Process for preparing human immune globulin by membrane chromatography and protein

Abstract

The invention relates to the field of biological pharmacy, and provides a process for preparing human immune globulin by membrane chromatography, which comprises the following steps: (1) dissolving plasma components II and III by using water for injection; (2) octanoic acid precipitation; (3) low-temperature precipitation; (4) membrane chromatography; (5) ultrafiltration and nano-membrane filtration; and (6) concentrating and diluting. The invention also provides the human immune globulin prepared by the process. According to the method, the membrane chromatography technology is adopted to replace traditional gel chromatography in the extraction and separation process, the chromatography loading capacity and flow speed are improved, the chromatography time is shortened, meanwhile, the purity of the human immunoglobulin for subcutaneous injection is guaranteed, and the preparation cost is reduced.

Description

Technical Field

[0001] The present invention relates to the field of biopharmaceuticals, and in particular to a process for preparing human immunoglobulin by membrane chromatography and the protein. Background Art

[0002] Immunoglobulin G is the main component of immunoglobulin in serum, accounting for 70% to 75%. It is synthesized and secreted by B lymphocytes and has antiviral, antibacterial, and immune regulation functions. It is mainly used for: ① Kawasaki disease; ② idiopathic thrombocytopenic purpura; ③ aplastic anemia; ④ primary and secondary immunodeficiency diseases; ⑤ other indications, diabetes, autoimmune anemia, AIDS, etc.

[0003] At present, the main purification method for the preparation of the fourth-generation intravenous human immunoglobulin is gel chromatography, which can effectively remove impurities such as IgM, IgA, Alb, etc. However, the use of traditional gel chromatography technology has disadvantages such as high cost, low protein loading, low chromatography flow rate, and long process time.

[0004] Membrane chromatography technology is a high-efficiency separation technology developed based on traditional column chromatography. It uses membrane as the stationary phase and separates different components in the sample in the mobile phase through the pores on the membrane. It has the characteristics of high throughput, high flow rate, high loading capacity, easy operation and low cost.

[0005] Based on this, there is an urgent need to design a human immunoglobulin preparation process based on membrane chromatography technology. Summary of the invention

[0006] The technical problem to be solved by the present invention is to provide a process and protein for preparing human immunoglobulin by membrane chromatography. The present invention adopts membrane chromatography technology to replace traditional gel chromatography in the extraction and separation process, thereby improving the chromatography loading capacity and flow rate, shortening the chromatography time, and ensuring the purity of subcutaneous human immunoglobulin and reducing the preparation cost.

[0007] The present invention adopts the following technical solutions to solve the above technical problems:

[0008] A process for preparing human immunoglobulin by membrane chromatography, comprising the following steps:

[0009] (1) Dissolution

[0010] Use water for injection to dissolve plasma components II+III;

[0011] (2) Caprylic acid precipitation

[0012] Treating the solution of step (1) with an octanoate to precipitate impurities, and filtering to obtain a filtrate;

[0013] (3) Low temperature precipitation

[0014] Ultrafilter the filtrate obtained in step (2) to a protein content of 1% to 4%, adjust the pH to 5.6 to 6.6, control the conductivity to 1.0 to 3.0 ms / cm, let stand, and filter to obtain a filtrate;

[0015] (4) Membrane chromatography

[0016] The pH of the filtrate obtained in step (3) is adjusted to 4.5-5.5, the conductivity is controlled to 0.5-3.5 ms / cm, and the filtrate is purified by membrane chromatography, and the flow-through is collected;

[0017] (5) Ultrafiltration, nano-membrane filtration

[0018] The flow-through liquid is concentrated by ultrafiltration to a protein content of more than 5%, dialyzed with 1.5-2.5% glycine in equal volumes, the pH is adjusted to 3.6-4.6, sterilized and filtered with a sterilizing filter element, and filtered with a nano-membrane for virus removal;

[0019] (6) Concentrated and diluted

[0020] Dialyze with 1.5-2.5% glycine and concentrate to 22%; adjust the protein content to 18%-20% with 1.5-2.5% glycine, adjust the pH to 3.5-5.0, sterilize and package after dilution, and place the product in a low pH incubator for incubation.

[0021] As one of the preferred embodiments of the present invention, in the step (1), 7 to 18 times of water for injection is used to dissolve the plasma component II+III precipitate, the injection water temperature is 2 to 8°C, the dissolution temperature is 2 to 20°C, and the dissolution time is 1 to 3 hours.

[0022] As one of the preferred embodiments of the present invention, in the step (2), after adding 0.5-2M glacial acetic acid to the solution of step (1) to adjust the pH to 3.8-4.8, a sodium octanoate solution with a mass fraction of 10% is added to the solution to adjust the sodium octanoate concentration to 10mM-40mM; then, the pH is adjusted to 3.5-5.5 and stirred at 2-20°C for 1-2h; finally, the solution is filtered to obtain a filtrate.

[0023] As one of the preferred embodiments of the present invention, in the step (3), the filtrate obtained in the step (2) is subjected to ultrafiltration and dialysis until the protein content is 1% to 4%, the pH is adjusted to 5.6 to 6.6, the conductivity is controlled at 1.0 to 3.0 ms / cm, the mixture is allowed to stand for 2 to 8 hours, and filtered through a 0.45 um filter element to obtain a filtrate.

[0024] As one of the preferred embodiments of the present invention, in step (4), Purcise Q strong anionic membrane is used as the membrane chromatography membrane, and the specific chromatography steps are:

[0025] ① Use pH 5.0-6.5, 180-300mM NaAc buffer to balance the Purcise Q strong anion membrane for 2-5 column volumes, with a flow rate not exceeding 1100cm / h;

[0026] ② Load the sample at a flow rate not exceeding 1100 cm / h, and collect the flow-through liquid after the UV peak;

[0027] ④ After loading, wash with 18-30 mM NaAc buffer at pH 5.0-6.5 for 2-4 column volumes; mix the washing solution with the loading flow-through solution as the sample solution for subsequent steps.

[0028] As one of the preferred embodiments of the present invention, in the step (5), the flow-through liquid of step (4) is concentrated to more than 5% by ultrafiltration using a 30KD ultrafiltration membrane, and is dialyzed with 1.5-2.5% glycine at an equal volume; then, the pH is adjusted to 3.6-4.6, and the virus is removed by filtration using a 20nm filter element to obtain a protein solution after nanofiltration.

[0029] As one of the preferred embodiments of the present invention, in the step (6), the protein solution after nanofiltration in the step (5) is dialyzed 2 to 4 times with 1.5 to 2.5% glycine of equal volume, the pH is adjusted to 3.5 to 5.0, and the protein content is concentrated to 22%; then, the protein content is diluted to 18 to 20% with 1.5 to 2.5% glycine, and the pH is adjusted to 3.5 to 5.0; sterile filtration is performed and the sample is packaged, and the packaged sample is placed in an incubation chamber at a pH of 4.0 to 4.5 for incubation.

[0030] A human immunoglobulin prepared by the process for preparing human immunoglobulin by the membrane chromatography method.

[0031] The advantages of the present invention compared to the prior art are:

[0032] (1) The present invention uses caprylic acid precipitation method to treat components II+III, and optimizes the caprylic acid precipitation chromatography process parameters to effectively remove foreign proteins (non-Ig proteins) and lipids, thereby improving the purity of IgG;

[0033] (2) The present invention uses membrane chromatography medium to replace traditional gel medium; wherein, the membrane chromatography medium is based on the principle of convective mass transfer, so that the charged biological molecules can be bound once passing through the membrane, and has a relatively large pore size channel, presenting a high load and flow rate at low pressure, so that the membrane chromatography has the characteristics of high binding efficiency and short chromatography time; compared with traditional anion gel chromatography, the flow rate of human immunoglobulin chromatography prepared by membrane chromatography is 3 to 5 times faster, the efficiency of chromatography is greatly improved, the load is increased from 80g / L to more than 200g / L, and the cost is lower; at the same time, IgA, IgM, Alb and other components are effectively removed by membrane chromatography, the IgG purity reaches ≥99%, IgG monomer + dimer ≥99%, and the IgA residual amount is <22mg / L, which improves the quality of the product, ensures the safety of the product, and reduces clinical risks;

[0034] (3) In the process of ultrafiltration and concentration, glycine is added to both the protein solution and the dialysate as a protein protectant to reduce the effect of ultrafiltration shear on the protein structure. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 is a process flow chart of preparing human immunoglobulin by membrane chromatography in Examples 1 to 4;

[0036] Figure 2 This is the experimental spectrum of Purcise Q membrane chromatography in Example 2. DETAILED DESCRIPTION

[0037] The embodiments of the present invention are described in detail below. The embodiments are implemented on the premise of the technical solution of the present invention, and detailed implementation methods and specific operation processes are given, but the protection scope of the present invention is not limited to the following embodiments. At the same time, the reagent products and experimental methods used in the following embodiments, unless otherwise specified, are conventional reagents or methods in the art and will not be repeated.

[0038] Example 1

[0039] A membrane chromatography process for preparing human immunoglobulin in this embodiment is as follows: Figure 1 As shown, the following steps are included:

[0040] (1) Dissolution

[0041] Weigh 1 kg of plasma component II+III precipitate and dissolve it with 7 times injection water (2°C) at a dissolution temperature of 2°C and a dissolution time of 1 hour.

[0042] (2) Caprylic acid precipitation

[0043] After adding 0.5M glacial acetic acid to the solution of step (1) to adjust the pH to 3.8, add a 10% sodium octanoate solution to the solution to adjust the sodium octanoate concentration to 10mM; then, use acetic acid to adjust the pH to 3.5, control the temperature of the solution at 2°C and stir for 1h; finally, filter press to obtain a filtrate.

[0044] (3) Low temperature precipitation

[0045] The filtrate obtained in step (2) is subjected to ultrafiltration and dialysis until the protein content is 1%, the pH is adjusted to 5.6, the conductivity is controlled at 1.0 ms / cm, and the mixture is allowed to stand for 2 hours and filtered through a 0.45 um filter element to obtain a filtrate.

[0046] (4) Membrane chromatography

[0047] The pH of the filtrate obtained in step (3) was adjusted to 4.5, the conductivity was controlled to 0.5 ms / cm, and the product was purified by membrane chromatography (using PurciseQ strong anionic membrane, column volume 0.45 ml), and the chromatography steps were as follows:

[0048] ① Use pH 5.0, 180mM NaAc buffer to balance the Purcise Q strong anion membrane for 2 column volumes, with a flow rate not exceeding 1100cm / h;

[0049] ② Load the sample at a flow rate not exceeding 1100 cm / h, and collect the flow-through liquid after the UV peak;

[0050] ④ After loading, wash with pH 5.0, 18 mM NaAc buffer for 2 column volumes; mix the washing solution with the loading flow-through solution as the sample for the subsequent steps.

[0051] (5) Ultrafiltration, nano-membrane filtration

[0052] The sample solution (flow-through + washing solution) of step (4) was concentrated to a protein content of 5% by ultrafiltration using a 30KD ultrafiltration membrane, and dialyzed against an equal volume of 1.5% glycine; then, the pH was adjusted to 3.6, and sterile filtration was performed using a sterilizing filter element, and virus removal filtration was performed using a 20nm nanomembrane.

[0053] (6) Concentrated and diluted

[0054] The protein solution after nanofiltration in step (5) is dialyzed twice with 1.5% glycine in equal volumes, the pH is adjusted to 3.5, and concentrated to a protein content of 22%; then, the protein content is diluted to 18% with 1.5% glycine, and the pH is adjusted to 3.5; sterile filtration is performed and the sample is placed in an incubator at pH 4.0 for incubation.

[0055] Example 2

[0056] A membrane chromatography process for preparing human immunoglobulin in this embodiment is as follows: Figure 1 As shown, the following steps are included:

[0057] (1) Dissolution

[0058] Weigh 1 kg of plasma component II+III precipitate and dissolve it with 10 times injection water (4°C) at a dissolution temperature of 4°C and a dissolution time of 2 h.

[0059] (2) Caprylic acid precipitation

[0060] After adding 2M glacial acetic acid to the solution of step (1) to adjust the pH to 4.0, add a 10% sodium octanoate solution to the solution to adjust the sodium octanoate concentration to 10mM; then, use acetic acid to adjust the pH to 4.0, control the temperature of the solution at 4°C and stir for 2h; finally, filter press to obtain a filtrate.

[0061] (3) Low temperature precipitation

[0062] The filtrate obtained in step (2) was subjected to ultrafiltration and dialysis until the protein content was 1.5%, the pH was adjusted to 6.0, the conductivity was controlled at 1.0 ms / cm, and the mixture was allowed to stand for 2 h and filtered with a 0.45 um filter element to obtain a filtrate.

[0063] (4) Membrane chromatography

[0064] The pH of the filtrate obtained in step (3) was adjusted to 5.0, the conductivity was controlled at 1.3 ms / cm, and the product was purified by membrane chromatography (using PurciseQ strong anionic membrane, column volume 0.45 ml), and the chromatography steps were as follows:

[0065] ① Use pH 5.0, 300mM NaAc buffer to balance the Purcise Q strong anion membrane for 3 column volumes, with a flow rate not exceeding 1100cm / h;

[0066] ② Load the sample at a flow rate not exceeding 1100 cm / h, and collect the flow-through liquid after the UV peak;

[0067] ④ After loading, wash with pH 5.0, 25mM NaAc buffer for 2 to 43 column volumes; mix the washing solution with the loading flow-through solution as the sample solution for subsequent steps.

[0068] Figure 2 This is the Purcise Q membrane chromatography experiment spectrum of this example.

[0069] (5) Ultrafiltration, nano-membrane filtration

[0070] The sample solution (flow-through + washing solution) of step (4) was concentrated to a protein content of 5% by ultrafiltration using a 30KD ultrafiltration membrane, and dialyzed seven times with 1.7% glycine in equal volumes; then, the pH was adjusted to 4.5 using 1M hydrochloric acid; after sterilization filtration using a sterilization filter element, virus removal filtration was performed using a 20nm nanomembrane to obtain a nanofiltered protein solution.

[0071] (6) Concentrated and diluted

[0072] The protein solution after nanofiltration in step (5) is dialyzed three times with equal volumes of 1.7% glycine, the pH is adjusted to 4.0, and the solution is concentrated to a protein content of 22%; then, the protein content is diluted to 20% with 1.7% glycine, and the pH is adjusted to 4.0; the solution is sterilized and filtered and packaged, and the packaged samples are placed in an incubation chamber at pH 4.0 for incubation.

[0073] Example 3

[0074] A membrane chromatography process for preparing human immunoglobulin in this embodiment is as follows: Figure 1 As shown, the following steps are included:

[0075] (1) Dissolution

[0076] Weigh 1 kg of plasma component II+III precipitate and dissolve it with 10 times injection water (4°C) at a dissolution temperature of 4°C and a dissolution time of 2 h.

[0077] (2) Caprylic acid precipitation

[0078] After adding 1M glacial acetic acid to the solution of step (1) to adjust the pH to 4.5, add a 10% sodium octanoate solution to the solution to adjust the sodium octanoate concentration to 20mM; then, use acetic acid to adjust the pH to 4.5, control the temperature of the solution at 4°C and stir for 1.5h; finally, filter press to obtain a filtrate.

[0079] (3) Low temperature precipitation

[0080] The filtrate obtained in step (2) is subjected to ultrafiltration and dialysis until the protein content is 2%, the pH is adjusted to 6.0, the conductivity is controlled at 2.0 ms / cm, and the mixture is allowed to stand for 5 hours and filtered with a 0.45 um filter element to obtain a filtrate.

[0081] (4) Membrane chromatography

[0082] The pH of the filtrate obtained in step (3) was adjusted to 5.0, the conductivity was controlled at 2.0 ms / cm, and the product was purified by membrane chromatography (using PurciseQ strong anionic membrane, column volume 0.45 ml), and the chromatography steps were as follows:

[0083] ① Use pH 6.0, 200mM NaAc buffer to balance the Purcise Q strong anion membrane for 3 column volumes, with a flow rate not exceeding 1100cm / h;

[0084] ② Load the sample at a flow rate not exceeding 1100 cm / h, and collect the flow-through liquid after the UV peak;

[0085] ④ After loading, wash with pH 5.5, 20mM NaAc buffer for 3 column volumes; mix the washing solution with the loading flow-through solution as the sample solution for subsequent steps.

[0086] (5) Ultrafiltration, nano-membrane filtration

[0087] The sample solution (flow-through + washing solution) of step (4) was concentrated to a protein content of 5% by ultrafiltration using a 30KD ultrafiltration membrane, and dialyzed against an equal volume of 2.0% glycine; then, the pH was adjusted to 4.0, and sterile filtration was performed using a sterilizing filter element, and virus removal filtration was performed using a 20nm nanomembrane.

[0088] (6) Concentrated and diluted

[0089] The protein solution after nanofiltration in step (5) is dialyzed three times with 2.0% glycine at an equal volume, the pH is adjusted to 4.0, and the solution is concentrated to a protein content of 22%. Next, the protein content is diluted to 19% with 2.0% glycine, and the pH is adjusted to 4.0. The solution is sterilized and filtered and packaged, and the packaged samples are placed in an incubator at pH 4.3 for incubation.

[0090] Example 4

[0091] A membrane chromatography process for preparing human immunoglobulin in this embodiment is as follows: Figure 1 As shown, the following steps are included:

[0092] (1) Dissolution

[0093] Weigh 1 kg of plasma component II+III precipitate and dissolve it in 17 times water for injection (4°C) at 20°C for 3 hours.

[0094] (2) Caprylic acid precipitation

[0095] After adding 2M glacial acetic acid to the solution of step (1) to adjust the pH to 4.8, add a 10% sodium octanoate solution to the solution to make the sodium octanoate concentration 40mM; then, use acetic acid to adjust the pH to 5.5, stir at 20°C for 2h; finally, filter and obtain a filtrate.

[0096] (3) Low temperature precipitation

[0097] The filtrate obtained in step (2) is subjected to ultrafiltration and dialysis until the protein content is 4%, the pH is adjusted to 6.6, the conductivity is controlled at 3.0 ms / cm, and the mixture is allowed to stand for 8 hours and filtered with a 0.45 um filter element to obtain a filtrate.

[0098] (4) Membrane chromatography

[0099] The pH of the filtrate obtained in step (3) was adjusted to 5.5, the conductivity was controlled to 3.5 ms / cm, and the product was purified by membrane chromatography (using PurciseQ strong anionic membrane, column volume 0.45 ml), and the chromatography steps were as follows:

[0100] ① Use pH 6.5, 300mM NaAc buffer to balance the Purcise Q strong anion membrane for 5 column volumes, with a flow rate not exceeding 1100cm / h;

[0101] ② Load the sample at a flow rate not exceeding 1100 cm / h, and collect the flow-through liquid after the UV peak;

[0102] ④ After loading, use pH 6.5, 30mM NaAc buffer to wash for 4 column volumes; mix the washing solution with the loading flow-through solution as the sample solution for the subsequent steps.

[0103] (5) Ultrafiltration, nano-membrane filtration

[0104] The sample solution (flow-through + washing solution) of step (4) was concentrated to a protein content of 5% by ultrafiltration using a 30KD ultrafiltration membrane, and dialyzed against an equal volume of 2.5% glycine; then, the pH was adjusted to 4.6, and sterile filtration was performed using a sterilizing filter element, and virus removal filtration was performed using a 20nm nanomembrane.

[0105] (6) Concentrated and diluted

[0106] The protein solution after nanofiltration in step (5) is dialyzed four times with 2.5% glycine at an equal volume, the pH is adjusted to 5.0, and the solution is concentrated to a protein content of 22%. Next, the protein content is diluted to 20% with 2.5% glycine, and the pH is adjusted to 5.0. The solution is sterilized and filtered and packaged, and the packaged samples are placed in an incubator at pH 4.5 for incubation.

[0107] Comparative Example

[0108] The human immunoglobulin preparation process of this comparative example is basically the same as that of Example 2, with the main difference being that in step (4), "conventional gel chromatography" is used instead of "membrane chromatography".

[0109] The protein preparation process of the present invention (taking Example 2 as an example) was compared with that of the comparative example, and the results are shown in Table 1.

[0110] Table 1 Comparison of protein preparation process data between the present invention and the comparative example

[0111]

[0112] From the results in Table 1, it can be seen that compared with traditional gel chromatography, the flow rate of human immunoglobulin prepared by membrane chromatography is faster, the efficiency of the layer is greatly improved, and the loading capacity is increased from 80 mg / ml to 220 mg / ml; at the same time, IgA, IgM, Alb and other components are effectively removed by membrane chromatography, the IgG purity reaches ≥99%, IgG monomer + dimer ≥99%, and the residual IgA is <22 mg / L.

[0113] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A process for preparing human immunoglobulin by membrane chromatography, characterized in that: The steps include: (1) Dissolution Use water for injection to dissolve plasma components II+III; (2) Caprylic acid precipitation Treating the solution of step (1) with an octanoate to precipitate impurities, and filtering to obtain a filtrate; (3) Low temperature precipitation Ultrafilter the filtrate obtained in step (2) to a protein content of 1% to 4%, adjust the pH to 5.6 to 6.6, control the conductivity to 1.0 to 3.0 ms / cm, let stand, and filter to obtain a filtrate; (4) Membrane chromatography The pH of the filtrate obtained in step (3) is adjusted to 4.5-5.5, the conductivity is controlled to 0.5-3.5 ms / cm, and the filtrate is purified by membrane chromatography, and the flow-through is collected; (5) Ultrafiltration, nano-membrane filtration The flow-through liquid is concentrated by ultrafiltration to a protein content of more than 5%, dialyzed with 1.5-2.5% glycine in equal volumes, the pH is adjusted to 3.6-4.6, sterilized and filtered with a sterilizing filter element, and filtered with a nano-membrane for virus removal; (6) Concentrated and diluted Dialyze with 1.5-2.5% glycine and concentrate to 22%; adjust the protein content to 18%-20% with 1.5-2.5% glycine, adjust the pH to 3.5-5.0, sterilize and package after dilution, and place the product in a low pH incubator for incubation.

2. The process for preparing human immunoglobulin by membrane chromatography according to claim 1, characterized in that: In the step (1), 7 to 18 times of injection water is used to dissolve the plasma component II+III precipitate, the injection water temperature is 2 to 8°C, the dissolution temperature is 2 to 20°C, and the dissolution time is 1 to 3 hours.

3. The process for preparing human immunoglobulin by membrane chromatography according to claim 1, characterized in that: In the step (2), 0.5-2M glacial acetic acid is added to the solution of step (1) to adjust the pH to 3.8-4.8, and then a sodium octanoate solution with a mass fraction of 10% is added to the solution to adjust the sodium octanoate concentration to 10mM-40mM; then, the pH is adjusted to 3.5-5.5, and stirred at 2-20°C for 1-2h; finally, the solution is filtered to obtain a filtrate.

4. The process for preparing human immunoglobulin by membrane chromatography according to claim 1, characterized in that: In the step (3), the filtrate obtained in the step (2) is subjected to ultrafiltration and dialysis until the protein content is 1% to 4%, the pH is adjusted to 5.6 to 6.6, the conductivity is controlled at 1.0 to 3.0 ms / cm, and the mixture is allowed to stand for 2 to 8 hours, and filtered through a 0.45 um filter element to obtain a filtrate.

5. The process for preparing human immunoglobulin by membrane chromatography according to claim 1, characterized in that: In the step (4), Purcise Q strong anionic membrane is used as the membrane chromatography membrane, and the specific chromatography steps are: ① Use pH 5.0-6.5, 180-300mM NaAc buffer to balance the Purcise Q strong anion membrane for 2-5 column volumes, with a flow rate not exceeding 1100cm / h; ② Load the sample at a flow rate not exceeding 1100 cm / h, and collect the flow-through liquid after the UV peak; ④ After loading, wash with 18-30 mM NaAc buffer at pH 5.0-6.5 for 2-4 column volumes; mix the washing solution with the loading flow-through solution as the sample solution for subsequent steps.

6. The process for preparing human immunoglobulin by membrane chromatography according to claim 1, characterized in that: In the step (5), the flow-through liquid of step (4) is concentrated to more than 5% by ultrafiltration using a 30KD ultrafiltration membrane, and is dialyzed with 1.5-2.5% glycine in equal volumes; then, the pH is adjusted to 3.6-4.6, and the virus is removed by filtration using a 20nm filter element to obtain a protein solution after nanofiltration.

7. The process for preparing human immunoglobulin by membrane chromatography according to claim 1, characterized in that: In the step (6), the protein solution after nanofiltration in step (5) is dialyzed 2 to 4 times with equal volumes of 1.5 to 2.5% glycine, the pH is adjusted to 3.5 to 5.0, and the protein content is concentrated to 22%; then, the protein content is diluted to 18 to 20% with 1.5 to 2.5% glycine, and the pH is adjusted to 3.5 to 5.0; sterile filtration is performed and the sample is packaged, and the packaged sample is placed in an incubation chamber at a pH of 4.0 to 4.5 for incubation.

8. A human immunoglobulin prepared by the process for preparing human immunoglobulin by membrane chromatography as claimed in any one of claims 1 to 7.

Images