CN104479011A - Method for preparing intravenous immunoglobulin - Google Patents

Abstract

The invention provides a method for preparing the intravenous immunoglobulin. The method particularly includes the steps of dissolving component I+II+III or component II+III sediments; separating component I+III-1 or component III-1 sediments; separating component I+III-2 or component III-2 sediments; separating component II sediments; dissolving and filtering the component II sediments; conducting ultrafiltration, dialyzing and concentrating; conducting purification and filtering; conducting ultrafiltration dialysis, concentrating and preparation. By means of the method, steps and step parameters of the traditional process method are improved, specific combinations of the steps and the step parameters can be used for effectively extracting the human immunoglobulin for the intravenous injections from the component I+II+III or component II+III sediments, the technical problems that the human immunoglobulin for the intravenous injections is low in purity, and the content of polymer and the like is slightly high are solved, the pharmacy risk of people is reduced, and the pharmacy safety of people is guaranteed.

Description

A kind of preparation method of quiet note human normal immunoglobulin

Technical field

The present invention relates to field of biological pharmacy, particularly relate to a kind of preparation method of quiet note human normal immunoglobulin.

Background technology

Immunoglobulin (Ig) is a kind of glycoprotein being synthesized by bone-marrow-derived lymphocyte, secrete, and be mainly present in the recycle system, cell surface also has distribution.The major function of immunoglobulin (Ig) is to resist the infection of various pathogenic micro-organism to human body.Be mainly used in: 1. primary and secondary immunodeficiency disease; 2. idiopathic thrombocytopenic purpura; 3. mucocutaneous lymphnode syndrome; 4. the treatment of acquired immune deficiency syndrome (AIDS); 5. other indications, as diabetes, rheumatoid arthritis, myasthenia gravis, autoimmune anemia, aplastic anemia etc.Immunoglobulin (Ig) production technique is a kind of cold ethanol protein separating technology researched and developed by medical college of Harvard University professor Cohn, this technology regulates the top 5 factor of cold ethanol method by multistep: ionic strength, pH value, temperature, alcohol concn and protein content realize being separated the component I I in I+II+III (or component I I+III) precipitation, then obtains quiet note human normal immunoglobulin through purifying further.This traditional production technique adopts two-step approach to extract quiet note human normal immunoglobulin, and its maximum defect is that the quiet note human normal immunoglobulin purity of producing is on the low side, and polymer equal size is higher, easily causes the untoward reaction after infusion.Because multimeric molecule amount wherein can cause anaphylactoid reaction, anticomplementary activity and blood pressure drops more greatly, its therapeutic domain is goed deep in continuous increase along with what study, as tetter, organ transplantation, tumour, leukemia etc., some client need life-time service or single-bolus high-dose infusion, such risk will increase greatly.

Summary of the invention

The technical problem to be solved in the present invention is to provide one, and to prepare purity high, the preparation method of the quiet note human normal immunoglobulin that Content of polymer, anticomplementary activity, Prekallikrein content are lower.

The invention provides a kind of preparation method of quiet note human normal immunoglobulin, comprise the steps:

Step one: component I+II+III or component I I+III resolution of precipitate

The aqueous solution of component I+II+III precipitation or component I I+III precipitation SODIUM PHOSPHATE, MONOBASIC is dissolved completely;

Step 2: separated portion I+III-1 precipitates or component III-1 precipitates

Regulate said components I+II+III resolution of precipitate liquid or component I I+III resolution of precipitate liquid pH value to 5.00 ~ 5.20, adjust the temperature to-0.5 ~ 0.0 DEG C, using the ethanolic soln of less than-20 DEG C 95% to be adjusted to ethanol final concentration is again 7.5 ~ 8.5%, and adjusted to ph to 5.00 ~ 5.20, adjust the temperature to-3.0 DEG C ~-2.0 DEG C, stirring makes it react completely, diatomite 3.5 ~ 4.5kg and perlite 2.0 ~ 3.0kg is added by every 1000kg suspension, stir, start suspension press filtration, controlled filter pressure is 0 ~ 0.3MPa, filtration velocity is 20 ~ 90kg/min, filtrate temperature-3.0 ~-1.0 DEG C, collect component I+III-1 supernatant liquor or component III-1 supernatant liquor,

Step 3: separated portion I+III-2 precipitates or component III-2 precipitates

Adjustment component I+III-1 supernatant liquor or component III-1 supernatant liquor pH value to 5.10 ~ 5.30, ethanol final concentration to 14 ~ 16% is regulated with the ethanol of less than-20 DEG C 95%, and adjusted to ph to 5.10 ~ 5.30, temperature of reaction is to-5.0 DEG C ~-4.0 DEG C, stirring makes it react completely, diatomite 2.0 ~ 3.0kg and perlite 2.0 ~ 3.0kg is added by every 1000kg suspension, stir, start suspension press filtration, controlled filter pressure is 0 ~ 0.3MPa, filtration velocity is 20 ~ 90kg/min, filtrate temperature-5.0 ~-3.0 DEG C, collect component I+III-2 supernatant liquor or component III-2 supernatant liquor,

Step 4: separated portion II precipitates

Every 1000KgI+III-2 supernatant liquor or component III-2 supernatant liquor add the sodium chloride solution 18.4 ~ 22.4kg of 25 ~ 35%, adjust ph to 6.90 ~ 7.10, alcohol concn to 19 ~ 21% is regulated with the ethanol of less than-20 DEG C 95%, adjusted to ph to 6.90 ~ 7.10, regulate temperature of reaction to-8.0 ~-6.0 DEG C, stirring makes it react completely, perlite 0.5 ~ 1.0kg is added by every 1000kg suspension, stir, start suspension press filtration, controlled filter pressure is 0.300MPa, filtration velocity is 20 ~ 90kg/min, filtrate temperature-7.5 ~-5.5 DEG C, supernatant discharges, collect component I I precipitation,

Step 5: component I I resolution of precipitate, filtration

The component I I of collection precipitation is added water for injection be stirred to and dissolve completely, adjusted to ph to 6.90 ~ 7.10, adjust the temperature to 0.0 ~ 4.0 DEG C, continue to stir, dissolve suspension by every 1000kg and add diatomite 1.0 ~ 2.0kg and perlite 1.0 ~ 2.0kg, stir, start press filtration, controlled filter pressure is 0.200MPa, filtration velocity is 10 ~ 60kg/min, filtrate temperature 0.0 ~ 5.0 DEG C, the isolated filtrate of press filtration, adjusted filtrate pH value to 3.80 ~ 4.10;

Step 6: ultrafiltration, dialysis, concentrated

Filtered liquid being concentrated into protein content is 8 ~ 12%, then continuous equivalent dialysis is carried out with the water for injection of 2 ~ 5 DEG C, 4 ~ 6 times volumes, proteins concentrate content to 8 ~ 9%, then adjusted to ph to 6.50 ~ 7.30, controlling protein content with water for injection dilution is 50.0 ~ 100.0g/L;

Step 7: purifying, filtration

Goods after step 6 is concentrated water for injection diluted protein matter content to 1.5 ~ 2.0% of 0.0 ~ 4.0 DEG C, SODIUM PHOSPHATE, MONOBASIC 1.0 ~ 3.0kg and sodium-chlor 0.2 ~ 1.0kg is added at every 1000kg goods after dilution, adjusted to ph to 6.90 ~ 7.10, adjust the temperature to 0.0 ~ 0.5 DEG C, alcohol concn to 1.5 ~ 2.5% is regulated with the ethanol of less than-20 DEG C 95%, adjust the temperature to-2.0 ~-1.0 DEG C, stirring makes it react completely, perlite 1.0 ~ 2.0kg is added by every 1000kg suspension, stir, start press filtration, controlled filter pressure is 0.200MPa, filtration velocity is 10 ~ 60kg/min, filtrate temperature-2.0 ~-1.0 DEG C, regulate filtrate pH value to 3.80 ~ 4.10, adjust the temperature to-2.0 ~-1.0 DEG C,

Step 8: ultrafiltration dialysis, concentrated, preparation

Be 8 ~ 12% by the filtrate of step 7 after pH and temperature regulate through ultrafiltration and concentration to protein content, then continuous equivalent dialysis is carried out with water for injection, proteins concentrate content to 8 ~ 9%, often rise by end article and add maltose containing maltose 95 ~ 105g, adjust ph to 3.90 ~ 4.30, controlling final protein content with water for injection dilution is 5.20 ~ 5.50%, carry out again degerming, except virus filtration, incubated at low pH and degerming packing, namely obtain quiet note human normal immunoglobulin work in-process.

The aqueous solution of SODIUM PHOSPHATE, MONOBASIC described in described step one is 6 ~ 8 times of component I+II+III or component I I+III Sediment weight, and concentration is 0.01mol/L, and churning time is more than 6 hours.

The solvent being used for regulating pH value in described step 2, three, four and five is 1mol/L acetum or 0.5mol/L sodium hydroxide solution.

In described step 6, filtered liquid to be concentrated into protein content be 8 ~ 12% is adopt film specification to be that 30KD ultra-fine filter concentrates.

The invention provides a kind of method of producing quiet note human normal immunoglobulin, this method has made improvement for each step of Conventional processing methods and each step parameter, the particular combination of each step and each step parameter effectively can extract quiet note human normal immunoglobulin from I+II+III (or component I I+I II) precipitation, solve quiet note human normal immunoglobulin purity on the low side, the technical barrier that polymer equal size is higher, reduce people's drug risk, ensure people's drug safety.

Accompanying drawing explanation

Fig. 1 is the production technological process that feed composition I+II+III (or component I I+III) of the present invention is precipitated.

Fig. 2 is traditional quiet note human normal immunoglobulin production technological process.

Fig. 3 is quiet note human normal immunoglobulin production technological process of the present invention.

Embodiment

Feed composition I+II+III in the present invention precipitates and II+III precipitation can adopt the most conventional explained hereafter, such as, can prepare according to the following steps of Fig. 1:

One, prepared by component I+II+III

1 blood plasma metering: by merge melt after human normal plasma (gathered by blood-collecting station and through twice inspection and meet window phase management requirement) metering.

2 adjustment protein contents: regulate plasma proteins content to 4.8 ~ 5.3% with 0.9% sodium chloride solution.

3 adjust ph: with pH4.0 Acetic acid-sodium acetate damping fluid adjust ph to 6.20 ~ 6.30.

4 regulate alcohol concn: add 95% ethanol and regulate ethanol final concentration to 20 ± 1%.

5 repetition measurements and adjusted to ph: after adding 95% ethanol, repetition measurement pH value should be 6.60 ~ 6.70.As pH value is less than 6.60, then apply 0.5mol/L sodium hydroxide and regulate so far in scope; As pH value is greater than 6.70, then apply pH4.0 Acetic acid-sodium acetate damping fluid and regulate so far in scope.

6 regulate temperature of reaction: regulate temperature of reaction to-5.0 ~-4.0 DEG C, temperature continues stirring reaction more than 2 hours after arriving target value, and obtain component I+II+III suspension, at this moment its protein content is 3.5 ~ 4.5%.

7 add flocculating aids: every 1000kg component I+II+III suspension adds diatomite 3.5 ~ 4.5kg, perlite 2 ~ 3kg, after adding above-mentioned flocculating aids, continue stirring more than 10 minutes.

8 suspension press filtrations: assemble filter plate on a filter press, hydraulic pressure 190 ~ 240bar is regulated to compress pressure filter, with 20% balance liquid, balance cooling is carried out to whole press filtration pipeline and pressure filter, setting purge time 20 ~ 40 minutes, filter plate is dried up with precooling clean compressed air, make exhaust temperature reach-5.0 ~-3.0 DEG C, component FI+II+III press filtration precooling compressed air temperature be in steps-7.0 ~-3.0 DEG C.Start suspension press filtration, controlled filter speed is 20 ~ 90kg/min, and check filtrate clarity, intake pressure is 0.300MPa, and filtrate temperature is-5.0 ~-3.0 DEG C.

9 tank decks are washed: after suspension press filtration completes, and carry out clean and reuse with 20% balance liquid 50 ~ 100kg to the tank body of suspension tank, washing lotion is target approach tank after pressure filter.After tank deck washes into, setting purge time 30 ~ 60 minutes, dries up filter plate with precooling clean compressed air; Then carry out clean and reuse through the bypass of suspension tank to pressure filter with 20% balance liquid, Ethylene recov target approach tank, terminates after fluid 50 ~ 150kg.Setting purge time 30 ~ 150 minutes, dries up filter plate with precooling clean compressed air.

10 collecting precipitations: unclamp pressure filter, collect component I+II+III precipitation, collect rear placement less than-30 DEG C freezers and preserve.

Two, component I I+III precipitation

1 centrifugal cryoprecipitate

1.1 blood plasma meterings: melt the blood plasma metering after merging.

1.2 centrifugal cryoprecipitates: blood plasma is sent to centrifugal by transport pipe, use whizzer to carry out cryoprecipitate centrifugation, go out liquid temp and control at 0.5 ~ 2.5 DEG C, fluid speed is less than 2.5kg/min, collects centrifugal supernatant.

2 component I make, centrifugal

The making of 2.1 component I

2.1.1 the supernatant metering after centrifugal cryoprecipitate.

2.1.2 adjust ph: regulate pH to 7.00 ~ 7.20 with pH4.0 Acetic acid-sodium acetate damping fluid.

2.1.3 alcohol concn is regulated: regulate alcohol concn to 8.0 ± 0.5% with 95% ethanol.

2.1.4 repetition measurement and adjusted to ph: after adding 95% ethanol, repetition measurement pH value should be 7.00 ~ 7.20.As pH value is less than 7.00, then apply 0.5mol/L sodium hydroxide solution and regulate so far in scope; As pH value is greater than 7.20, then apply pH4.0 Acetic acid-sodium acetate damping fluid and regulate so far in scope.

2.1.5 temperature of reaction is regulated: regulate temperature of reaction to-2.5 ~-1.5 DEG C, stirring reaction more than 30 minutes after temperature arrival target value.

2.1.6 protein content is checked: final protein content should be 4.5 ~ 5.5%.

2.2 component I are centrifugal: centrifugal fluid speed is that every bench centrifuge is less than 2.0kg/min, and check fluid clarity, going out liquid temp is-2.0 ~-1.0 DEG C, collects centrifugal supernatant.

3 component I I+III make, press filtration

3.1 component I I+III makes

3.1.1 the supernatant metering after centrifugal component I.

3.1.2 protein content is adjusted: with 8% balance liquid, protein content is adjusted to 4.00 ~ 5.00%.

3.1.3 adjust ph: regulate pH to 6.40 ~ 6.50 with pH4.0 Acetic acid-sodium acetate damping fluid.

3.1.4 alcohol concn is regulated: regulate alcohol concn to 20 ± 1% with 95% ethanol.

3.1.5 repetition measurement and adjust ph: after adding 95% ethanol, repetition measurement pH value should be 6.60 ~ 6.70.As pH value is less than 6.60, then apply 0.5mol/L sodium hydroxide and regulate so far in scope; As pH value is greater than 6.70, then apply pH4.0 Acetic acid-sodium acetate damping fluid and regulate so far in scope.

3.1.6 temperature of reaction is regulated: regulate temperature of reaction to-5.0 ~-4.0 DEG C, temperature continues stirring reaction more than 2 hours after arriving target value.

3.1.7 protein content is checked: final protein content should be 3.0 ~ 4.5%.

3.2 component I I+III precipitate press filtration

Except the drainage dosage added is different, all the other operation stepss consistent with " step 2 component I+II+III precipitates press filtration " in above-mentioned " the feed composition I+II+III production stage in the present invention " (wherein flocculating aids dosage is that every 1000kg component I I+III suspension adds diatomite 5.5 ± 0.5kg, perlite 3.5 ± 0.5kg).

Utilize feed composition I+II+III to precipitate and II+III precipitation quiet note human normal immunoglobulin work in-process, technical process as shown in Figure 3, comprises the steps:

Step one: component I+II+III (or component I I+III) resolution of precipitate

The aqueous solution containing 0.01mol/L SODIUM PHOSPHATE, MONOBASIC of 0.0 ~ 4.0 DEG C of component I+II+III (or component I I+III) precipitation precipitation 6 ~ 8 times of weight being stirred more than 6 hours to dissolving completely, obtaining component I+II+III (or component I I+III) resolution of precipitate liquid.

Wherein the compound method of the aqueous solution containing 0.01mol/L SODIUM PHOSPHATE, MONOBASIC of 0.0 ~ 4.0 DEG C is: take 1.38kg SODIUM PHOSPHATE, MONOBASIC by every 1000kg water for injection and add to be stirred to and dissolve completely.

Step 2: separated portion I+III-1 (or component III-1) precipitates

Said components I+II+III (or component I I+III) resolution of precipitate liquid pH value to 5.00 ~ 5.20 are regulated with 1mol/L acetum,-0.5 ~ 0.0 DEG C is cooled to-15 DEG C of refrigerants, being adjusted to ethanol final concentration with less than-20 DEG C 95% (v/v) ethanolic solns is 7.5 ~ 8.5% (v/v), and with 1mol/L acetum or 0.5mol/L sodium hydroxide solution adjusted to ph to 5.00 ~ 5.20, regulate last suspension temperature to-3.0 DEG C ~-2.0 DEG C with-15 DEG C of refrigerants, with stirring velocity be 50 ~ 100rpm stir 2 ~ 3 hours, diatomite 3.5 ~ 4.5kg and perlite 2.0 ~ 3.0kg is added by every 1000kg suspension after reacting completely, be that 50 ~ 100rpm stirs 30 minutes with stirring velocity, with 8% balance liquid 800 ~ 1200Kg, balance cooling is carried out to whole press filtration pipeline and pressure filter, setting purge time 20 ~ 40 minutes, (German Begerow company produces to dry up filter plate with-4.0 ~ 0.0 DEG C of precooling clean compressed air, model: PX50, specification 680*779mm), exhaust temperature is made to reach-3.0 ~-1.0 DEG C.Start suspension press filtration, controlled filter pressure is 0 ~ 0.3MPa, filtration velocity is 20 ~ 90kg/min, filtrate temperature-3.0 ~-1.0 DEG C, suspension has pressed setting purge time 30 ~ 60 minutes, filter plate is dried up with precooling clean compressed air, with 8% balance liquid 800 ~ 1000Kg, clean and reuse is carried out to pressure filter, Ethylene recov is incorporated to press filtration supernatant tank, setting purge time 30 ~ 150 minutes, re-use-4.0 ~ 0.0 DEG C of precooling clean compressed air to dry up, collect component I+III-1 (or component III-1) supernatant liquor, component I+III-1 (or component III-1) is discarded after precipitating autoclaving,

8% balance liquid compound method used is: 95% (v/v) ethanol 77.7kg that every 1000kg water for injection adds less than-20 DEG C obtains the ethanolic soln of 8% (v/v); Then in the ethanolic soln of every 1000kg 8%, Sodium phosphate dibasic 3.58kg is added; With Glacial acetic acid adjust ph to 4.90 ~ 5.10;-3.0 ~-2.0 DEG C are adjusted the temperature to-15 DEG C of refrigerants.

Step 3: separated portion I+III-2 (or component III-2) precipitates

With 0.5mol/L sodium hydroxide solution adjustment component I+III-1 (or component III-1) supernatant liquor pH value to 5.10 ~ 5.30, ethanol final concentration to 14 ~ 16% (v/v) is regulated with less than-20 DEG C 95% (v/v) ethanol, and with 1mol/L acetic acid or 0.5mol/L sodium hydroxide adjusted to ph to 5.10 ~ 5.30, regulate temperature of reaction to-5.0 DEG C ~-4.0 DEG C with-15 DEG C of refrigerants, with stirring velocity be 50 ~ 100rpm stir 2 ~ 3 hours, diatomite 2.0 ~ 3.0kg and perlite 2.0 ~ 3.0kg is added by every 1000kg suspension after reacting completely, be that 50 ~ 100rpm stirs 30 minutes with stirring velocity, with 15% balance liquid 800 ~ 1200Kg, balance cooling is carried out to whole press filtration pipeline and pressure filter, setting purge time 20 ~ 40 minutes, (German Begerow company produces to dry up filter plate with-6.5 ~-2.5 DEG C of precooling clean compressed airs, model: PX50, specification 680*779mm), exhaust temperature is made to reach-5.0 ~-3.0 DEG C.Start suspension press filtration, controlled filter pressure is 0 ~ 0.3MPa, filtration velocity is 20 ~ 90kg/min, filtrate temperature-5.0 ~-3.0 DEG C, suspension has pressed setting purge time 30 ~ 60 minutes, filter plate is dried up with precooling clean compressed air, with 15% balance liquid 800 ~ 1000Kg, clean and reuse is carried out to pressure filter, Ethylene recov is incorporated to press filtration supernatant tank, setting purge time 30 ~ 150 minutes, re-use-6.5 ~-2.5 DEG C of precooling clean compressed airs to dry up, collect component I+III-2 (or component III-2) supernatant liquor, component I+III-2 (or component III-2) precipitates autoclaving and discards,

15% balance liquid compound method used is: every 1000kg water for injection adds the ethanol that less than-20 DEG C 95% (v/v) ethanol 158.3kg obtain 15% (v/v); Then in every 1000kg 15% ethanol, Sodium phosphate dibasic 3.58kg is added; With Glacial acetic acid adjust ph to 5.10 ~ 5.30;-5.0 ~-4.0 DEG C are adjusted the temperature to-15 DEG C of refrigerants;

Step 4: separated portion II precipitates

Take the weight of component I+III-2 (or component III-2) supernatant liquor, every 1000Kg supernatant liquor adds 25 ~ 35% (m/v) sodium chloride solution, 18.4 ~ 22.4kg, with 0.5mol/L sodium hydroxide solution adjust ph to 6.90 ~ 7.10, alcohol concn to 19 ~ 21% (v/v) is regulated with less than-20 DEG C 95% (v/v) ethanol, repetition measurement adjusted to ph to 6.90 ~ 7.10 (with 1mol/L acetic acid or the adjustment of 0.5mol/L sodium hydroxide), regulate temperature of reaction to-8.0 ~-6.0 DEG C with-15 DEG C of refrigerants, continue stirring reaction 2 ~ 3 hours, its stirring velocity is 50 ~ 100RPM, perlite 0.5 ~ 1.0kg is added by every 1000kg suspension after reacting completely, stir 30 minutes, its stirring velocity is 50 ~ 100RPM, with 20% (v/v) ethanol, 1000 ~ 2000Kg, balance cooling is carried out to whole press filtration pipeline and pressure filter, setting purge time 20 ~ 40 minutes, (German Begerow company produces to dry up filter plate with-10.0 ~-6.0 DEG C of precooling clean compressed airs, model: PX50, specification 680*779mm), exhaust temperature is made to reach-7.5 ~-5.5 DEG C, start suspension press filtration, controlled filter pressure is 0.300MPa, filtration velocity is 20 ~ 90kg/min, filtrate temperature-7.5 ~-5.5 DEG C, supernatant directly discharges through ethanol emission pipeline, when component I I suspension tank weighs less than 200kg, press filtration supernatant no longer discharges and directly enters suspension tank thus form a circulation line, after circulation 10min, supernatant starts to continue through the discharge of ethanol emission pipeline, after the whole press filtration of suspension is complete, setting purge time is 30 ~ 150 minutes, filter plate is dried up with-10.0 ~-6.0 DEG C of precooling clean compressed airs, collect component I I precipitation,

20% ethanol compound method used is: 95% (v/v) ethanol 225.5kg that every 1000kg water for injection adds less than-20 DEG C obtains the ethanol of 20% (v/v), adjusts the temperature to-8.5 ~-6.5 DEG C with-15 DEG C of refrigerants;

Step 5: component I I resolution of precipitate, filtration

The water for injection of 0.0 ~ 4.0 DEG C of component I I precipitation precipitation 5 ~ 7 times of weight of collecting is dissolved, stir under stir speed (S.S.) is 50 ~ 100rpm and dissolve completely, with 0.5mol/L sodium hydroxide solution or 1mol/L acetum adjusted to ph to 6.90 ~ 7.10 to precipitation for more than 4 hours, 0.0 ~ 4.0 DEG C is adjusted the temperature to-15 DEG C of refrigerants, continue stirring reaction 2 ~ 3 hours under stir speed (S.S.) is 50 ~ 100rpm, dissolve completely and add diatomite 1.0 ~ 2.0kg and perlite 1.0 ~ 2.0kg by every 1000kg dissolving suspension, stir 30 minutes under stir speed (S.S.) is 50 ~ 100rpm, with 0.0 ~ 4.0 DEG C of water for injection 300 ~ 500kg, balance cooling is carried out to whole press filtration pipeline and pressure filter, setting purge time 10 ~ 30 minutes, (German Begerow company produces to dry up filter plate with 0.0 ~ 4.0 DEG C of precooling clean compressed air, model: Steril S100, specification: 349*384mm), exhaust temperature is made to reach 0.0 ~ 4.0 DEG C, start press filtration, controlled filter pressure is 0.200MPa, filtration velocity is 10 ~ 60kg/min, filtrate temperature 0.0 ~ 5.0 DEG C, dissolve the complete setting purge time of hydraulic pressure 10 ~ 30 minutes, press filtration separate dissolved suspension, with 50 ~ 200kg, 0.0 ~ 4.0 DEG C of water for injection, clean and reuse is carried out to pressure filter, filter plate is dried up with 0.0 ~ 4.0 DEG C of precooling clean compressed air, Ethylene recov is incorporated to filtrate tank, setting purge time is 10 ~ 30 minutes, filter plate is dried up with 0.0 ~ 4.0 DEG C of precooling clean compressed air, filtrate pH value to 3.80 ~ 4.10 are regulated with 1mol/L acetum, filtering-depositing autoclaving is discarded,

Step 6: ultrafiltration, dialysis, concentrated

Be that filtered liquid is concentrated into protein content is 8 ~ 12% (m/v) to 30KD ultra-fine filter by film specification, then continuous equivalent dialysis is carried out with the water for injection of 2 ~ 5 DEG C, 4 ~ 6 times volumes, proteins concentrate content to 8 ~ 9% (m/v), again with NaOH solution adjustment goods pH value to 6.50 ~ 7.30 of 0.5mol/L, controlling protein content with water for injection dilution is 50.0 ~ 100.0g/L;

Step 7: purifying, filtration

Goods after concentrated are transferred to sphaeroprotein treatment tank, by water for injection diluted protein matter content to 1.5 ~ 2.0% (m/v) of 0.0 ~ 4.0 DEG C, SODIUM PHOSPHATE, MONOBASIC 1.38kg and sodium-chlor 0.585kg is added at every 1000kg goods after dilution, with 0.5mol/L sodium hydroxide solution or 1mol/L acetum adjusted to ph to 6.90 ~ 7.10, 0.0 ~ 0.5 DEG C is adjusted the temperature to-15 DEG C of refrigerants, alcohol concn to 1.5 ~ 2.5% (v/v) is regulated with less than-20 DEG C 95% (v/v) ethanol, regulate outlet temperature to-2.0 ~-1.0 DEG C with-15 DEG C of refrigerants, stirring reaction is continued 2 ~ 3 hours under stir speed (S.S.) is 50 ~ 100rpm, perlite 1.0 ~ 2.0kg is added by every 1000kg suspension after reacting completely, stir 30 minutes under stir speed (S.S.) is 50 ~ 100rpm, with the 2% ethanol 300 ~ 500kg of-2.0 ~-1.0 DEG C, balance cooling is carried out to whole press filtration pipeline and pressure filter, setting purge time 10 ~ 30 minutes, (German Begerow company produces to dry up filter plate with-2.0 ~ 2.0 DEG C of precooling clean compressed airs, model: Steril S100, specification: 349*384mm), exhaust temperature is made to reach-2.0 ~-1.0 DEG C, start press filtration, controlled filter pressure is 0.200MPa, filtration velocity is 10 ~ 60kg/min, filtrate temperature-2.0 ~-1.0 DEG C, the complete setting purge time of suspension press filtration is 10 ~ 30 minutes, with 50 ~ 200kg 2% ethanol, clean and reuse is carried out to pressure filter, Ethylene recov is incorporated to filtrate tank, filter plate is dried up with-2.0 ~ 2.0 DEG C of precooling clean compressed airs, filtering supernatant pH value to 3.80 ~ 4.10 are regulated with 0.5mol/L hydrochloric acid soln, regulate outlet temperature to-2.0 ~-1.0 DEG C,

2% ethanol compound method used is: every 1000kg water for injection adds less than-20 DEG C 95% (v/v) ethanol 18.2kg, adjusts the temperature to-2.0 ~-1.0 DEG C with-15 DEG C of refrigerants;

Step 8: ultrafiltration dialysis, concentrated, preparation

Be 8 ~ 12% (m/v) (intake pressure≤4.0bar by the filtrate of step 7 after pH and temperature regulate through ultrafiltration and concentration to protein content, return pressure≤2.0bar), then continuous equivalent dialysis is carried out with the water for injection of 2 ~ 5 DEG C of 10 ~ 13 times of volumes, proteins concentrate content to 8 ~ 9% (m/v), often rise by end article and add maltose containing maltose 95 ~ 105g, with 0.5mol/L hydrochloric acid soln or 0.5mol/L sodium hydroxide solution adjust ph to 3.90 ~ 4.30, controlling final protein content with water for injection dilution is 5.20 ~ 5.50% (m/v), carry out degerming again, except virus filtration, incubated at low pH and degerming packing, namely quiet note human normal immunoglobulin work in-process are obtained.

Product checking: the present invention all detects the half-finished protein content of described quiet note human normal immunoglobulin prepared, purity, pH value, residual ethanol content, Content of polymer, ACA (anticomplementary activity), PKA (Prekallikrein) content, and contrast with national standard (version " Chinese Pharmacopoeia " in 2010) and prior art, its result is as shown in table 1.

Embodiment 1

Step one: component I+II+III resolution of precipitate

By the aqueous solution (the phosphoric acid sodium dihydrogen 0.54kg containing 0.54kg SODIUM PHOSPHATE, MONOBASIC of 0.7 DEG C of component I+II+III56.3kg with 394.54kg, water for injection 394.0kg) stir more than 6 hours to dissolving completely, obtain component I+II+III resolution of precipitate liquid 450.8kg.

Step 2: separated portion I+III-1 precipitates

Said components I+II+III resolution of precipitate liquid pH value to 5.03 is regulated with 1mol/L acetum 3.0kg,-0.4 DEG C is cooled to-15 DEG C of refrigerants, add 95% (v/v) ethanolic soln 28.2kg of-20.8 DEG C again, and by 0.5mol/L sodium hydroxide solution adjusted to ph to 5.12, regulate last suspension temperature to-2.6 DEG C with-15 DEG C of refrigerants, be that 90rpm stirs 2 hours with stirring velocity, diatomite 1.9kg and perlite 1.2kg is added after reacting completely, be that 80rpm stirs 30 minutes with stirring velocity, with 8% balance liquid 927Kg, balance cooling is carried out to whole press filtration pipeline and pressure filter, setting purge time 29 minutes, (German Begerow company produces to dry up filter plate with-1.7 DEG C of precooling clean compressed airs, model: PX50, specification 680*779mm), exhaust temperature is made to reach-1.8 DEG C.Start suspension press filtration, controlled filter pressure is 0.20MPa, filtration velocity is 79kg/min, filtrate temperature-2.0 DEG C, suspension has pressed setting purge time 49 minutes, filter plate is dried up with precooling clean compressed air, with 8% balance liquid 927Kg, clean and reuse is carried out to pressure filter, Ethylene recov is incorporated to press filtration supernatant tank, setting purge time 82 minutes, re-use-2.2 DEG C of precooling clean compressed airs to dry up, collect component I+III-1 supernatant liquor 1372.7kg, component I+III-1 is discarded after precipitating 39.4kg autoclaving;

8% balance liquid compound method used is: 95% (v/v) ethanol 77.7kg that every 1000kg water for injection adds less than-20 DEG C obtains the ethanolic soln of 8% (v/v); Then in the ethanolic soln of every 1000kg 8%, Sodium phosphate dibasic 3.58kg is added; By Glacial acetic acid adjust ph to 5.01;-2.8 DEG C are adjusted the temperature to-15 DEG C of refrigerants.

Step 3: separated portion I+III-2 precipitates

Component I+III-1 supernatant liquor pH value to 5.24 is adjusted with 0.5mol/L sodium hydroxide solution 0.7kg, add 95% (v/v) ethanol 102.5kg of-21.3 DEG C again, and by 0.5mol/L sodium hydroxide adjusted to ph to 5.20, regulate temperature of reaction to-4.9 DEG C with-15 DEG C of refrigerants, be that 77rpm stirs 2.9 hours with stirring velocity, diatomite 3.7kg and perlite 3.7kg is added after reacting completely, be that 75rpm stirs 30 minutes with stirring velocity, with 15% balance liquid 982Kg, balance cooling is carried out to whole press filtration pipeline and pressure filter, setting purge time 29 minutes, (German Begerow company produces to dry up filter plate with-5.0 DEG C of precooling clean compressed airs, model: PX50, specification 680*779mm), exhaust temperature is made to reach-3.8 DEG C.Start suspension press filtration, controlled filter pressure is 0.21MPa, filtration velocity is 78kg/min, filtrate temperature-4.1 DEG C, suspension has pressed setting purge time 46 minutes, filter plate is dried up with precooling clean compressed air, with 15% balance liquid 879Kg, clean and reuse is carried out to pressure filter, Ethylene recov is incorporated to press filtration supernatant tank, setting purge time 99 minutes, re-use-4.5 DEG C of precooling clean compressed airs to dry up, collect component I+III-2 supernatant liquor 2345.4kg, component I+III-2 precipitates 16.9kg autoclaving and discards;

15% balance liquid compound method used is: every 1000kg water for injection adds the ethanol that less than-20 DEG C 95% (v/v) ethanol 158.3kg obtain 15% (v/v); Then in every 1000kg 15% ethanol, Sodium phosphate dibasic 3.58kg is added; By Glacial acetic acid adjust ph to 5.25;-4.8 DEG C are adjusted the temperature to-15 DEG C of refrigerants;

Step 4: separated portion II precipitates

30% (m/v) sodium chloride solution 47.8kg is added in said components I+III-2 supernatant liquor, by 0.5mol/L sodium hydroxide solution 37.5kg adjust ph to 7.07, add 95% (v/v) ethanol 154.9kg of-20.1 DEG C again, by 1mol/L acetic acid adjusted to ph to 7.00, regulate temperature of reaction to-7.2 DEG C with-15 DEG C of refrigerants, continue stirring reaction 2.3 hours, its stirring velocity is 70RPM, perlite 1.9kg is added after reacting completely, stir 30 minutes, its stirring velocity is 70RPM, with 20% (v/v) ethanol 1536Kg of-6.8 DEG C, balance cooling is carried out to whole press filtration pipeline and pressure filter, setting purge time 30 minutes, (German Begerow company produces to dry up filter plate with-8.4 DEG C of precooling clean compressed airs, model: PX50, specification 680*779mm), exhaust temperature is made to reach-7.0 DEG C, start suspension press filtration, controlled filter pressure is 0.15MPa, filtration velocity is 70kg/min, filtrate temperature-6.2 DEG C, supernatant directly discharges through ethanol emission pipeline, when component I I suspension tank weighs less than 200kg, press filtration supernatant no longer discharges and directly enters suspension tank thus form a circulation line, after circulation 10min, supernatant starts to continue through the discharge of ethanol emission pipeline, after the whole press filtration of suspension is complete, setting purge time is 100 minutes, filter plate is dried up with-7.7 DEG C of precooling clean compressed airs, collect component I I and precipitate 17.4kg,

20% ethanol compound method used is: 95% (v/v) ethanol 225.5kg that every 1000kg water for injection adds less than-20 DEG C obtains the ethanol of 20% (v/v), adjusts the temperature to-6.8 DEG C with-15 DEG C of refrigerants;

Step 5: component I I resolution of precipitate, filtration

The water for injection of 1.4 DEG C of component I I precipitation 104.7kg step 4 collected dissolves, and under stir speed (S.S.) is 72rpm, stirring 4.1 is little dissolves, by 0.5mol/L sodium hydroxide solution 1.4kg adjusted to ph to 7.03 completely up to precipitation, 2.0 DEG C are adjusted the temperature to-15 DEG C of refrigerants, continue stirring reaction 3 hours under stir speed (S.S.) is 74rpm, dissolve completely and add diatomite 0.19kg and perlite 0.19kg, stir 30 minutes under stir speed (S.S.) is 72rpm, with 1.3 DEG C of water for injection 432kg, balance cooling is carried out to whole press filtration pipeline and pressure filter, setting purge time 18 minutes, (German Begerow company produces to dry up filter plate with 1.9 DEG C of precooling clean compressed airs, model: Steril S100, specification: 349*384mm), exhaust temperature is made to reach 2.5 DEG C, start press filtration, controlled filter pressure is 0.12MPa, filtration velocity is 20kg/min, filtrate temperature 2.0 DEG C, dissolve the complete setting purge time of hydraulic pressure 24 minutes, press filtration separate dissolved suspension, with 130kg 1.0 DEG C of waters for injection, clean and reuse is carried out to pressure filter, filter plate is dried up with 1.5 DEG C of precooling clean compressed airs, Ethylene recov is incorporated to filtrate tank, setting purge time is 19 minutes, filter plate is dried up with 0.0 ~ 4.0 DEG C of precooling clean compressed air, be precipitated 1.4kg, filtrate 252.4kg, filtrate pH value to 4.00 is regulated with 1mol/L acetum 3.0kg, filtering-depositing autoclaving is discarded, at this moment filtrate protein content is 1.53%, total protein is 3.91kg.

Step 6: ultrafiltration, dialysis, concentrated

Be that to be concentrated into protein content be 10% (m/v) for filtrate that step 5 obtains by 30KD ultra-fine filter by film specification, heavy 39.1kg, then with 3.9 DEG C, the water for injection of 195.5kg carries out continuous equivalent dialysis, proteins concentrate content to 8.6% (m/v), goods pH value to 6.90 is adjusted again with the NaOH solution 2.3kg of 0.5mol/L, controlling protein content with water for injection dilution is 7.1%, and after dilution, weight is 55.1kg;

Step 7: purifying, filtration

Goods after concentrated are transferred to sphaeroprotein treatment tank, by water for injection diluted protein matter content to 1.83% (m/v) of 1.2 DEG C, after dilution, weight is 213.6kg, then SODIUM PHOSPHATE, MONOBASIC 0.29kg and sodium-chlor 0.13kg is added, by 0.5mol/L sodium hydroxide solution adjusted to ph to 6.97, 0.2 DEG C is adjusted the temperature to-15 DEG C of refrigerants, add 95% (v/v) ethanol 3.9kg of-21 DEG C again, regulate outlet temperature to-1.3 DEG C with-15 DEG C of refrigerants, stirring reaction is continued 2.4 hours under stir speed (S.S.) is 69rpm, perlite 0.33kg is added after reacting completely, stir 30 minutes under stir speed (S.S.) is 69rpm, with the 2% ethanol 424kg of-1.7 DEG C, balance cooling is carried out to whole press filtration pipeline and pressure filter, setting purge time 18 minutes, (German Begerow company produces to dry up filter plate with-1.4 DEG C of precooling clean compressed airs, model: Steril S100, specification: 349*384mm), exhaust temperature is made to reach-1.4 DEG C, start press filtration, controlled filter pressure is 0.12MPa, filtration velocity is 30kg/min, filtrate temperature-1.5 DEG C, the complete setting purge time of suspension press filtration is 20 minutes, with 139kg 2% ethanol, clean and reuse is carried out to pressure filter, Ethylene recov is incorporated to filtrate tank, filter plate is dried up with-1.4 DEG C of precooling clean compressed airs, filtering supernatant pH value to 3.95 is regulated with 0.5mol/L hydrochloric acid soln 2.3kg, regulate outlet temperature to-1.3 DEG C, be precipitated 1.6kg, filtrate 357.8kg, wherein filtrate protein content is 1.04%.

2% ethanol compound method used is: every 1000kg water for injection adds less than-20 DEG C 95% (v/v) ethanol 18.2kg, adjusts the temperature to-1.4 DEG C with-15 DEG C of refrigerants;

Step 8: ultrafiltration dialysis, concentrated, preparation

Be 9.6% (m/v) (weight is 38.7kg) (intake pressure 3.9bar by the filtrate of step 7 after pH and temperature regulate through ultrafiltration and concentration to protein content, return pressure 1.8bar), then continuous equivalent dialysis is carried out with the water for injection of the 426kg of 3.7 DEG C, proteins concentrate content to 8.64% (total protein is 3.71kg) (m/v), add maltose 6.64kg, by 0.5mol/L hydrochloric acid soln adjust ph to 3.97, controlling final protein content with water for injection dilution is 5.33% (m/v), obtaining weight after preparation is 69.7kg, yield is the blood plasma of 6.3kg/kL.Carry out after having prepared degerming, except virus filtration, incubated at low pH and degerming packing, namely obtain quiet note human normal immunoglobulin work in-process.

Product checking:

The half-finished protein content of described quiet note human normal immunoglobulin is 5.28%, its purity is 98.9%, pH value is 4.00, wherein residual ethanol content is for being less than 0.025g/100ml, Content of polymer is 1.1%, ACA (anticomplementary activity) is for 6IU/ml, PKA (Prekallikrein) content is for being less than 10%.

Embodiment 2

Step one: component I+II+III resolution of precipitate

Component I+II+III is precipitated the 56.0kg aqueous solution (phosphoric acid sodium dihydrogen 0.54kg, water for injection 391.7kg) of the phosphoric acid sodium dihydrogen of 1.1 DEG C and stir more than 6 hours to dissolving completely, obtain component I+II+III resolution of precipitate liquid 448.2kg.

Step 2: separated portion I+III-1 precipitates

Said components I+II+III resolution of precipitate liquid pH value to 5.10 is regulated with 1mol/L acetum 2.9kg,-0.3 DEG C is cooled to-15 DEG C of refrigerants, add 95% (v/v) ethanolic soln 28.0kg of-20.5 DEG C again, and by 0.5mol/L sodium hydroxide solution adjusted to ph to 5.13, regulate last suspension temperature to-2.9 DEG C with-15 DEG C of refrigerants, be that 82rpm stirs 2.8 hours with stirring velocity, diatomite 1.9kg and perlite 1.2kg is added after reacting completely, be that 89rpm stirs 30 minutes with stirring velocity, with 8% balance liquid 1044Kg, balance cooling is carried out to whole press filtration pipeline and pressure filter, setting purge time 26 minutes, (German Begerow company produces to dry up filter plate with-2.7 DEG C of precooling clean compressed airs, model: PX50, specification 680*779mm), exhaust temperature is made to reach-2.0 DEG C.Start suspension press filtration, controlled filter pressure is 0.23MPa, filtration velocity is 75kg/min, filtrate temperature-1.9 DEG C, suspension has pressed setting purge time 48 minutes, filter plate is dried up with precooling clean compressed air, with 8% balance liquid 930Kg, clean and reuse is carried out to pressure filter, Ethylene recov is incorporated to press filtration supernatant tank, setting purge time 100 minutes, re-use-1.2 DEG C of precooling clean compressed airs to dry up, collect component I+III-1 supernatant liquor 1373.1kg, component I+III-1 is discarded after precipitating 39.2kg autoclaving;

8% balance liquid compound method used is: 95% (v/v) ethanol 77.7kg that every 1000kg water for injection adds less than-20 DEG C obtains the ethanolic soln of 8% (v/v); Then in the ethanolic soln of every 1000kg 8%, Sodium phosphate dibasic 3.58kg is added; By Glacial acetic acid adjust ph to 5.04;-2.6 DEG C are adjusted the temperature to-15 DEG C of refrigerants.

Step 3: separated portion I+III-2 precipitates

Component I+III-1 supernatant liquor pH value to 5.17 is adjusted with 0.5mol/L sodium hydroxide solution 0.7kg, add 95% (v/v) ethanol 102.5kg of-20.4 DEG C again, and by 0.5mol/L sodium hydroxide adjusted to ph to 5.21, regulate temperature of reaction to-4.0 DEG C with-15 DEG C of refrigerants, be that 72rpm stirs 2.8 hours with stirring velocity, diatomite 3.7kg and perlite 3.7kg is added after reacting completely, be that 77rpm stirs 30 minutes with stirring velocity, with 15% balance liquid 924Kg, balance cooling is carried out to whole press filtration pipeline and pressure filter, setting purge time 32 minutes, (German Begerow company produces to dry up filter plate with-4.5 DEG C of precooling clean compressed airs, model: PX50, specification 680*779mm), exhaust temperature is made to reach-3.9 DEG C.Start suspension press filtration, controlled filter pressure is 0.21MPa, filtration velocity is 67kg/min, filtrate temperature-4.3 DEG C, suspension has pressed setting purge time 43 minutes, filter plate is dried up with precooling clean compressed air, with 15% balance liquid 897Kg, clean and reuse is carried out to pressure filter, Ethylene recov is incorporated to press filtration supernatant tank, setting purge time 81 minutes, re-use-4.2 DEG C of precooling clean compressed airs to dry up, collect component I+III-2 supernatant liquor 2363.9kg, component I+III-2 precipitates 16.8kg autoclaving and discards;

15% balance liquid compound method used is: every 1000kg water for injection adds the ethanol that less than-20 DEG C 95% (v/v) ethanol 158.3kg obtain 15% (v/v); Then in every 1000kg 15% ethanol, Sodium phosphate dibasic 3.58kg is added; By Glacial acetic acid adjust ph to 5.23;-4.7 DEG C are adjusted the temperature to-15 DEG C of refrigerants;

Step 4: separated portion II precipitates

30% (m/v) sodium chloride solution 48.2kg is added in said components I+III-2 supernatant liquor, by 0.5mol/L sodium hydroxide solution 37.8kg adjust ph to 7.04, add 95% (v/v) ethanol 156.1kg of-21.0 DEG C again, by 1mol/L acetic acid adjusted to ph to 7.01, regulate temperature of reaction to-6.9 DEG C with-15 DEG C of refrigerants, continue stirring reaction 3.0 hours, its stirring velocity is 78RPM, perlite 2.0kg is added after reacting completely, stir 30 minutes, its stirring velocity is 78RPM, with 20% (v/v) ethanol 1473Kg of-7.0 DEG C, balance cooling is carried out to whole press filtration pipeline and pressure filter, setting purge time 30 minutes, (German Begerow company produces to dry up filter plate with-7.8 DEG C of precooling clean compressed airs, model: PX50, specification 680*779mm), exhaust temperature is made to reach-6.8 DEG C, start suspension press filtration, controlled filter pressure is 0.18MPa, filtration velocity is 64kg/min, filtrate temperature-6.5 DEG C, supernatant directly discharges through ethanol emission pipeline, when component I I suspension tank weighs less than 200kg, press filtration supernatant no longer discharges and directly enters suspension tank thus form a circulation line, after circulation 10min, supernatant starts to continue through the discharge of ethanol emission pipeline, after the whole press filtration of suspension is complete, setting purge time is 119 minutes, filter plate is dried up with-8.4 DEG C of precooling clean compressed airs, collect component I I and precipitate 17.3kg,

20% ethanol compound method used is: 95% (v/v) ethanol 225.5kg that every 1000kg water for injection adds less than-20 DEG C obtains the ethanol of 20% (v/v), adjusts the temperature to-6.0 DEG C with-15 DEG C of refrigerants;

Step 5: component I I resolution of precipitate, filtration

The water for injection of 1.1 DEG C of component I I precipitation 104.1kg step 4 collected dissolves, and under stir speed (S.S.) is 77rpm, stirring 4.7 is little dissolves, by 0.5mol/L sodium hydroxide solution 1.4kg adjusted to ph to 6.98 completely up to precipitation, 1.4 DEG C are adjusted the temperature to-15 DEG C of refrigerants, continue stirring reaction 2.9 hours under stir speed (S.S.) is 79rpm, dissolve completely and add diatomite 0.18kg and perlite 0.18kg, stir 30 minutes under stir speed (S.S.) is 76rpm, with 1.8 DEG C of water for injection 371kg, balance cooling is carried out to whole press filtration pipeline and pressure filter, setting purge time 22 minutes, (German Begerow company produces to dry up filter plate with 2.3 DEG C of precooling clean compressed airs, model: Steril S100, specification: 349*384mm), exhaust temperature is made to reach 1.7 DEG C, start press filtration, controlled filter pressure is 0.13MPa, filtration velocity is 20kg/min, filtrate temperature 2.9 DEG C, dissolve the complete setting purge time of hydraulic pressure 19 minutes, press filtration separate dissolved suspension, with 1.6 DEG C of waters for injection of 122kg, clean and reuse is carried out to pressure filter, filter plate is dried up with 1.5 DEG C of precooling clean compressed airs, Ethylene recov is incorporated to filtrate tank, setting purge time is 25 minutes, filter plate is dried up with 0.0 ~ 4.0 DEG C of precooling clean compressed air, be precipitated 1.3kg, filtrate 243.7kg, filtrate pH value to 3.93 is regulated with 1mol/L acetum 2.9kg, filtering-depositing autoclaving is discarded, at this moment filtrate protein content is 1.57%, total protein is 3.87kg.

Step 6: ultrafiltration, dialysis, concentrated

Be that to be concentrated into protein content be 10.2% (m/v) for filtrate that step 5 obtains by 30KD ultra-fine filter by film specification, heavy 37.9kg, then with 4.0 DEG C, the water for injection of 189.7kg carries out continuous equivalent dialysis, proteins concentrate content to 8.8% (m/v), goods pH value to 6.91 is adjusted again with the NaOH solution 2.2kg of 0.5mol/L, controlling protein content with water for injection dilution is 7.3%, and after dilution, weight is 53.0kg;

Step 7: purifying, filtration

Goods after concentrated are transferred to sphaeroprotein treatment tank, by water for injection diluted protein matter content to 1.83% (m/v) of 1.0 DEG C, after dilution, weight is 211.4kg, then SODIUM PHOSPHATE, MONOBASIC 0.29kg and sodium-chlor 0.12kg is added, by 1mol/L acetum adjusted to ph to 6.93, 0.4 DEG C is adjusted the temperature to-15 DEG C of refrigerants, add 95% (v/v) ethanol 3.8kg of-20.2 DEG C again, regulate outlet temperature to-1.5 DEG C with-15 DEG C of refrigerants, stirring reaction is continued 2.8 hours under stir speed (S.S.) is 66rpm, perlite 0.32kg is added after reacting completely, stir 30 minutes under stir speed (S.S.) is 63rpm, with the 2% ethanol 351kg of-1.4 DEG C, balance cooling is carried out to whole press filtration pipeline and pressure filter, setting purge time 24 minutes, (German Begerow company produces to dry up filter plate with-0.7 DEG C of precooling clean compressed air, model: Steril S100, specification: 349*384mm), exhaust temperature is made to reach-1.6 DEG C, start press filtration, controlled filter pressure is 0.11MPa, filtration velocity is 30kg/min, filtrate temperature-1.5 DEG C, the complete setting purge time of suspension press filtration is 21 minutes, with 2% ethanol of 134kg, clean and reuse is carried out to pressure filter, Ethylene recov is incorporated to filtrate tank, filter plate is dried up with-1.5 DEG C of precooling clean compressed airs, filtering supernatant pH value to 3.93 is regulated with 0.5mol/L hydrochloric acid soln 2.2kg, regulate outlet temperature to-1.5 DEG C, be precipitated 1.6kg, filtrate 350.5kg, wherein filtrate protein content is 1.05%.

2% ethanol compound method used is: every 1000kg water for injection adds less than-20 DEG C 95% (v/v) ethanol 18.2kg, adjusts the temperature to-1.4 DEG C with-15 DEG C of refrigerants;

Step 8: ultrafiltration dialysis, concentrated, preparation

Be 10.3% (m/v) (weight is 35.7kg) (intake pressure 3.3bar by the filtrate of step 7 after pH and temperature regulate through ultrafiltration and concentration to protein content, return pressure 1.0bar), then continuous equivalent dialysis is carried out with the water for injection of the 393kg of 3.2 DEG C, proteins concentrate content to 8.43% (total protein is 3.68kg) (m/v), add maltose 6.54kg, by 0.5mol/L hydrochloric acid soln adjust ph to 3.96, controlling final protein content with water for injection dilution is 5.35% (m/v), obtaining weight after preparation is 68.7kg, yield is 6.2kg/kL blood plasma.Carry out after having prepared degerming, except virus filtration, incubated at low pH and degerming packing, namely obtain quiet note human normal immunoglobulin work in-process.

Product checking:

The half-finished protein content of described quiet note human normal immunoglobulin is 5.34%, its purity is 98.8%, pH value is 4.01, wherein residual ethanol content is for being less than 0.025g/100ml, Content of polymer is 1.2%, ACA (anticomplementary activity) is for 8IU/ml, PKA (Prekallikrein) content is for being less than 10%.

Embodiment 3

Step one: component I+II+III resolution of precipitate

Component I+II+III is precipitated the 54.6kg aqueous solution (phosphoric acid sodium dihydrogen 0.53kg, water for injection 382.1kg) of the phosphoric acid sodium dihydrogen of 3.1 DEG C and stir more than 6 hours to dissolving completely, obtain component I+II+III resolution of precipitate liquid 437.3kg.

Step 2: separated portion I+III-1 precipitates

Said components I+II+III resolution of precipitate liquid pH value to 5.06 is regulated with 1mol/L acetum 2.9kg,-0.2 DEG C is cooled to-15 DEG C of refrigerants, add 95% (v/v) ethanolic soln 27.3kg of-20.8 DEG C again, and by 0.5mol/L sodium hydroxide solution adjusted to ph to 5.19, regulate last suspension temperature to-3.0 DEG C with-15 DEG C of refrigerants, be that 83rpm stirs 2.1 hours with stirring velocity, diatomite 1.9kg and perlite 1.2kg is added after reacting completely, be that 80rpm stirs 30 minutes with stirring velocity, with 8% balance liquid 919Kg, balance cooling is carried out to whole press filtration pipeline and pressure filter, setting purge time 34 minutes, (German Begerow company produces to dry up filter plate with-2.5 DEG C of precooling clean compressed airs, model: PX50, specification 680*779mm), exhaust temperature is made to reach-2.2 DEG C.Start suspension press filtration, controlled filter pressure is 0.24MPa, filtration velocity is 63kg/min, filtrate temperature-2.4 DEG C, suspension has pressed setting purge time 49 minutes, filter plate is dried up with precooling clean compressed air, with 8% balance liquid 897Kg, clean and reuse is carried out to pressure filter, Ethylene recov is incorporated to press filtration supernatant tank, setting purge time 89 minutes, re-use-1.6 DEG C of precooling clean compressed airs to dry up, collect component I+III-1 supernatant liquor 1329.3kg, component I+III-1 is discarded after precipitating 38.2kg autoclaving;

8% balance liquid compound method used is: 95% (v/v) ethanol 77.7kg that every 1000kg water for injection adds less than-20 DEG C obtains the ethanolic soln of 8% (v/v); Then in the ethanolic soln of every 1000kg 8%, Sodium phosphate dibasic 3.58kg is added; By Glacial acetic acid adjust ph to 5.11;-2.1 DEG C are adjusted the temperature to-15 DEG C of refrigerants.

Step 3: separated portion I+III-2 precipitates

Component I+III-1 supernatant liquor pH value to 5.20 is adjusted with 0.5mol/L sodium hydroxide solution 0.7kg, add 95% (v/v) ethanol 99.3kg of-21.6 DEG C again, and by 0.5mol/L sodium hydroxide adjusted to ph to 5.23, regulate temperature of reaction to-4.5 DEG C with-15 DEG C of refrigerants, be that 72rpm stirs 2.5 hours with stirring velocity, diatomite 3.6kg and perlite 3.6kg is added after reacting completely, be that 75rpm stirs 30 minutes with stirring velocity, with 15% balance liquid 1012Kg, balance cooling is carried out to whole press filtration pipeline and pressure filter, setting purge time 35 minutes, (German Begerow company produces to dry up filter plate with-5.0 DEG C of precooling clean compressed airs, model: PX50, specification 680*779mm), exhaust temperature is made to reach-3.5 DEG C.Start suspension press filtration, controlled filter pressure is 0.24MPa, filtration velocity is 71kg/min, filtrate temperature-4.1 DEG C, suspension has pressed setting purge time 40 minutes, filter plate is dried up with precooling clean compressed air, with 15% balance liquid 890Kg, clean and reuse is carried out to pressure filter, Ethylene recov is incorporated to press filtration supernatant tank, setting purge time 99 minutes, re-use-4.8 DEG C of precooling clean compressed airs to dry up, collect component I+III-2 supernatant liquor 2310.0kg, component I+III-2 precipitates 16.4kg autoclaving and discards;

15% balance liquid compound method used is: every 1000kg water for injection adds the ethanol that less than-20 DEG C 95% (v/v) ethanol 158.3kg obtain 15% (v/v); Then in every 1000kg 15% ethanol, Sodium phosphate dibasic 3.58kg is added; By Glacial acetic acid adjust ph to 5.23;-4.3 DEG C are adjusted the temperature to-15 DEG C of refrigerants;

Step 4: separated portion II precipitates

30% (m/v) sodium chloride solution 47.1kg is added in said components I+III-2 supernatant liquor, by 0.5mol/L sodium hydroxide solution 37.0kg adjust ph to 7.07, add 95% (v/v) ethanol 152.6kg of-20.1 DEG C again, by 1mol/L acetic acid adjusted to ph to 7.07, regulate temperature of reaction to-7.2 DEG C with-15 DEG C of refrigerants, continue stirring reaction 2.3 hours, its stirring velocity is 78RPM, perlite 1.9kg is added after reacting completely, stir 30 minutes, its stirring velocity is 78RPM, with 20% (v/v) ethanol 1477Kg of-6.4 DEG C, balance cooling is carried out to whole press filtration pipeline and pressure filter, setting purge time 30 minutes, (German Begerow company produces to dry up filter plate with-7.9 DEG C of precooling clean compressed airs, model: PX50, specification 680*779mm), exhaust temperature is made to reach-6.8 DEG C, start suspension press filtration, controlled filter pressure is 0.20MPa, filtration velocity is 79kg/min, filtrate temperature-6.4 DEG C, supernatant directly discharges through ethanol emission pipeline, when component I I suspension tank weighs less than 200kg, press filtration supernatant no longer discharges and directly enters suspension tank thus form a circulation line, after circulation 10min, supernatant starts to continue through the discharge of ethanol emission pipeline, after the whole press filtration of suspension is complete, setting purge time is 119 minutes, filter plate is dried up with-8.1 DEG C of precooling clean compressed airs, collect component I I and precipitate 16.9kg,

20% ethanol compound method used is: 95% (v/v) ethanol 225.5kg that every 1000kg water for injection adds less than-20 DEG C obtains the ethanol of 20% (v/v), adjusts the temperature to-6.4 DEG C with-15 DEG C of refrigerants;

Step 5: component I I resolution of precipitate, filtration

The water for injection of 1.5 DEG C of component I I precipitation 101.5kg step 4 collected dissolves, and under stir speed (S.S.) is 72rpm, stirring 4.9 is little dissolves, by 0.5mol/L sodium hydroxide solution 1.3kg adjusted to ph to 7.04 completely up to precipitation, 1.6 DEG C are adjusted the temperature to-15 DEG C of refrigerants, continue stirring reaction 2.3 hours under stir speed (S.S.) is 74rpm, dissolve completely and add diatomite 0.18kg and perlite 0.18kg, stir 30 minutes under stir speed (S.S.) is 76rpm, with 1.5 DEG C of water for injection 408kg, balance cooling is carried out to whole press filtration pipeline and pressure filter, setting purge time 15 minutes, (German Begerow company produces to dry up filter plate with 2.3 DEG C of precooling clean compressed airs, model: Steril S100, specification: 349*384mm), exhaust temperature is made to reach 2.0 DEG C, start press filtration, controlled filter pressure is 0.10MPa, filtration velocity is 29kg/min, filtrate temperature 2.4 DEG C, dissolve the complete setting purge time of hydraulic pressure 19 minutes, press filtration separate dissolved suspension, with 1.1 DEG C of waters for injection of 128kg, clean and reuse is carried out to pressure filter, filter plate is dried up with 2.1 DEG C of precooling clean compressed airs, Ethylene recov is incorporated to filtrate tank, setting purge time is 18 minutes, filter plate is dried up with 0.0 ~ 4.0 DEG C of precooling clean compressed air, be precipitated 1.4kg, filtrate 246.7kg, filtrate pH value to 3.94 is regulated with 1mol/L acetum 3.0kg, filtering-depositing autoclaving is discarded, at this moment filtrate protein content is 1.59%, total protein is 3.98kg.

Step 6: ultrafiltration, dialysis, concentrated

Be that to be concentrated into protein content be 10.3% (m/v) for filtrate that step 5 obtains by 30KD ultra-fine filter by film specification, heavy 38.6kg, then with 3.6 DEG C, the water for injection of 193.0kg carries out continuous equivalent dialysis, proteins concentrate content to 8.5% (m/v), goods pH value to 6.99 is adjusted again with the NaOH solution 2.2kg of 0.5mol/L, controlling protein content with water for injection dilution is 7.9%, and after dilution, weight is 50.3kg;

Step 7: purifying, filtration

Goods after concentrated are transferred to sphaeroprotein treatment tank, by water for injection diluted protein matter content to 1.74% (m/v) of 1.1 DEG C, after dilution, weight is 228.5kg, then SODIUM PHOSPHATE, MONOBASIC 0.32kg and sodium-chlor 0.13kg is added, by 1mol/L acetum adjusted to ph to 6.93, 0.1 DEG C is adjusted the temperature to-15 DEG C of refrigerants, add 95% (v/v) ethanol 4.2kg of-21.7 DEG C again, regulate outlet temperature to-1.8 DEG C with-15 DEG C of refrigerants, stirring reaction is continued 2.1 hours under stir speed (S.S.) is 62rpm, perlite 0.35kg is added after reacting completely, stir 30 minutes under stir speed (S.S.) is 69rpm, with the 2% ethanol 359kg of-1.5 DEG C, balance cooling is carried out to whole press filtration pipeline and pressure filter, setting purge time 15 minutes, (German Begerow company produces to dry up filter plate with-0.8 DEG C of precooling clean compressed air, model: Steril S100, specification: 349*384mm), exhaust temperature is made to reach-1.5 DEG C, start press filtration, controlled filter pressure is 0.15MPa, filtration velocity is 26kg/min, filtrate temperature-1.7 DEG C, the complete setting purge time of suspension press filtration is 19 minutes, with 2% ethanol of 148kg, clean and reuse is carried out to pressure filter, Ethylene recov is incorporated to filtrate tank, filter plate is dried up with-0.6 DEG C of precooling clean compressed air, filtering supernatant pH value to 3.94 is regulated with 0.5mol/L hydrochloric acid soln 2.2kg, regulate outlet temperature to-1.6 DEG C, be precipitated 1.7kg, filtrate 381.8kg, wherein filtrate protein content is 0.99%.

2% ethanol compound method used is: every 1000kg water for injection adds less than-20 DEG C 95% (v/v) ethanol 18.2kg, adjusts the temperature to-1.5 DEG C with-15 DEG C of refrigerants;

Step 8: ultrafiltration dialysis, concentrated, preparation

Be 9.9% (m/v) (weight is 38.2kg) (intake pressure 3.2bar by the filtrate of step 7 after pH and temperature regulate through ultrafiltration and concentration to protein content, return pressure 1.5bar), then continuous equivalent dialysis is carried out with the water for injection of the 420kg of 3.3 DEG C, proteins concentrate content to 8.79% (m/v) (total protein is 3.78kg), add maltose 6.72kg, by 0.5mol/L sodium hydroxide solution adjust ph to 4.04, controlling final protein content with water for injection dilution is 5.35% (m/v), obtaining weight after preparation is 70.6kg, yield is the blood plasma of 6.4kg/kL.Carry out after having prepared degerming, except virus filtration, incubated at low pH and degerming packing, namely obtain quiet note human normal immunoglobulin work in-process.

Product checking:

The half-finished protein content of described quiet note human normal immunoglobulin is 5.28%, its purity is 98.8%, pH value is 4.03, wherein residual ethanol content is for being less than 0.025g/100ml, Content of polymer is 1.0%, ACA (anticomplementary activity) is for 6IU/ml, PKA (Prekallikrein) content is for being less than 10%.

Embodiment 4

Step one: component I I+III resolution of precipitate

Component I I+III is precipitated the 55.3kg aqueous solution (phosphoric acid sodium dihydrogen 0.53kg, water for injection 386.8kg) of the phosphoric acid sodium dihydrogen of 3.9 DEG C and stir more than 6 hours to dissolving completely, obtain component I I+III resolution of precipitate liquid 442.6kg.

Step 2: separated portion III-1 precipitates

Said components II+III resolution of precipitate liquid pH value to 5.01 is regulated with 1mol/L acetum 2.9kg,-0.5 DEG C is cooled to-15 DEG C of refrigerants, add 95% (v/v) ethanolic soln 27.7kg of-22.0 DEG C again, and by 0.5mol/L sodium hydroxide solution adjusted to ph to 5.18, regulate last suspension temperature to-2.5 DEG C with-15 DEG C of refrigerants, be that 90rpm stirs 2.5 hours with stirring velocity, diatomite 1.9kg and perlite 1.2kg is added after reacting completely, be that 80rpm stirs 30 minutes with stirring velocity, with 8% balance liquid 1002Kg, balance cooling is carried out to whole press filtration pipeline and pressure filter, setting purge time 26 minutes, (German Begerow company produces to dry up filter plate with-3.0 DEG C of precooling clean compressed airs, model: PX50, specification 680*779mm), exhaust temperature is made to reach-2.2 DEG C.Start suspension press filtration, controlled filter pressure is 0.25MPa, filtration velocity is 69kg/min, filtrate temperature-1.6 DEG C, suspension has pressed setting purge time 46 minutes, filter plate is dried up with precooling clean compressed air, with 8% balance liquid 931Kg, clean and reuse is carried out to pressure filter, Ethylene recov is incorporated to press filtration supernatant tank, setting purge time 80 minutes, re-use-2.3 DEG C of precooling clean compressed airs to dry up, collect component III-1 supernatant liquor 1368.6kg, component III-1 is discarded after precipitating 38.7kg autoclaving;

8% balance liquid compound method used is: 95% (v/v) ethanol 77.7kg that every 1000kg water for injection adds less than-20 DEG C obtains the ethanolic soln of 8% (v/v); Then in the ethanolic soln of every 1000kg 8%, Sodium phosphate dibasic 3.58kg is added; By Glacial acetic acid adjust ph to 5.00;-2.2 DEG C are adjusted the temperature to-15 DEG C of refrigerants.

Step 3: separated portion III-2 precipitates

Component III-1 supernatant liquor pH value to 5.16 is adjusted with 0.5mol/L sodium hydroxide solution 0.7kg, add 95% (v/v) ethanol 102.2kg of-20.2 DEG C again, and by 0.5mol/L sodium hydroxide adjusted to ph to 5.22, regulate temperature of reaction to-4.9 DEG C with-15 DEG C of refrigerants, be that 79rpm stirs 2.4 hours with stirring velocity, diatomite 3.7kg and perlite 3.7kg is added after reacting completely, be that 75rpm stirs 30 minutes with stirring velocity, with 15% balance liquid 917Kg, balance cooling is carried out to whole press filtration pipeline and pressure filter, setting purge time 32 minutes, (German Begerow company produces to dry up filter plate with-4.2 DEG C of precooling clean compressed airs, model: PX50, specification 680*779mm), exhaust temperature is made to reach-4.5 DEG C.Start suspension press filtration, controlled filter pressure is 0.23MPa, filtration velocity is 71kg/min, filtrate temperature-4.4 DEG C, suspension has pressed setting purge time 42 minutes, filter plate is dried up with precooling clean compressed air, with 15% balance liquid 851Kg, clean and reuse is carried out to pressure filter, Ethylene recov is incorporated to press filtration supernatant tank, setting purge time 94 minutes, re-use-4.3 DEG C of precooling clean compressed airs to dry up, collect component III-2 supernatant liquor 2313.2kg, component III-2 precipitates 16.6kg autoclaving discards;

15% balance liquid compound method used is: every 1000kg water for injection adds the ethanol that less than-20 DEG C 95% (v/v) ethanol 158.3kg obtain 15% (v/v); Then in every 1000kg 15% ethanol, Sodium phosphate dibasic 3.58kg is added; By Glacial acetic acid adjust ph to 5.16;-4.0 DEG C are adjusted the temperature to-15 DEG C of refrigerants;

Step 4: separated portion II precipitates

30% (m/v) sodium chloride solution 47.2kg is added in said components component III-2 supernatant liquor, by 0.5mol/L sodium hydroxide solution 37.0kg adjust ph to 7.07, add 95% (v/v) ethanol 152.8kg of-20.0 DEG C again, by 1mol/L acetic acid adjusted to ph to 7.06, regulate temperature of reaction to-7.5 DEG C with-15 DEG C of refrigerants, continue stirring reaction 2.3 hours, its stirring velocity is 79RPM, perlite 1.9kg is added after reacting completely, stir 30 minutes, its stirring velocity is 79RPM, with 20% (v/v) ethanol 1517Kg of-6.1 DEG C, balance cooling is carried out to whole press filtration pipeline and pressure filter, setting purge time 30 minutes, (German Begerow company produces to dry up filter plate with-7.6 DEG C of precooling clean compressed airs, model: PX50, specification 680*779mm), exhaust temperature is made to reach-6.3 DEG C, start suspension press filtration, controlled filter pressure is 0.20MPa, filtration velocity is 61kg/min, filtrate temperature-6.3 DEG C, supernatant directly discharges through ethanol emission pipeline, when component I I suspension tank weighs less than 200kg, press filtration supernatant no longer discharges and directly enters suspension tank thus form a circulation line, after circulation 10min, supernatant starts to continue through the discharge of ethanol emission pipeline, after the whole press filtration of suspension is complete, setting purge time is 107 minutes, filter plate is dried up with-7.8 DEG C of precooling clean compressed airs, collect component I I and precipitate 17.1kg,

20% ethanol compound method used is: 95% (v/v) ethanol 225.5kg that every 1000kg water for injection adds less than-20 DEG C obtains the ethanol of 20% (v/v), adjusts the temperature to-6.1 DEG C with-15 DEG C of refrigerants;

Step 5: component I I resolution of precipitate, filtration

The water for injection of 1.5 DEG C of component I I precipitation 102.8kg step 4 collected dissolves, and under stir speed (S.S.) is 76rpm, stirring 4.6 is little dissolves, by 0.5mol/L sodium hydroxide solution 1.3kg adjusted to ph to 6.97 completely up to precipitation, 1.8 DEG C are adjusted the temperature to-15 DEG C of refrigerants, continue stirring reaction 2.9 hours under stir speed (S.S.) is 80rpm, dissolve completely and add diatomite 0.18kg and perlite 0.18kg, stir 30 minutes under stir speed (S.S.) is 76rpm, with 1.8 DEG C of water for injection 350kg, balance cooling is carried out to whole press filtration pipeline and pressure filter, setting purge time 15 minutes, (German Begerow company produces to dry up filter plate with 1.9 DEG C of precooling clean compressed airs, model: Steril S100, specification: 349*384mm), exhaust temperature is made to reach 2.3 DEG C, start press filtration, controlled filter pressure is 0.11MPa, filtration velocity is 29kg/min, filtrate temperature 2.4 DEG C, dissolve the complete setting purge time of hydraulic pressure 22 minutes, press filtration separate dissolved suspension, with 1.6 DEG C of waters for injection of 118kg, clean and reuse is carried out to pressure filter, filter plate is dried up with 2.1 DEG C of precooling clean compressed airs, Ethylene recov is incorporated to filtrate tank, setting purge time is 25 minutes, filter plate is dried up with 0.0 ~ 4.0 DEG C of precooling clean compressed air, be precipitated 1.5kg, filtrate 238.2kg, filtrate pH value to 3.90 is regulated with 1mol/L acetum 2.9kg, filtering-depositing autoclaving is discarded, at this moment filtrate protein content is 1.65%, total protein is 3.98kg.

Step 6: ultrafiltration, dialysis, concentrated

Be that to be concentrated into protein content be 10.2% (m/v) for filtrate that step 5 obtains by 30KD ultra-fine filter by film specification, heavy 39.0kg, then with 3.4 DEG C, the water for injection of 195.2kg carries out continuous equivalent dialysis, proteins concentrate content to 8.9% (m/v), goods pH value to 6.99 is adjusted again with the NaOH solution 2.1kg of 0.5mol/L, controlling protein content with water for injection dilution is 8.0%, and after dilution, weight is 49.8kg;

Step 7: purifying, filtration

Goods after concentrated are transferred to sphaeroprotein treatment tank, by water for injection diluted protein matter content to 1.85% (m/v) of 1.6 DEG C, after dilution, weight is 215.2kg, then SODIUM PHOSPHATE, MONOBASIC 0.30kg and sodium-chlor 0.13kg is added, by 1mol/L acetum adjusted to ph to 6.97, 0.2 DEG C is adjusted the temperature to-15 DEG C of refrigerants, add 95% (v/v) ethanol 3.9kg of-21.8 DEG C again, regulate outlet temperature to-1.5 DEG C with-15 DEG C of refrigerants, stirring reaction is continued 2.3 hours under stir speed (S.S.) is 68rpm, perlite 0.33kg is added after reacting completely, stir 30 minutes under stir speed (S.S.) is 60rpm, with the 2% ethanol 371kg of-1.7 DEG C, balance cooling is carried out to whole press filtration pipeline and pressure filter, setting purge time 23 minutes, (German Begerow company produces to dry up filter plate with-1.2 DEG C of precooling clean compressed airs, model: Steril S100, specification: 349*384mm), exhaust temperature is made to reach-1.5 DEG C, start press filtration, controlled filter pressure is 0.13MPa, filtration velocity is 23kg/min, filtrate temperature-1.7 DEG C, the complete setting purge time of suspension press filtration is 15 minutes, with 2% ethanol of 137kg, clean and reuse is carried out to pressure filter, Ethylene recov is incorporated to filtrate tank, filter plate is dried up with-0.6 DEG C of precooling clean compressed air, filtering supernatant pH value to 3.94 is regulated with 0.5mol/L hydrochloric acid soln 2.1kg, regulate outlet temperature to-1.6 DEG C, be precipitated 1.6kg, filtrate 357.2kg, wherein filtrate protein content is 1.06%.

2% ethanol compound method used is: every 1000kg water for injection adds less than-20 DEG C 95% (v/v) ethanol 18.2kg, adjusts the temperature to-1.3 DEG C with-15 DEG C of refrigerants;

Step 8: ultrafiltration dialysis, concentrated, preparation

Be 9.8% (m/v) (weight is 38.6kg) (intake pressure 3.9bar by the filtrate of step 7 after pH and temperature regulate through ultrafiltration and concentration to protein content, return pressure 1.7bar), then continuous equivalent dialysis is carried out with the water for injection of the 425kg of 3.1 DEG C, proteins concentrate content to 8.60% (total protein is 3.78kg) (m/v), add maltose 6.85kg, by 0.5mol/L sodium hydroxide solution adjust ph to 3.99, controlling final protein content with water for injection dilution is 5.26% (m/v), obtaining weight after preparation is 71.9kg, yield is the blood plasma of 6.4kg/kL.Carry out after having prepared degerming, except virus filtration, incubated at low pH and degerming packing, namely obtain quiet note human normal immunoglobulin work in-process.

Product checking:

The half-finished protein content of described quiet note human normal immunoglobulin is 5.30%, its purity is 99.0%, pH value is 3.95, wherein residual ethanol content is for being less than 0.025g/100ml, Content of polymer is 1.3%, ACA (anticomplementary activity) is for 6IU/ml, PKA (Prekallikrein) content is for being less than 10%.

Embodiment 5

Step one: component I I+III resolution of precipitate

Component I I+III is precipitated the 54.8kg aqueous solution (phosphoric acid sodium dihydrogen 0.53kg, water for injection 383.9kg) of the phosphoric acid sodium dihydrogen of 1.4 DEG C and stir more than 6 hours to dissolving completely, obtain component I I+III resolution of precipitate liquid 439.3kg.

Step 2: separated portion I+III-1 (or component III-1) precipitates

Said components II+III resolution of precipitate liquid pH value to 5.03 is regulated with 1mol/L acetum 2.9kg,-0.2 DEG C is cooled to-15 DEG C of refrigerants, add 95% (v/v) ethanolic soln 27.5kg of-21.4 DEG C again, and by 0.5mol/L sodium hydroxide solution adjusted to ph to 5.16, regulate last suspension temperature to-2.6 DEG C with-15 DEG C of refrigerants, be that 84rpm stirs 2.9 hours with stirring velocity, diatomite 1.9kg and perlite 1.2kg is added after reacting completely, be that 86rpm stirs 30 minutes with stirring velocity, with 8% balance liquid 910Kg, balance cooling is carried out to whole press filtration pipeline and pressure filter, setting purge time 26 minutes, (German Begerow company produces to dry up filter plate with-2.6 DEG C of precooling clean compressed airs, model: PX50, specification 680*779mm), exhaust temperature is made to reach-1.9 DEG C.Start suspension press filtration, controlled filter pressure is 0.25MPa, filtration velocity is 62kg/min, filtrate temperature-1.6 DEG C, suspension has pressed setting purge time 42 minutes, filter plate is dried up with precooling clean compressed air, with 8% balance liquid 889Kg, clean and reuse is carried out to pressure filter, Ethylene recov is incorporated to press filtration supernatant tank, setting purge time 93 minutes, re-use-2.8 DEG C of precooling clean compressed airs to dry up, collect component III-1 supernatant liquor 1323.3kg, component III-1 is discarded after precipitating 38.4kg autoclaving;

8% balance liquid compound method used is: 95% (v/v) ethanol 77.7kg that every 1000kg water for injection adds less than-20 DEG C obtains the ethanolic soln of 8% (v/v); Then in the ethanolic soln of every 1000kg 8%, Sodium phosphate dibasic 3.58kg is added; By Glacial acetic acid adjust ph to 5.13;-2.8 DEG C are adjusted the temperature to-15 DEG C of refrigerants.

Step 3: separated portion III-2 precipitates

Component III-1 supernatant liquor pH value to 5.21 is adjusted with 0.5mol/L sodium hydroxide solution 0.7kg, add 95% (v/v) ethanol 98.8kg of-20.6 DEG C again, and by 0.5mol/L sodium hydroxide adjusted to ph to 5.23, regulate temperature of reaction to-4.5 DEG C with-15 DEG C of refrigerants, be that 78rpm stirs 2.7 hours with stirring velocity, diatomite 3.6kg and perlite 3.6kg is added after reacting completely, be that 77rpm stirs 30 minutes with stirring velocity, with 15% balance liquid 933Kg, balance cooling is carried out to whole press filtration pipeline and pressure filter, setting purge time 32 minutes, (German Begerow company produces to dry up filter plate with-4.2 DEG C of precooling clean compressed airs, model: PX50, specification 680*779mm), exhaust temperature is made to reach-3.6 DEG C.Start suspension press filtration, controlled filter pressure is 0.20MPa, filtration velocity is 60kg/min, filtrate temperature-4.0 DEG C, suspension has pressed setting purge time 49 minutes, filter plate is dried up with precooling clean compressed air, with 15% balance liquid 886Kg, clean and reuse is carried out to pressure filter, Ethylene recov is incorporated to press filtration supernatant tank, setting purge time 90 minutes, re-use-4.0 DEG C of precooling clean compressed airs to dry up, collect component III-2 supernatant liquor 2299.4kg, component III-2 precipitates 16.5kg autoclaving discards;

15% balance liquid compound method used is: every 1000kg water for injection adds the ethanol that less than-20 DEG C 95% (v/v) ethanol 158.3kg obtain 15% (v/v); Then in every 1000kg 15% ethanol, Sodium phosphate dibasic 3.58kg is added; By Glacial acetic acid adjust ph to 5.21;-4.9 DEG C are adjusted the temperature to-15 DEG C of refrigerants;

Step 4: separated portion II precipitates

30% (m/v) sodium chloride solution 46.9kg is added in said components component III-2 supernatant liquor, by 0.5mol/L sodium hydroxide solution 36.8kg adjust ph to 7.10, add 95% (v/v) ethanol 151.9kg of-20.7 DEG C again, by 0.5mol/L sodium hydroxide adjusted to ph to 7.02, regulate temperature of reaction to-7.2 DEG C with-15 DEG C of refrigerants, continue stirring reaction 2.7 hours, its stirring velocity is 72RPM, perlite 1.9kg is added after reacting completely, stir 30 minutes, its stirring velocity is 72RPM, with 20% (v/v) ethanol 1501Kg of-6.4 DEG C, balance cooling is carried out to whole press filtration pipeline and pressure filter, setting purge time 30 minutes, (German Begerow company produces to dry up filter plate with-7.9 DEG C of precooling clean compressed airs, model: PX50, specification 680*779mm), exhaust temperature is made to reach-6.0 DEG C, start suspension press filtration, controlled filter pressure is 0.20MPa, filtration velocity is 72kg/min, filtrate temperature-6.7 DEG C, supernatant directly discharges through ethanol emission pipeline, when component I I suspension tank weighs less than 200kg, press filtration supernatant no longer discharges and directly enters suspension tank thus form a circulation line, after circulation 10min, supernatant starts to continue through the discharge of ethanol emission pipeline, after the whole press filtration of suspension is complete, setting purge time is 106 minutes, filter plate is dried up with-8.3 DEG C of precooling clean compressed airs, collect component I I and precipitate 17.0kg,

20% ethanol compound method used is: 95% (v/v) ethanol 225.5kg that every 1000kg water for injection adds less than-20 DEG C obtains the ethanol of 20% (v/v), adjusts the temperature to-6.0 DEG C with-15 DEG C of refrigerants;

Step 5: component I I resolution of precipitate, filtration

The water for injection of 1.9 DEG C of component I I precipitation 102.0kg step 4 collected dissolves, and under stir speed (S.S.) is 78rpm, stirring 5.0 is little dissolves, by 0.5mol/L sodium hydroxide solution 1.3kg adjusted to ph to 6.95 completely up to precipitation, 1.9 DEG C are adjusted the temperature to-15 DEG C of refrigerants, continue stirring reaction 3.0 hours under stir speed (S.S.) is 74rpm, dissolve completely and add diatomite 0.18kg and perlite 0.18kg, stir 30 minutes under stir speed (S.S.) is 71rpm, with 1.6 DEG C of water for injection 368kg, balance cooling is carried out to whole press filtration pipeline and pressure filter, setting purge time 15 minutes, (German Begerow company produces to dry up filter plate with 1.9 DEG C of precooling clean compressed airs, model: Steril S100, specification: 349*384mm), exhaust temperature is made to reach 1.8 DEG C, start press filtration, controlled filter pressure is 0.10MPa, filtration velocity is 30kg/min, filtrate temperature 2.8 DEG C, dissolve the complete setting purge time of hydraulic pressure 18 minutes, press filtration separate dissolved suspension, with 1.7 DEG C of waters for injection of 117kg, clean and reuse is carried out to pressure filter, filter plate is dried up with 2.5 DEG C of precooling clean compressed airs, Ethylene recov is incorporated to filtrate tank, setting purge time is 15 minutes, filter plate is dried up with 0.0 ~ 4.0 DEG C of precooling clean compressed air, be precipitated 1.4kg, filtrate 236.3kg, filtrate pH value to 4.00 is regulated with 1mol/L acetum 2.8kg, filtering-depositing autoclaving is discarded, at this moment filtrate protein content is 1.65%, total protein is 3.93kg.

Step 6: ultrafiltration, dialysis, concentrated

Be that to be concentrated into protein content be 10.4% (m/v) for filtrate that step 5 obtains by 30KD ultra-fine filter by film specification, heavy 37.8kg, then with 3.5 DEG C, the water for injection of 189.1kg carries out continuous equivalent dialysis, proteins concentrate content to 8.7% (m/v), goods pH value to 6.97 is adjusted again with the NaOH solution 2.1kg of 0.5mol/L, controlling protein content with water for injection dilution is 7.9%, and after dilution, weight is 49.8kg;

Step 7: purifying, filtration

Goods after concentrated are transferred to sphaeroprotein treatment tank, by water for injection diluted protein matter content to 1.69% (m/v) of 1.5 DEG C, after dilution, weight is 232.8kg, then SODIUM PHOSPHATE, MONOBASIC 0.32kg and sodium-chlor 0.14kg is added, by 1mol/L acetum adjusted to ph to 6.96, 0.5 DEG C is adjusted the temperature to-15 DEG C of refrigerants, add 95% (v/v) ethanol 4.2kg of-21.8 DEG C again, regulate outlet temperature to-1.4 DEG C with-15 DEG C of refrigerants, stirring reaction is continued 2.3 hours under stir speed (S.S.) is 67rpm, perlite 0.36kg is added after reacting completely, stir 30 minutes under stir speed (S.S.) is 61rpm, with the 2% ethanol 384kg of-1.7 DEG C, balance cooling is carried out to whole press filtration pipeline and pressure filter, setting purge time 16 minutes, (German Begerow company produces to dry up filter plate with-1.5 DEG C of precooling clean compressed airs, model: Steril S100, specification: 349*384mm), exhaust temperature is made to reach-1.3 DEG C, start press filtration, controlled filter pressure is 0.10MPa, filtration velocity is 27kg/min, filtrate temperature-1.7 DEG C, the complete setting purge time of suspension press filtration is 24 minutes, with 2% ethanol of 129kg, clean and reuse is carried out to pressure filter, Ethylene recov is incorporated to filtrate tank, filter plate is dried up with-1.2 DEG C of precooling clean compressed airs, filtering supernatant pH value to 3.95 is regulated with 0.5mol/L hydrochloric acid soln 2.1kg, regulate outlet temperature to-1.7 DEG C, be precipitated 1.8kg, filtrate 367.1kg, wherein filtrate protein content is 1.02%.

2% ethanol compound method used is: every 1000kg water for injection adds less than-20 DEG C 95% (v/v) ethanol 18.2kg, adjusts the temperature to-1.6 DEG C with-15 DEG C of refrigerants;

Step 8: ultrafiltration dialysis, concentrated, preparation

Be 10.4% (m/v) (weight is 35.9kg) (intake pressure 3.4bar by the filtrate of step 7 after pH and temperature regulate through ultrafiltration and concentration to protein content, return pressure 1.9bar), then continuous equivalent dialysis is carried out with the water for injection of the 395kg of 3.6 DEG C, proteins concentrate content to 8.49% (m/v) (total protein is 3.74kg), add maltose 6.70kg, by 0.5mol/L sodium hydroxide solution adjust ph to 4.01, controlling final protein content with water for injection dilution is 5.31% (m/v), obtaining weight after preparation is 70.4kg, yield is the blood plasma of 6.3kg/kL.Carry out after having prepared degerming, except virus filtration, incubated at low pH and degerming packing, namely obtain quiet note human normal immunoglobulin work in-process.

Product checking:

The half-finished protein content of described quiet note human normal immunoglobulin is 5.28%, its purity is 99.0%, pH value is 4.02, wherein residual ethanol content is for being less than 0.025g/100ml, Content of polymer is 1.2%, ACA (anticomplementary activity) is for 8IU/ml, PKA (Prekallikrein) content is for being less than 10%.

Embodiment 6

Step one: component I I+III resolution of precipitate

Component I I+III is precipitated the 55.4kg aqueous solution (phosphoric acid sodium dihydrogen 0.53kg, water for injection 387.7kg) of the phosphoric acid sodium dihydrogen of 1.3 DEG C and stir more than 6 hours to dissolving completely, obtain component I I+III resolution of precipitate liquid 443.6kg.

Step 2: separated portion III-1 precipitates

Said components II+III resolution of precipitate liquid pH value to 5.03 is regulated with 1mol/L acetum 2.9kg,-0.2 DEG C is cooled to-15 DEG C of refrigerants, add 95% (v/v) ethanolic soln 27.7kg of-20.8 DEG C again, and by 0.5mol/L sodium hydroxide solution adjusted to ph to 5.18, regulate last suspension temperature to-3.0 DEG C with-15 DEG C of refrigerants, be that 86rpm stirs 2.4 hours with stirring velocity, diatomite 1.9kg and perlite 1.2kg is added after reacting completely, be that 86rpm stirs 30 minutes with stirring velocity, with 8% balance liquid 1022Kg, balance cooling is carried out to whole press filtration pipeline and pressure filter, setting purge time 26 minutes, (German Begerow company produces to dry up filter plate with-2.6 DEG C of precooling clean compressed airs, model: PX50, specification 680*779mm), exhaust temperature is made to reach-1.6 DEG C.Start suspension press filtration, controlled filter pressure is 0.21MPa, filtration velocity is 74kg/min, filtrate temperature-2.2 DEG C, suspension has pressed setting purge time 42 minutes, filter plate is dried up with precooling clean compressed air, with 8% balance liquid 856Kg, clean and reuse is carried out to pressure filter, Ethylene recov is incorporated to press filtration supernatant tank, setting purge time 95 minutes, re-use-2.9 DEG C of precooling clean compressed airs to dry up, collect component III-1 supernatant liquor 1294.5kg, component III-1 is discarded after precipitating 38.8kg autoclaving;

8% balance liquid compound method used is: 95% (v/v) ethanol 77.7kg that every 1000kg water for injection adds less than-20 DEG C obtains the ethanolic soln of 8% (v/v); Then in the ethanolic soln of every 1000kg 8%, Sodium phosphate dibasic 3.58kg is added; By Glacial acetic acid adjust ph to 5.01;-2.5 DEG C are adjusted the temperature to-15 DEG C of refrigerants.

Step 3: separated portion III-2 precipitates

Component III-1 supernatant liquor pH value to 5.24 is adjusted with 0.5mol/L sodium hydroxide solution 0.6kg, add 95% (v/v) ethanol 96.7kg of-21.8 DEG C again, and by 1mol/L acetic acid adjusted to ph to 5.23, regulate temperature of reaction to-5.0 DEG C with-15 DEG C of refrigerants, be that 77rpm stirs 2.0 hours with stirring velocity, diatomite 3.5kg and perlite 3.5kg is added after reacting completely, be that 79rpm stirs 30 minutes with stirring velocity, with 15% balance liquid 997Kg, balance cooling is carried out to whole press filtration pipeline and pressure filter, setting purge time 30 minutes, (German Begerow company produces to dry up filter plate with-4.4 DEG C of precooling clean compressed airs, model: PX50, specification 680*779mm), exhaust temperature is made to reach-4.1 DEG C.Start suspension press filtration, controlled filter pressure is 0.20MPa, filtration velocity is 71kg/min, filtrate temperature-3.9 DEG C, suspension has pressed setting purge time 45 minutes, filter plate is dried up with precooling clean compressed air, with 15% balance liquid 933Kg, clean and reuse is carried out to pressure filter, Ethylene recov is incorporated to press filtration supernatant tank, setting purge time 87 minutes, re-use-4.0 DEG C of precooling clean compressed airs to dry up, collect component III-2 supernatant liquor 2315.2kg, component III-2 precipitates 16.6kg autoclaving discards;

15% balance liquid compound method used is: every 1000kg water for injection adds the ethanol that less than-20 DEG C 95% (v/v) ethanol 158.3kg obtain 15% (v/v); Then in every 1000kg 15% ethanol, Sodium phosphate dibasic 3.58kg is added; By Glacial acetic acid adjust ph to 5.17;-4.1 DEG C are adjusted the temperature to-15 DEG C of refrigerants;

Step 4: separated portion II precipitates

30% (m/v) sodium chloride solution 47.2kg is added in said components component III-2 supernatant liquor, by 0.5mol/L sodium hydroxide solution 37.0kg adjust ph to 7.09, add 95% (v/v) ethanol 152.9kg of-20.5 DEG C again, by 1mol/L acetic acid adjusted to ph to 7.04, regulate temperature of reaction to-7.5 DEG C with-15 DEG C of refrigerants, continue stirring reaction 2.6 hours, its stirring velocity is 74RPM, perlite 1.9kg is added after reacting completely, stir 30 minutes, its stirring velocity is 74RPM, with 20% (v/v) ethanol 1587Kg of-6.0 DEG C, balance cooling is carried out to whole press filtration pipeline and pressure filter, setting purge time 30 minutes, (German Begerow company produces to dry up filter plate with-8.3 DEG C of precooling clean compressed airs, model: PX50, specification 680*779mm), exhaust temperature is made to reach-6.6 DEG C, start suspension press filtration, controlled filter pressure is 0.16MPa, filtration velocity is 65kg/min, filtrate temperature-6.0 DEG C, supernatant directly discharges through ethanol emission pipeline, when component I I suspension tank weighs less than 200kg, press filtration supernatant no longer discharges and directly enters suspension tank thus form a circulation line, after circulation 10min, supernatant starts to continue through the discharge of ethanol emission pipeline, after the whole press filtration of suspension is complete, setting purge time is 105 minutes, filter plate is dried up with-8.0 DEG C of precooling clean compressed airs, collect component I I and precipitate 17.2kg,

20% ethanol compound method used is: 95% (v/v) ethanol 225.5kg that every 1000kg water for injection adds less than-20 DEG C obtains the ethanol of 20% (v/v), adjusts the temperature to-6.5 DEG C with-15 DEG C of refrigerants;

Step 5: component I I resolution of precipitate, filtration

The water for injection of 1.1 DEG C of component I I precipitation 103.0kg step 4 collected dissolves, and under stir speed (S.S.) is 71rpm, stirring 5.0 is little dissolves, by 0.5mol/L sodium hydroxide solution 1.3kg adjusted to ph to 7.01 completely up to precipitation, 1.6 DEG C are adjusted the temperature to-15 DEG C of refrigerants, continue stirring reaction 2.3 hours under stir speed (S.S.) is 74rpm, dissolve completely and add diatomite 0.18kg and perlite 0.18kg, stir 30 minutes under stir speed (S.S.) is 79rpm, with 1.0 DEG C of water for injection 408kg, balance cooling is carried out to whole press filtration pipeline and pressure filter, setting purge time 15 minutes, (German Begerow company produces to dry up filter plate with 1.6 DEG C of precooling clean compressed airs, model: Steril S100, specification: 349*384mm), exhaust temperature is made to reach 2.1 DEG C, start press filtration, controlled filter pressure is 0.15MPa, filtration velocity is 23kg/min, filtrate temperature 2.0 DEG C, dissolve the complete setting purge time of hydraulic pressure 25 minutes, press filtration separate dissolved suspension, with 1.9 DEG C of waters for injection of 112kg, clean and reuse is carried out to pressure filter, filter plate is dried up with 2.4 DEG C of precooling clean compressed airs, Ethylene recov is incorporated to filtrate tank, setting purge time is 22 minutes, filter plate is dried up with 0.0 ~ 4.0 DEG C of precooling clean compressed air, be precipitated 1.5kg, filtrate 232.5kg, filtrate pH value to 3.97 is regulated with 1mol/L acetum 2.8kg, filtering-depositing autoclaving is discarded, at this moment filtrate protein content is 1.67%, total protein is 3.93kg.

Step 6: ultrafiltration, dialysis, concentrated

Be that to be concentrated into protein content be 10.2% (m/v) for filtrate that step 5 obtains by 30KD ultra-fine filter by film specification, heavy 38.5kg, then with 3.0 DEG C, the water for injection of 192.7kg carries out continuous equivalent dialysis, proteins concentrate content to 8.6% (m/v), goods pH value to 6.91 is adjusted again with the NaOH solution 2.1kg of 0.5mol/L, controlling protein content with water for injection dilution is 7.8%, and after dilution, weight is 50.4kg;

Step 7: purifying, filtration

Goods after concentrated are transferred to sphaeroprotein treatment tank, by water for injection diluted protein matter content to 1.81% (m/v) of 1.4 DEG C, after dilution, weight is 217.1kg, then SODIUM PHOSPHATE, MONOBASIC 0.30kg and sodium-chlor 0.13kg is added, by 0.5mol/L sodium hydroxide solution adjusted to ph to 6.92, 0.0 DEG C is adjusted the temperature to-15 DEG C of refrigerants, add 95% (v/v) ethanol 4.0kg of-21.4 DEG C again, regulate outlet temperature to-1.5 DEG C with-15 DEG C of refrigerants, stirring reaction is continued 2.1 hours under stir speed (S.S.) is 67rpm, perlite 0.33kg is added after reacting completely, stir 30 minutes under stir speed (S.S.) is 69rpm, with the 2% ethanol 382kg of-1.6 DEG C, balance cooling is carried out to whole press filtration pipeline and pressure filter, setting purge time 16 minutes, (German Begerow company produces to dry up filter plate with-0.5 DEG C of precooling clean compressed air, model: Steril S100, specification: 349*384mm), exhaust temperature is made to reach-1.7 DEG C, start press filtration, controlled filter pressure is 0.15MPa, filtration velocity is 30kg/min, filtrate temperature-1.3 DEG C, the complete setting purge time of suspension press filtration is 19 minutes, with 2% ethanol of 124kg, clean and reuse is carried out to pressure filter, Ethylene recov is incorporated to filtrate tank, filter plate is dried up with-0.7 DEG C of precooling clean compressed air, filtering supernatant pH value to 3.98 is regulated with 0.5mol/L hydrochloric acid soln 2.1kg, regulate outlet temperature to-1.3 DEG C, be precipitated 1.7kg, filtrate 346.2kg, wherein filtrate protein content is 1.08%.

2% ethanol compound method used is: every 1000kg water for injection adds less than-20 DEG C 95% (v/v) ethanol 18.2kg, adjusts the temperature to-1.6 DEG C with-15 DEG C of refrigerants;

Step 8: ultrafiltration dialysis, concentrated, preparation

Be 10.2% (m/v) (weight is 36.6kg) (intake pressure 3.7bar by the filtrate of step 7 after pH and temperature regulate through ultrafiltration and concentration to protein content, return pressure 1.0bar), then continuous equivalent dialysis is carried out with the water for injection of the 403kg of 3.1 DEG C, proteins concentrate content to 8.82% (m/v) (total protein is 3.73kg), add maltose 6.75kg, by 0.5mol/L sodium hydroxide solution adjust ph to 4.02, controlling final protein content with water for injection dilution is 5.27% (m/v), obtaining weight after preparation is 70.8kg, yield is the blood plasma of 6.3kg/kL.Carry out after having prepared degerming, except virus filtration, incubated at low pH and degerming packing, namely obtain quiet note human normal immunoglobulin work in-process.

Product checking:

The half-finished protein content of described quiet note human normal immunoglobulin is 5.33%, its purity is 98.8%, pH value is 4.02, wherein residual ethanol content is for being less than 0.025g/100ml, Content of polymer is 1.5%, ACA (anticomplementary activity) is for 8IU/ml, PKA (Prekallikrein) content is for being less than 10%.

Table 1

As can be seen from detecting step and the result of above embodiment, the present invention adopts cold ethanol method production technique, the top 5 factor of cold ethanol method is regulated: ionic strength, pH value, temperature, alcohol concn and protein content realize being separated the component I I in I+II+III (or component I I+III) precipitation by multistep, quiet note human normal immunoglobulin finished product is obtained through purifying further, technological operation is simple, yield rate is high, steady quality, application risk is low, thus ensure that people's drug safety.

Claims (4)

1. a preparation method for quiet note human normal immunoglobulin, is characterized in that, comprise the steps:

Step one: component I+II+III or component I I+III resolution of precipitate

The aqueous solution of component I+II+III precipitation or component I I+III precipitation SODIUM PHOSPHATE, MONOBASIC is dissolved completely;

Step 2: separated portion I+III-1 precipitates or component III-1 precipitates

Regulate said components I+II+III resolution of precipitate liquid or component I I+III resolution of precipitate liquid pH value to 5.00 ~ 5.20, adjust the temperature to-0.5 ~ 0.0 DEG C, using the ethanolic soln of less than-20 DEG C 95% to be adjusted to ethanol final concentration is again 7.5 ~ 8.5%, and adjusted to ph to 5.00 ~ 5.20, adjust the temperature to-3.0 DEG C ~-2.0 DEG C, stirring makes it react completely, diatomite 3.5 ~ 4.5kg and perlite 2.0 ~ 3.0kg is added by every 1000kg suspension, stir, start suspension press filtration, controlled filter pressure is 0 ~ 0.3MPa, filtration velocity is 20 ~ 90kg/min, filtrate temperature-3.0 ~-1.0 DEG C, collect component I+III-1 supernatant liquor or component III-1 supernatant liquor,

Step 3: separated portion I+III-2 precipitates or component III-2 precipitates

Adjustment component I+III-1 supernatant liquor or component III-1 supernatant liquor pH value to 5.10 ~ 5.30, ethanol final concentration to 14 ~ 16% is regulated with the ethanol of less than-20 DEG C 95%, and adjusted to ph to 5.10 ~ 5.30, temperature of reaction is to-5.0 DEG C ~-4.0 DEG C, stirring makes it react completely, diatomite 2.0 ~ 3.0kg and perlite 2.0 ~ 3.0kg is added by every 1000kg suspension, stir, start suspension press filtration, controlled filter pressure is 0 ~ 0.3MPa, filtration velocity is 20 ~ 90kg/min, filtrate temperature-5.0 ~-3.0 DEG C, collect component I+III-2 supernatant liquor or component III-2 supernatant liquor,

Step 4: separated portion II precipitates

The I+III-2 supernatant liquor of every 1000Kg or component III-2 supernatant liquor add the sodium chloride solution 18.4 ~ 22.4kg of 25 ~ 35%, adjust ph to 6.90 ~ 7.10, alcohol concn to 19 ~ 21% is regulated with the ethanol of less than-20 DEG C 95%, adjusted to ph to 6.90 ~ 7.10, regulate temperature of reaction to-8.0 ~-6.0 DEG C, stirring makes it react completely, perlite 0.5 ~ 1.0kg is added by every 1000kg suspension, stir, start suspension press filtration, controlled filter pressure is 0.300MPa, filtration velocity is 20 ~ 90kg/min, filtrate temperature-7.5 ~-5.5 DEG C, supernatant discharges, collect component I I precipitation,

Step 5: component I I resolution of precipitate, filtration

The component I I of collection precipitation is added water for injection be stirred to and dissolve completely, adjusted to ph to 6.90 ~ 7.10, adjust the temperature to 0.0 ~ 4.0 DEG C, continue to stir, dissolve suspension by every 1000kg and add diatomite 1.0 ~ 2.0kg and perlite 1.0 ~ 2.0kg, stir, start press filtration, controlled filter pressure is 0.200MPa, filtration velocity is 10 ~ 60kg/min, filtrate temperature 0.0 ~ 5.0 DEG C, the isolated filtrate of press filtration, adjusted filtrate pH value to 3.80 ~ 4.10;

Step 6: ultrafiltration, dialysis, concentrated

Filtered liquid being concentrated into protein content is 8 ~ 12%, then continuous equivalent dialysis is carried out with the water for injection of 2 ~ 5 DEG C, 4 ~ 6 times volumes, proteins concentrate content to 8 ~ 9%, then adjusted to ph to 6.50 ~ 7.30, controlling protein content with water for injection dilution is 50.0 ~ 100.0g/L;

Step 7: purifying, filtration

Goods after step 6 is concentrated water for injection diluted protein matter content to 1.5 ~ 2.0% of 0.0 ~ 4.0 DEG C, SODIUM PHOSPHATE, MONOBASIC 1.0 ~ 3.0kg and sodium-chlor 0.2 ~ 1.0kg is added at every 1000kg goods after dilution, adjusted to ph to 6.90 ~ 7.10, adjust the temperature to 0.0 ~ 0.5 DEG C, alcohol concn to 1.5 ~ 2.5% is regulated with the ethanol of less than-20 DEG C 95%, adjust the temperature to-2.0 ~-1.0 DEG C, stirring makes it react completely, perlite 1.0 ~ 2.0kg is added by every 1000kg suspension, stir, start press filtration, controlled filter pressure is 0.200MPa, filtration velocity is 10 ~ 60kg/min, filtrate temperature-2.0 ~-1.0 DEG C, regulate filtrate pH value to 3.80 ~ 4.10, adjust the temperature to-2.0 ~-1.0 DEG C,

Step 8: ultrafiltration dialysis, concentrated, preparation

Be 8 ~ 12% by the filtrate of step 7 after pH and temperature regulate through ultrafiltration and concentration to protein content, then continuous equivalent dialysis is carried out with water for injection, proteins concentrate content to 8 ~ 9%, often rise by end article and add maltose containing maltose 95 ~ 105g, adjust ph to 3.90 ~ 4.30, controlling final protein content with water for injection dilution is 5.20 ~ 5.50%, carry out again degerming, except virus filtration, incubated at low pH and degerming packing, namely obtain quiet note human normal immunoglobulin work in-process.

2. the preparation method of quiet note human normal immunoglobulin according to claim 1, it is characterized in that, the aqueous solution of SODIUM PHOSPHATE, MONOBASIC described in step one is 6 ~ 8 times of component I+II+III or component I I+III Sediment weight, and concentration is 0.01mol/L, and churning time is more than 6 hours.

3. the preparation method of quiet note human normal immunoglobulin according to claim 1, is characterized in that, the solvent being used for regulating pH value in step 2, three, four and five is 1mol/L acetum or 0.5mol/L sodium hydroxide solution.

4. the preparation method of quiet note human normal immunoglobulin according to claim 1, is characterized in that, in described step 6, filtered liquid to be concentrated into protein content be 8 ~ 12% is adopt film specification to be that 30KD ultra-fine filter concentrates.