CN116023466B - Method for purifying PEG modified recombinant human interferon beta 1b protein - Google Patents

Abstract

The present invention relates to a method for purifying PEG-singly modified interferon beta protein, and more particularly, to a method for purifying PEG-singly modified human interferon beta 1b protein, and a protein purified product obtained by the method. The method can realize the effective separation of the PEG mono-modified interferon-beta protein from free PEG, unmodified protein, multi-point modified protein and other impurities, thereby obtaining the PEG mono-modified protein purified product with high purity, low free PEG residual quantity and low cyano sodium borohydride residual quantity.

Description

Method for purifying PEG modified recombinant human interferon beta 1b protein

Technical Field

The invention belongs to the field of protein purification, and relates to a method for stably and efficiently extracting PEG modified recombinant human interferon beta 1b protein (IFN-beta 1 b), in particular to separation of PEG single modified IFN-beta 1b serving as a target product from free modifier, multi-modified or unmodified protein and other impurities.

Background

Interferon-beta (IFN- β) is a first-line drug for the treatment of relapsing-remitting Multiple Sclerosis (MS), and treatment of MS by IFN- β is based on its immunomodulatory effects, and IFN- β reduces the number of bone marrow dendritic cells in peripheral blood, down regulates antigen presentation by APC, reduces T cell responses, not only reduces the number of episodes in relapsing-remitting MS patients, but also effectively delays the progression of disease in relapsing-remitting and secondary remitting MS patients. In addition, IFN-beta has strong antiviral infection and anti-tumor effect, and has obvious clinical curative effect on hepatitis B, hepatitis C and viral diseases thereof. Interferon beta-1 b (IFN-beta 1 b) is a genetically engineered protein drug obtained by changing Cys17 into Ser17 and deleting Met1 from human fibroblast-derived IFN-beta through point mutation, and rhIFN-beta 1b is approved to be marketed in the United states in 1993, but the product is not yet available in China.

The pharmacological and pharmaceutical properties of the modified polyethylene glycol (PEG) coupled pharmaceutical protein can be obviously changed, and the modified polyethylene glycol molecule can shield the antigenic site of the pharmaceutical protein, so that the immunogenicity of the prototype protein can be obviously reduced, in addition, the molecular weight of the modified pharmaceutical protein is increased, the filtering effect of glomerulus can be reduced, the modified pharmaceutical protein has lower in vivo clearance rate, and the prototype protein and in vivo half-life are effectively improved. Especially for cytokine drugs, the half-life of the drug in vivo is short, and the half-life in vivo can be obviously improved after polyethylene glycol modification, so that the dosage and the injection times of the drug are effectively reduced. In addition, the protein can be modified by polyethylene glycol to improve the hydration radius, so that the water solubility of the protein is improved, and the stability of some protein medicines with stronger hydrophobicity is improved.

The interferon beta 1b protein is a strong hydrophobin, so that a surfactant is unavoidable in the preparation and purification of the protein and modification of polyethylene glycol, and Sodium Dodecyl Sulfate (SDS) is generally used for preparing a purified interferon beta 1b protein buffer. In the presence of Sodium Dodecyl Sulfate (SDS), the ion exchange process cannot be performed because neither the conjugated nor unconjugated PEG interferon beta 1b proteins bind to the ion exchange filler. The gel filtration purification technology is simply used, so that the PEG molecules of unconjugated protein cannot be removed, the gel filtration purification effect is affected by the existence of a large number of PEG molecules, and single-molecule PEG modified IFN beta 1b cannot be effectively separated from other components. In general, the protein solution obtained after the PEG modification process is complex in composition and comprises unmodified IFN- β1b prototype protein, single-molecule PEG modified IFN- β1b protein, two or more PEG molecule modified IFN- β1b proteins, and unconjugated PEG molecules. In view of this, there is a need in the art for how to effectively separate single molecule PEG-modified IFN- β1b proteins from other components.

Disclosure of Invention

The invention provides a method for purifying PEG single-modified IFN-beta (PEG-IFN-beta), in particular to a method for purifying PEG single-modified recombinant human interferon beta 1b protein (PEG-rhIFN-beta 1 b), and more particularly relates to a method for stably and efficiently extracting PEG-modified recombinant human interferon beta 1b protein.

The PEG mono-modified IFN- β or recombinant human interferon β 1b proteins to be purified by the methods of the present invention can be prepared using a PEG modifier and sodium cyanoborohydride. For example, a recombinant human interferon beta 1b protein to be modified (e.g., a recombinant human interferon beta 1b protein purified by fermentation and renatured) is first prepared; then modifying recombinant human interferon beta 1b protein by using a PEG modifier; in the modification process, sodium cyanoborohydride is used as a reducing agent to activate the PEG modifier, so that the N-terminal amino group of the protein is fully modified. However, sodium cyanoborohydride is a highly toxic compound that needs to be removed stepwise in a subsequent purification process.

The PEG modifier described above is an activated form or an activated derivative of PEG, for example, polyethylene glycol activated form having an N-terminal amino group reactivity (for example, methoxypolyethylene glycol propionaldehyde (mPEG-ALD)), but is not limited thereto, and PEG modifier may also be mPEG-SC (succinimidyl carbonate), mPEG-SCM (succinimidyl acetate), mPEG-SPA (succinimidyl propionate), mPEG-OTS (p-toluenesulfonate), mPEG butyraldehyde, and the like.

The target product to be purified in the present invention is IFN- β or IFN- β1b having a PEG single modification at the N-terminus (hereinafter referred to simply as "PEG single modification" IFN- β or IFN- β1b, "single molecule PEG modification" IFN- β or IFN- β1b, or PEG-IFN- β or PEG-IFN- β1b).

The method of the invention can remove process impurities such as sodium cyanoborohydride and free PEG in the PEG modification process in the presence of SDS, and remove SDS, thereby realizing the purification of single modified PEG interferon beta 1b protein with high purity and high activity.

To achieve the above object, a first aspect of the present invention provides a method for purifying polyethylene glycol (PEG) singly modified IFN- β protein, comprising the steps of:

s1) desalting gel filtration chromatography of a mixture containing PEG single modified IFN- β protein using an elution buffer having a pH of 2.0 to 5.0, and collecting protein peaks to obtain a first collection solution;

s2) adding a salt solution containing sodium chloride into the first collection liquid obtained in the step S1 for salting out, centrifugally separating out a precipitate, and dissolving the precipitate with a buffer solution to obtain a compound solution; in the salt solution containing sodium chloride, the molar ratio of sodium chloride to total salt is more than 70%, preferably more than 80%, more preferably more than 90%, most preferably 100%;

s3) performing gel filtration chromatography on the complex solution obtained in the step S2, and collecting protein peaks to obtain a second collection liquid containing PEG single modified IFN-beta protein;

s4) filtering and concentrating the second collection liquid by adopting a filter membrane with a molecular weight cut-off value of less than 30 kD to obtain a concentrated liquid; and

s5) carrying out desalting gel filtration chromatography on the concentrated solution, and collecting protein peaks to obtain purified PEG single modified IFN-beta protein.

In the method of the present invention, the mixture containing PEG mono-modified IFN- β protein used in step S1) may be a reaction product mixture obtained by modifying IFN- β protein with a PEG modifier, for example, a reaction product mixture obtained by modifying IFN- β protein with a PEG modifier in the presence of sodium cyanoborohydride, or may be a concentrate or a crude extract thereof; in addition to the PEG mono-modified IFN- β protein, the reaction product mixture or concentrate or crude extract thereof typically contains unconjugated free PEG modifier, unmodified IFN- β protein, two or more PEG molecule modified IFN- β proteins, sodium cyanoborohydride, SDS, and other impurities.

In the method of the present invention, the pH of the elution buffer in step S1) may be 2.3 to 4.0, preferably 2.5 to 3.5, and the elution buffer may be a citric acid buffer.

In the method of the present invention, the sodium chloride concentration in the sodium chloride-containing salt solution used in step S2) may be from 2.5 to M to saturation, preferably from 3M to 4M, more preferably from 3M to 3.5M, and the volume of the sodium chloride-containing salt solution may be from 0.5 to 1.5 times, from 0.8 to 1.2 times, preferably 1 time, the volume of the first collection liquid.

In the method of the present invention, it is preferable to perform gel filtration chromatography using an agarose gel column in step S3).

In the method of the present invention, in step S4), the OD of the buffer used in the second collection solution is used ₂₈₀ At baseline, concentration was carried out to the OD of the concentrate ₂₈₀ Greater than 1.100, preferably greater than 1.200, and more preferably greater than 1.300.

In the methods of the invention, the IFN- β protein may be a human wild-type IFN- β protein or a human IFN- β1b protein, or other animal IFN- β protein, or a chemically modified, site-directed mutated or recombinant variant of an IFN- β protein.

In another aspect of the invention, there is provided a purified PEG mono-modified IFN- β protein product obtained by the purification method of the invention described above.

The molecular weight of the purified PEG mono-modified IFN- β proteins of the present invention can be from 30 to 50 kDa, preferably from 35 to 45 kDa, 38 to 40 kDa, more preferably about 39 kDa, depending on the molecular weight of the PEG used.

The purified PEG mono-modified IFN- β protein obtained by the method of the present invention has a free PEG residual content of 0.2w/v% or less, preferably 0.18w/v% or less, 0.17w/v% or less, more preferably 0.15w/v% or less or 0.14w/v% or less.

The purified PEG mono-modified IFN- β protein obtained by the method of the present invention has a sodium cyanoborohydride content of 0.050 mmol/L or less, preferably 0.045 mmol/L or less, more preferably 0.040 mmol/L or less.

The HPLC purity of the purified PEG single modified IFN- β protein obtained by the method of the present invention can be 95% or more, 96% or more, preferably 97% or more, 98% or more, more preferably 99% or more, or 99.5% or more.

Drawings

FIG. 1 is an iodine-stained SDS-PAGE of an aliquot of pure protein purified by the method of one embodiment of the present invention, wherein lane 1 is a protein molecular weight Marker, lane 2 is an unmodified IFN- β protein, lane 3 is a PEG standard Marker, lanes 4-6 are three separate batches of PEG-modified and pre-purified products shown in Table 1, and lanes 8-10 are products of lanes 4-6, respectively, purified by the method of the present invention;

FIG. 2 is a Coomassie-stained SDS-PAGE of an pool of pure protein purified by the method of one embodiment of the present invention, wherein lane 1 is a protein molecular weight Marker, lane 2 is an unmodified IFN- β protein, lane 3 is a PEG standard Marker, lanes 4-6 are three separate batches of PEG-modified and pre-purified products shown in Table 1, and lanes 8-10 are products of lanes 4-6, respectively, purified by the method of the present invention;

FIG. 3 is a SEC-HPLC analysis of three batches of PEG single modified protein pure product shown in Table 1 obtained by the method of one embodiment of the present invention.

Detailed Description

The steps of the method of purifying PEG single modified IFN- β protein of the present invention will now be described in detail using recombinant human interferon- β1b protein as an example.

The resulting product mixture after modification of recombinant human interferon beta 1b protein with PEG contains various components, such as unmodified IFN-beta 1b prototype protein, single molecule PEG modified IFN-beta 1b protein (i.e., PEG single modified IFN-beta 1b protein), IFN-beta 1b protein modified with more than two PEG molecules, unconjugated free PEG molecules (activated form), sodium cyanoborohydride, SDS, and the like.

Step S1, desalting gel filtration chromatography

First, desalting gel filtration chromatography of the above product mixture at a pH of 2.0 to 5.0, preferably 2.3 to 4.0, more preferably 2.5 to 3.5, is effective to remove sodium cyanoborohydride, thereby performing preliminary purification of PEG-singly-modified recombinant human interferon beta 1b protein. If the pH value of the desalting gel filtration chromatography process is more than 5.0, the free PEG and the target product cannot be effectively separated; if the pH is less than 2.0, irreversible denaturation of the protein occurs, resulting in a decrease in biological activity.

The gel filtration chromatography medium used in this step is not particularly limited, and may be appropriately selected by those skilled in the art. In one embodiment, the gel filtration chromatography column may be a G25 protein desalting column, the loading may be 5% -20% column volume (cV), and the flow rate may be 10 cm/h-30 cm/h.

In one embodiment, the elution buffer used for gel filtration chromatography may be 50 mM citrate buffer (pH 3.0), but the present invention is not limited thereto as long as it is a buffer commonly used in the art that can provide the above pH range.

S2, salting out

Adding a salt solution containing sodium chloride to the protein collection solution obtained in step S1 (referred to as "first collection solution") for salting out effectively precipitates PEG mono-modified IFN- β1b protein while leaving impurities (e.g., residual free PEG) soluble in the salt solution containing sodium chloride substantially in the supernatant. This step utilizes not only the salting-out effect of the protein, but also the additional acceleration of the precipitation/crystallization of the free PEG in the salt solution, thereby synergistically effecting the precipitation and separation of the PEG-mono-modified IFN- β1b protein under suitable salting-out conditions.

The sodium chloride concentration of the sodium chloride-containing salt solution used in this step may be 2.5 to M to saturation, preferably 3M to 4M, more preferably 3M to 3.5M, and the volume of the salt solution may be 0.5 to 1.5 times, 0.8 to 1.2 times, preferably 1 time the volume of the first collection liquid. In the sodium chloride-containing salt solution, the molar ratio of sodium chloride to the total salt is 70% or more, preferably 80% or more, more preferably 90% or more, and most preferably 95% or more, 99% or more, or 100%. Herein, the term "salt solution containing sodium chloride" refers to a salt solution in which sodium chloride is dissolved, but the salt solution may be dissolved with other salts than sodium chloride; the term "sodium chloride solution" refers to a solution composed of sodium chloride and water, but may contain trace amounts of unavoidable impurities.

The inventors found that if a solution of a salt such as ammonium sulfate, sodium sulfate or potassium chloride is used, a part of free PEG precipitates together with the protein, and the objective of removing PEG without separation is achieved.

Then, the precipitate generated by salting-out is collected and separated by centrifugation, and the precipitate is dissolved with a suitable buffer to obtain a double solution. Suitable buffers may be buffers commonly used in the art for dissolving proteins, such as sodium acetate buffer, phosphate buffer, MES buffer, etc. of 10-100 mM, which may have a pH of 4.0-6.0, or 5.0-5.5, e.g., 5.5. In one embodiment, the salt solution used for salting out is an equal volume of sodium chloride solution with a concentration of not less than 3M, centrifugation is performed at 10000 g-21900 g for 0.5-3 hours, and the precipitate can be dissolved with 50 mM of sodium acetate buffer to obtain a complex solution.

Step S3, gel column chromatography

The salting-out precipitate complex solution obtained in step S2 is preferably subjected to gel filtration chromatography using an agarose gel column, thereby further purifying the PEG-singly modified IFN- β protein.

The agarose gel column used in this step may be of the SUPEROSE series, such as SUPEROSE 6, SUPEROSE 12, but is not limited thereto.

Furthermore, the inventors have unexpectedly found that if this step is performed using a polyacrylamide dextran-based gel column (e.g., sephacryl series) for filtration chromatography, the peak of PEG residue, the peak of PEG single-modified protein, and the peak of PEG double-site modified protein overlap each other, it is difficult to effectively separate the PEG single-modified protein peak, and the chromatography resolution is low, resulting in a possibility that the purity of the target product is lower than 95%.

In one embodiment, the conditions for agarose gel column chromatography may be: SUPEORE 12 column diameter to height ratio of 1:7 to 1:20, loading of 1-5% cV, flow rate of 10cm/h to 20cm/h, and/or eluent sodium acetate buffer (e.g., 50 mM).

After collection of the protein peaks, a second collection was obtained, which could be sampled for SDS-PAGE electrophoresis to confirm the identity and general purity of the purified product.

Step S4, ultrafiltration concentration and separation

And (3) performing ultrafiltration concentration on the second collected liquid obtained in the step (S3) by using a filter membrane with a molecular weight cut-off value of less than 30 kD to obtain a concentrated liquid. In one embodiment, the concentration factor may be 4-15, preferably 5-10, for example 7-9. In one embodiment, as used in the second collection fluidOD of buffer (e.g. sodium acetate) ₂₈₀ For baseline, concentration may be carried out to the OD of the concentrate ₂₈₀ Greater than 1.100, preferably greater than 1.200, and more preferably greater than 1.300.

The purpose of this step is to concentrate the purified product while also removing a portion of the small molecule impurities dissolved in the collection liquid.

The filter used in this step may be 20 or less kD or 10 or less kD. In view of filtration efficiency, a filter membrane having a cut-off value of 30 kD is preferably used.

Step S5, desalting chromatography

Gel filtration chromatography is performed on the concentrated PEG single modified interferon beta protein concentrate to remove salts, such as sodium acetate and SDS, and protein peaks are collected to obtain purified PEG single modified IFN-beta 1b protein.

The column used in this step may be a G25 protein desalting column, but is not limited thereto; the loading amount can be 1% -15% cV, and the chromatography buffer can be 10 mM phosphate buffer (pH 7.2), but is not limited thereto.

In each of the above steps, the collection and detection of the protein may be performed using a detector having a detection wavelength of 280 nm.

In one exemplary embodiment, the method of the present invention comprises: s1) measuring a PEG modified protein reaction solution, desalting and chromatography by a G25 column, wherein the chromatographic buffer solution is 50 mM citric acid buffer solution (pH 3.0), the flow rate is 20cm/h, the detection wavelength is 280nm, and a protein peak is collected; s2) slowly adding 3M to saturated sodium chloride solution into the protein peak collecting liquid to start salting out, fully stirring the salting-out suspension, standing for 30min, centrifuging at 16000 g for 30min, dissolving the precipitate after centrifugation with 50 mM sodium acetate buffer (pH 5.5), and sampling to detect the polyethylene glycol removal effect; s3) loading the salted-out solution on a SUPEROSE 12 column for chromatography, wherein the chromatographic buffer is 50 mM sodium acetate buffer (pH 5.5), the flow rate is 10cm/h, the detection wavelength is 280nm, and the protein peaks are collected for electrophoresis to detect the purity and then are combined; s4) ultrafiltering and concentrating the combined solution by using a 30 kD filter membrane until reaching OD ₂₈₀ Greater than 1.200AU; s5) loading the concentrated single modified protein into a G25 chromatographic column, wherein the chromatographic buffer solution is 10 mM phosphateBuffer solution (pH 7.2), flow rate of 10cm/h, detection wavelength of 280nm, and collecting desalted protein peak as PEG single modified protein pure product.

The inventors modified and purified three different batches of recombinant human interferon beta 1b using the methods of the above exemplary embodiments, collected three different batches of PEG single-modified protein products 20190527, 20200301, 20200426 and purified followed by the following purity verification.

FIGS. 1 and 2 are SDS-PAGE electrophoresis of an iodine-stained and Coomassie-stained collection of a purified protein product purified by the method according to the above-described exemplary embodiment of the present invention, wherein lanes 4-6 are three separate batches of PEG-modified and pre-purified products shown in Table 1, and lanes 8-10 are three separate batches of PEG-modified and purified products, respectively.

FIG. 3 shows SEC-HPLC detection results of three batches of PEG single modified protein pure products shown in Table 1 obtained by the method of the above-described exemplary embodiment of the present invention, and it can be seen that the purity of all three batches of pure products reaches more than 99%.

In addition, the residual amount of PEG single modified pure polyethylene glycol (PEG) is less than 1w/v% by RP-HPLC evaporative light scattering detector, and the residual sodium cyanoborohydride content is compounded with pharmacopoeia standards, as shown in the following table 1.

TABLE 1 purity and impurity residual quantity of purified pure product of the three batches of PEG mono-modified IFN- β1b product

Batch of	PEG residual content (w/v%)	SEC-HPLC purity	Sodium cyanoborohydride content
				20190527	0.2%	>99%	<0.0475 mmol/L
20200301	0.18%	>99%	<0.0475 mmol/L
				20200426	0.15%	>99%	<0.0475 mmol/L

Embodiments of the present invention are further described below by way of examples, which are to be understood as being for purposes of illustration and explanation only and are not intended to limit the scope of the present invention.

Another batch of PEG-modified product of recombinant human interferon beta 1b was used for purification in the following examples and comparative examples.

Example 1

1. Desalting gel filtration chromatography with G25 column with diameter of 50 mm and height of 80 cm, chromatographic column volume of 1680 ml, balancing with 50 mM citric acid buffer pH 3.0, loading PEG modified protein reaction solution of 85 ml (5.06% cV) at flow rate of 10cm/h, and collecting protein peak;

2. adding 3M sodium chloride solution with the same volume as the protein peak collecting solution for salting out, centrifuging 10000 g for 30min, discarding the supernatant to collect precipitate, and dissolving the precipitate by using 50 mM sodium acetate buffer solution with pH of 5.5;

3. chromatography of the above precipitated complex solution was performed using a SUPEROSE 12 gel filtration column, column diameter 50 mm, column height 89.5 cm, column volume 1760 ml, loading 18 ml (1.02% cV), elution with equilibration solution 50 mM sodium acetate buffer pH5.5, flow rate 10cm/h, detection wavelength 280nm, collection of protein peaks, detection of purity by electrophoresis, and combination;

4. combining, ultrafiltering with 30 kD membrane, concentrating until OD ₂₈₀ Greater than 1.200AU;

5. loading the concentrated solution into a G25 column with the column volume of 480 ml, the pH of the buffer solution of 10 mM phosphate buffer solution of 7.2, the flow rate of 10cm/h and the detection wavelength of 280nm, collecting desalted protein peak as PEG single modified pure protein, and obtaining the final product with the electrophoresis purity of 98.2%, the HPLC purity of 99.1%, the PEG residue of 0.14% and the cyano sodium borohydride residue of <0.0475 mmol/L.

Example 2

1. Desalting gel filtration chromatography with G25 column with diameter of 50 mm and height of 80 cm, chromatographic column volume of 1680 ml, balancing with 50 mM citric acid buffer pH 3.0, loading 168 ml (10% cV) PEG modified protein reaction solution, flow rate of 20cm/h, and collecting protein peak;

2. salting out was performed by adding 4M sodium chloride solution in the same volume as the protein peak collection solution, centrifuging 16000 g for 30min, discarding the supernatant, collecting the precipitate, and dissolving the precipitate with 50 mM sodium acetate buffer pH 5.5.

3. Chromatography of the above precipitated complex solution was performed using a SUPEROSE 12 gel filtration column, column diameter 50 mm, column height 89.5 cm, column volume 1760 ml, loading 53 ml (3.00% cV), elution with equilibration solution 50 mM sodium acetate buffer pH5.5, flow rate 15 cm/h, detection wavelength 280nm, collection of protein peaks, detection of purity by electrophoresis, and combination;

4. combining, ultrafiltering with 30 kD membrane, concentrating until OD ₂₈₀ Greater than 1.200AU;

5. loading the concentrated solution into a G25 column with the column volume of 480 ml, the pH of the buffer solution of 10 mM phosphate buffer solution of 7.2, the flow rate of 15 cm/h and the detection wavelength of 280nm, collecting desalted protein peak as PEG single modified pure protein, and obtaining the final product with the electrophoresis purity of 97.5%, the HPLC purity of 98.9%, the PEG residue of 0.17% and the cyano sodium borohydride residue of <0.0475 mmol/L.

Example 3

1. Desalting gel filtration chromatography with G25 column with diameter of 50 mm and height of 80 cm, chromatographic column volume of 1680 ml, equilibration with 50 mM citric acid buffer pH 3.0, loading 340 ml (20.24% cV) PEG modified protein reaction solution, flow rate of 30 cm/h, collecting protein peak;

2. salting out was performed by adding saturated sodium chloride solution in the same volume as the protein peak collection solution, centrifuging 21900 and g for 30min, collecting the precipitate by discarding the supernatant, and dissolving the precipitate by using 50 mM sodium acetate buffer pH 5.5.

3. Chromatography of the above precipitated complex solution was performed using a SUPEROSE 12 gel filtration column, column diameter 50 mm, column height 89.5 cm, column volume 1760 ml, loading 88 ml (5.00% cV), elution with equilibration solution 50 mM sodium acetate buffer pH5.5, flow rate 20cm/h, detection wavelength 280nm, collection of protein peaks, detection of purity by electrophoresis, and combination;

4. combining, ultrafiltering with 30 kD membrane, concentrating until OD ₂₈₀ Greater than 1.200AU;

5. loading the concentrated solution into a G25 column with the column volume of 480 ml, the pH of the buffer solution of 10 mM phosphate buffer solution of 7.2, the flow rate of 15 cm/h and the detection wavelength of 280nm, collecting desalted protein peak as PEG single modified pure protein, and obtaining the final product with the electrophoresis purity of 97.8%, the HPLC purity of 99.0%, the PEG residue of 0.2% and the cyano sodium borohydride residue of <0.0475 mmol/L.

Example 4

1. Desalting gel filtration chromatography with G25 column with diameter of 100 mm and height of 80 cm, wherein the volume of the column is 6820 ml, balancing with 50 mM citric acid buffer solution pH 3.0, loading PEG modified protein reaction solution of 685 ml (10.04% cV) at flow rate of 15 cm/h, and collecting protein peak;

2. salting out was performed by adding 3M sodium chloride solution in the same volume as the protein peak collection solution, centrifuging 16000 g for 30min, discarding the supernatant, collecting the precipitate, and dissolving the precipitate with 50 mM sodium acetate buffer pH 5.5.

3. Chromatography of the above precipitated complex solution was performed using a SUPEROSE 12 gel filtration column with a column diameter of 100 mm, a column height of 85 cm, a column volume of 6670 ml, a loading amount of 200 ml (3.00% cV), elution with a equilibration solution of 50 mM sodium acetate buffer pH5.5, a flow rate of 15 cm/h, a detection wavelength of 280nm, collection of protein peaks, detection of purity by electrophoresis, and combination;

4. after combination use 30Ultrafiltration and concentration with kD membrane until OD ₂₈₀ Greater than 1.200AU;

5. the concentrated solution is loaded on a G25 column, the diameter of the column is 100 mm, the height of the column is 85 cm, the volume 6670 ml, the loading amount is 670 ml, the pH of a phosphate buffer solution is 10 mM, the flow rate is 15 cm/h, the detection wavelength is 280nm, the peak of desalted protein is collected as PEG single modified pure protein, the electrophoresis purity of the final product is 97.6%, the HPLC purity is 98.8%, the PEG residue is 0.15%, and the sodium cyanoborohydride residue is <0.0475 mmol/L.

Example 5

1. Desalting gel filtration chromatography with G25 column with diameter of 100 mm and height of 80 cm, wherein the volume of the column is 6820 ml, balancing with 50 mM citric acid buffer solution pH 3.0, loading 1365 ml (20.01% cV) PEG modified protein reaction solution, and collecting protein peak at flow rate of 30 cm/h;

2. salting out was performed by adding saturated sodium chloride solution in the same volume as the protein peak collection solution, centrifuging 21900 and g for 30min, collecting the precipitate by discarding the supernatant, and dissolving the precipitate by using 50 mM sodium acetate buffer pH 5.5.

3. Chromatography of the above precipitated complex solution was performed using a SUPEROSE 12 gel filtration column with a column diameter of 100 mm, a column height of 85 cm, a column volume of 6670 ml, a loading amount of 335 ml (5.02% cV), elution with a equilibration solution of 50 mM sodium acetate buffer pH5.5, a flow rate of 20cm/h, a detection wavelength of 280nm, collection of protein peaks, detection of purity by electrophoresis, and combination;

4. combining, ultrafiltering with 30 kD membrane, concentrating until OD ₂₈₀ Greater than 1.200AU;

5. the concentrated solution is loaded on a G25 column, the diameter of the column is 100 mm, the height of the column is 85 cm, the volume is 6670 ml, the pH of the buffer solution is 10 mM phosphate buffer solution and 7.2, the flow rate is 20cm/h, the detection wavelength is 280nm, the peak of desalted protein is collected as PEG single modified pure protein, the electrophoresis purity of the final product is 96.5%, the HPLC purity is 98.0%, the PEG residue is 0.18%, and the sodium cyanoborohydride residue is <0.0475 mmol/L.

The purity and impurity content of the pure products obtained in examples 1 to 5 are shown in the following table:

TABLE 2 purity and impurity residual levels of purified pure product of PEG mono-modified IFN- β1b products of examples 1-5

	PEG residual (w/v%)	Electrophoretic purity	HPLC purity	Residual amount of sodium cyanoborohydride
					Example 1	0.14%	98.2%	99.1%	<0.0475 mmol/L
Example 2	0.17%	97.5%	98.9%	<0.0475 mmol/L
					Example 3	0.2%	97.8%	99.0%	<0.0475 mmol/L
Example 4	0.15%	97.6%	98.8%	<0.0475 mmol/L
					Example 5	0.18%	96.5%	98.0%	<0.0475 mmol/L

Comparative example 1

1. Desalting gel filtration chromatography with G25 column with diameter of 50 mm and height of 80 cm, chromatographic column volume of 1680 ml, balancing with 50 mM sodium acetate solution pH5.5, loading 90 ml (5.36% cV) PEG modified protein reaction solution, flow rate of 10cm/h, and collecting protein peak;

2. adding 3M sodium chloride solution with the same volume as the protein peak collecting solution for salting out, centrifuging 10000 g for 30min, wherein the supernatant and the sediment both contain a large amount of PEG single modified protein, and effective separation of free PEG and target products cannot be realized. Purification is stopped.

Results: the electrophoresis purity of PEG single-modified interferon-beta in the salting-out supernatant is 43.4%, the electrophoresis purity of PEG single-modified interferon-beta in the precipitation species is 91.1%, the proportion of PEG single-modified interferon-beta in the supernatant and the precipitation is about 60%, 40%, the recovery rate is low, and the purification can not be continued.

Comparative example 2

1. Desalting gel filtration chromatography with G25 column with diameter of 50 mm and height of 80 cm, chromatographic column volume of 1680 ml, balancing with 50 mM citric acid buffer pH 3.0, loading 90 ml (5.36% cV) PEG modified protein reaction solution at flow rate of 10cm/h, and collecting protein peak;

2. adding saturated ammonium sulfate solution with the same volume as the protein peak collecting solution for salting out, centrifuging 10000 g for 30min, respectively taking supernatant and sediment samples for detection, and finding that PEG single modified protein and free PEG are simultaneously precipitated.

Results: in the salting-out system, components such as PEG, PEG single-modified protein, PEG multi-site modified protein, unmodified protein and the like are precipitated, the PEG residue in the precipitate is more than 5%, and the single-modified protein and the free PEG cannot be separated.

Comparative example 3

1. Desalting gel filtration chromatography with G25 column with diameter of 50 mm and height of 80 cm, chromatographic column volume of 1680 ml, balancing with 50 mM citric acid buffer pH 3.0, loading PEG modified protein reaction solution of 85 ml (5.06% cV) at flow rate of 10cm/h, and collecting protein peak;

2. adding saturated sodium sulfate solution with the same volume as the protein peak collecting solution for salting out, centrifuging 10000 g for 30min, respectively taking supernatant and sediment samples for detection, and finding that PEG single modified protein and free PEG are simultaneously precipitated.

Results: in the salting-out system, components such as PEG, PEG single-modified protein, PEG multi-site modified protein, unmodified protein and the like are precipitated, the PEG residue in the precipitate is more than 5%, and the single-modified protein and the free PEG cannot be separated.

Comparative example 4

1. Desalting gel filtration chromatography with G25 column with diameter of 50 mm and height of 80 cm, chromatographic column volume of 1680 ml, balancing with 50 mM citric acid buffer pH 3.0, loading 90 ml (5.36% cV) PEG modified protein reaction solution at flow rate of 10cm/h, and collecting protein peak;

2. salting out was performed by adding saturated sodium chloride solution of the same volume as the protein peak collection solution, centrifuging at 10000, g for 30min, discarding the supernatant to collect the precipitate, and dissolving the precipitate with 50, mM sodium acetate buffer pH 5.5.

3. The above precipitated complex solution was chromatographed using a Sephacryl 200 HR gel filtration column, column diameter 16 mm, column height 85 cm, column volume 170 ml, loading 2 ml (1.18% cV), eluting with equilibration solution 50 mM acetate buffer pH5.5, flow rate 10cm/h, detection wavelength 280nm, protein peak collected, electrophoresis detection purity.

Results: the PEG single-modified interferon-beta protein is difficult to separate by Sephacryl 200 HR gel filtration chromatography, and in chromatographic protein peaks, the PEG residual peak, the PEG single-modified protein peak and the PEG double-site modified protein peak are overlapped, so that the chromatographic resolution is low, and the purity of the single-modified product is less than 95%.

The experimental results show that the specific combination of the steps of the method, particularly the pH value of the desalting gel filtration chromatography in the step 1 and the salting-out of sodium chloride in the step 2, can realize the effective separation of PEG single-modified interferon-beta protein from free PEG, unmodified protein and multi-point modified protein, thereby obtaining a single-modified protein purified product with high purity, low free PEG residual quantity and low cyano sodium borohydride residual quantity.

While the present disclosure has been described in detail with respect to the specific embodiments, those skilled in the art will recognize that the foregoing embodiments are illustrative and are not to be construed as limiting the invention, and that the invention is susceptible to several modifications and variations without departing from the spirit of the invention, and that the invention is to be construed as limited by the scope of the appended claims.

Claims (14)

1. A method for purifying a polyethylene glycol PEG mono-modified IFN- β protein, said method comprising the steps of:

s1) desalting gel filtration chromatography of a mixture containing PEG mono-modified IFN- β proteins using an elution buffer having a pH of 2.5 to 3.5, and collecting protein peaks to obtain a first collection solution;

s2) adding sodium chloride solution into the first collection liquid obtained in the step S1 for salting out, centrifugally separating out precipitate, and dissolving the precipitate by using buffer solution to obtain a compound solution; the sodium chloride concentration in the sodium chloride solution is 2.5. 2.5M to saturation;

s3) performing gel filtration chromatography on the complex solution obtained in the step S2 by using an agarose gel column, and collecting protein peaks to obtain a second collection liquid containing PEG single-modified IFN-beta protein;

s4) filtering and concentrating the second collecting liquid by adopting a filter membrane with a molecular weight cut-off value of less than 30 kDa to obtain concentrated liquid; and

s5) carrying out desalting gel filtration chromatography on the concentrated solution, and collecting protein peaks to obtain purified PEG single modified IFN-beta protein.

2. The method of claim 1, wherein the mixture comprising PEG mono-modified IFN- β protein is a reaction product mixture obtained after modification of IFN- β protein with a PEG modifier in the presence of sodium cyanoborohydride.

3. The method of claim 2, wherein the PEG modifier is selected from methoxy polyethylene glycol propionaldehyde mPEG-ALD, mPEG-SC, mPEG-SCM, mPEG-SPA, mPEG-OTS, and mPEG butyraldehyde.

4. The method of claim 1, wherein the elution buffer in step S1 is a citrate buffer.

5. The method of claim 1, wherein in step S2 the sodium chloride concentration in the sodium chloride solution is 3M to 4M and/or the volume of the sodium chloride solution is 0.5 to 1.5 times the volume of the first collection liquid.

6. The method of claim 1, wherein in step S2 the sodium chloride concentration in the sodium chloride solution is 3M to 3.5M and/or the volume of the sodium chloride solution is 0.8 to 1.2 times the volume of the first collection liquid.

7. The method of claim 1, wherein the agarose gel column used in step S3 is the SUPEROSE series.

8. The method according to claim 1, wherein the OD of the buffer used in the second collection solution is set to ₂₈₀ For baseline, the concentration in step S4 is carried out to the OD of the concentrate ₂₈₀ Greater than 1.100AU.

9. The method according to claim 1, wherein the OD of the buffer used in the second collection solution is set to ₂₈₀ For baseline, the concentration in step S4 is carried out to the OD of the concentrate ₂₈₀ Greater than 1.200AU.

10. The method according to claim 1, wherein the OD of the buffer used in the second collection solution is set to ₂₈₀ For baseline, the concentration in step S4 is carried out to the OD of the concentrate ₂₈₀ Greater than 1.300AU.

11. The method of any one of claims 1 to 10, wherein the IFN- β protein is a human natural IFN- β protein or a human IFN- β1b protein.

12. A PEG mono-modified IFN- β protein product made by the method of any one of claim 1 to 11, wherein,

the PEG mono-modified IFN- β protein has a molecular weight of 30 to 50 kDa;

the residual PEG content in the protein product is below 0.2 w/v%;

the content of sodium cyanoborohydride in the protein product is below 0.050 mmol/L; and is also provided with

The HPLC purity of the PEG single modified IFN-beta protein in the protein product is more than 95 percent.

13. The PEG single modified IFN- β protein product of claim 12 wherein,

the PEG mono-modified IFN- β protein has a molecular weight of 38 to 40 kDa;

the residual PEG content in the protein product is below 0.18 w/v%;

the content of sodium cyanoborohydride in the protein product is below 0.0475 mmol/L; and is also provided with

The HPLC purity of the PEG single modified IFN-beta protein in the protein product is more than 98 percent.

14. The PEG single modified IFN- β protein product of claim 12 wherein,

the molecular weight of the PEG single modified IFN-beta protein is 39 kDa;

the residual PEG content in the protein product is below 0.15 w/v%;

the content of sodium cyanoborohydride in the protein product is below 0.0475 mmol/L; and is also provided with

The HPLC purity of the PEG single modified IFN-beta protein in the protein product is more than 99 percent.

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