Intravenous Cytomegalovirus Human Immune Globulin and Manufacturing Method Thereof

A method for preparing intravenous cytomegalovirus human immune globulin, comprising the steps of:

• (a) preparing FI+II+III and FII+III deposits:
• preparing human plasma measured by enzyme linked immunosorbent assay, dissolving said human plasma at 2˜30° C., and mixing said human plasma, wherein said human plasma has a high titer of anti-CMV;
• preparing FI+II+III deposit;
• adjusting the protein content of said human plasma to 45˜55 mg/ml with saline, adjusting the pH of said human plasma to 6.0˜6.5 with glacial acetic acid, and adding 95% ethanol or ethanol to adjust the concentration of ethanol of said human plasma to 20˜25%, wherein the reaction temperature is −5.5˜−4.5° C., and then stirred for 4˜6 hours, after the reaction is finished, obtaining FI+II+III deposit by centrifuging or pressure filtering;
• preparing FI+II+III deposit;
• adjusting the protein content of said human plasma to 45˜55 mg/ml with saline, adjusting the pH of said human plasma to 6.8˜7.2 with glacial acetic acid, and adding 95% ethanol or ethanol to adjust the concentration of ethanol of said human plasma to 7.5˜8.5%, wherein the reaction temperature is −2.5˜−2.0° C., and then stirred for 4 hours, after the reaction is finished, removing FI deposit by centrifuging or pressure filtering to obtain the supernatant, adjusting the pH of said supernatant to 6.0˜6.5 with glacial acetic acid, adding 95% ethanol or ethanol to adjust the concentration of ethanol of said supernatant to 20˜25%, wherein the reaction temperature is −5.5˜−4.5° C., and then stirred for 4˜6 hours, after the reaction is finished, obtaining FII+III deposit by centrifuging or pressure filtering;
• (b) dissolving FI+II+III or FII+III deposit:
• dissolving FI+II+III or FII+III deposit with 20˜80 mM sodium acetate buffer solution, stirring for 8˜16 hours at 2˜8° C. to dissolve completely FI+II+III or FII+III deposit, separating the supernatant by centrifuging or pressure filtering, wherein the pH of said sodium acetate buffer solution is 4.8˜5.2, and the amount of said sodium acetate buffer solution is 0.9˜1.1 times said plasma;
• (c) depositing with caprylic acid:
• adjusting the pH of said supernatant to 4.5˜5.5 with 4 mol/L acetic acid or 0.5˜1 mol/L sodium hydroxide, adding caprylic acid until the concentration of caprylic acid is 10˜100 mmol/L, stirring for 1˜3 hours at 18˜25° C., and separating the supernatant by centrifuging or filtering;
• (d) inactivating virus with caprylic acid:
• filtering said supernatant with a 1.0 μm filter membrane, controlling the pressure to no more than 0.25 MPa, adjusting the pH of the filtrate to 4.5˜5.5 with 4 mol/L acetic acid or 0.5˜1 mol/L sodium hydroxide, adding water for infection or caprylic acid to adjust the concentration of caprylic acid of the suspension to 20˜80 mmol/L, stirring for 1˜2 hours at 20˜30° C., and separating the supernatant by centrifuging or filtering;
• (e) depositing with ethanol:
• adjusting the pH of said supernatant to 4.5˜5.5 with 0.5˜1 mol/L hydrochloric acid or sodium hydroxide, adding 95% ethanol or ethanol for precipitating until the concentration of ethanol is 12˜16%, stirring for 2˜8 hours at −4.0˜−2.5° C., and separating the supernatant by centrifuging or pressure filtering;
• (f) ultrafiltering:
• filtering said supernatant with a 0.45 μm filter membrane, controlling the pressure to no more than 0.25 MPa, concentrating the filtrate with 30 KD ultrafiltration membrane by 15˜20 times, ultrafiltering and dialyzing with 20˜60 mmol/L phosphate buffer solution, after ultrafiltering, controlling and obtaining a solution product containing 25˜40 mg/ml protein, wherein said phosphate buffer solution has a pH of 6.0˜7.1, and the volume of said phosphate buffer solution is 8˜10 times of said supernatant;
• (g) purifying with anion exchange chromatography:
• using a phosphate buffer solution as an equilibration buffer solution to balance the chromatography column, wherein said phosphate buffer solution has a pH of 6.0˜7.1, a concentration of 20˜60 mmol/L, wherein the volume of said phosphate buffer solution is 8˜10 times the volume of said chromatography column, calculating the amount of protein of the sample loaded to no more than 70˜80% of the maximum loading capacity per ml filler, after loading, collecting the penetrated solution and eluting with a 20˜60 mmol/L phosphate buffer solution having a pH of 6.0˜7.1 to remove other protein hung in said chromatography column, wherein said phosphate buffer solution contains 2 mol/L NaCl;
• (h) filtering with nano film filter to remove virus:
• adjusting the pH of the penetrated solution to 4.2˜5.0 with 0.5˜1 mol/L HCl, after pre-filtering with a 0.1 μm filter membrane, and removing the virus by Novasip DV20 nano film filter, controlling the pressure to no more than 0.25 Mpa;
• (i) Ultrafiltering:
• after removing the virus, adjusting the protein content of the filtrate to 80˜100 mg/ml by ultrafiltering with 30 KD ultrafiltration membranes, ultrafiltering with water for infection, wherein the water for infection is 8˜10 times of said filtrate, and after ultrafiltering, controlling and obtaining a product of solution containing the protein content of 80˜150 mg/ml;
• and
• (j) preparing:
• measuring the protein content of the ultrafiltrated filtrate, adjusting the protein content of product to 51˜55 mg/ml with water of infection, adding maltose malt sugar until the content of maltose malt sugar is 9˜11%, and adjusting the pH to 3.8˜4.2 with 0.5˜1 mol/L hydrochloric acid.