in the family Herpesviridae. The infection mortality rate of pregnant women, newborns, organ transplant patients and immunosuppressed patients caused by CMV can reach 50% to 80%. Because human immunoglobulin CMV-IgG can specifically neutralize cytomegalovirus, it is mainly used clinically to treat severe cytomegalovirus infection caused by pregnant women, newborns, immunosuppressed patients and organ transplantation. The natural infection rate of cytomegalovirus in the general population can reach more than 80%. The plasma containing high-titer cytomegalovirus IgG antibody is screened and collected from healthy people, and the cytomegalovirus human immunoglobuli...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/08C07K1/36C07K1/30C07K1/18A61K39/42A61P31/22
Inventor 张运佳郭采平丁玉江宋清爽张信王锦才吴恩应张佩陈玉琴李慧黄伟荣戴美兰周兰贞王春华罗姗黄倩云刘永娣吴开永
Owner SHENZHEN WEIGUANG BIOLOGICAL PROD
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