CN111111264A - Regeneration method of chromatographic stationary phase in step of preparing polypeptide - Google Patents

Regeneration method of chromatographic stationary phase in step of preparing polypeptide Download PDF

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CN111111264A
CN111111264A CN201911412151.7A CN201911412151A CN111111264A CN 111111264 A CN111111264 A CN 111111264A CN 201911412151 A CN201911412151 A CN 201911412151A CN 111111264 A CN111111264 A CN 111111264A
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glp
arg
lys
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徐文长
马国昌
徐飞虎
李博
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Hangzhou Jiuyuan Gene Engineering Co ltd
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    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
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    • B01D15/42—Selective adsorption, e.g. chromatography characterised by the development mode, e.g. by displacement or by elution
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    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/281—Sorbents specially adapted for preparative, analytical or investigative chromatography
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    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/30—Processes for preparing, regenerating, or reactivating
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    • B01J20/345—Regenerating or reactivating using a particular desorbing compound or mixture
    • B01J20/3475—Regenerating or reactivating using a particular desorbing compound or mixture in the liquid phase
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Abstract

The invention provides a chromatographic stationary phase regeneration method suitable for industrial application, which recovers the performance of a chromatographic stationary phase by contacting the chromatographic stationary phase with a regeneration solution containing at least one weak base, an organic solvent and less than 70% (v/v) of water, realizes the regeneration of the chromatographic stationary phase in a chromatographic purification cycle, can perform the next purification cycle after the chromatographic column is regenerated, can continuously perform a plurality of purification cycles without damaging the chromatographic stationary phase, and has simple, safe, economic and effective operation.

Description

Regeneration method of chromatographic stationary phase in step of preparing polypeptide
Technical Field
The present invention relates to the field of chromatographic purification. More particularly, the invention relates to methods of regenerating chromatographic stationary phases.
Background
Polypeptide drugs have been used in the treatment of various diseases such as diabetes, tumors, etc., and approved polypeptide drugs include insulin and its mutants, liraglutide, somaglutide, exenatide, goserelin, leuprorelin, etc., which have a global market sales of over $ 100 billion and play an important role in improving the quality of life and health of patients.
Since natural polypeptide drugs generally have short half-lives, it is generally necessary to improve their pharmacological properties by chemical modification, and the like, for example, typical variants are amide, saccharide, alkyl, acyl, ester, pegylation, and the like, and the related technical reports can refer to WO96/29342, WO98/08871, WO99/43708, WO00/34331, WO00/69911, CN97198413.1, CN200680006674.6, and the like.
In the preparation of polypeptide drugs, since the molecular weight of the drugs is smaller than that of common proteins, multi-step purification methods are often used in the whole purification process. Among them, reverse phase high pressure chromatography (RP-HPLC) is a preferred method for industrially isolating polypeptides, and has been proven to be versatile for large-scale purification of various polypeptides.
At the same time, only high purity polypeptide samples are sufficient for treatment to avoid adverse events upon administration to a patient. The aim of preparing high-purity polypeptide is achieved by applying reversed-phase chromatographic purification in the production process, the separation efficiency of the chromatographic column is gradually reduced along with the increase of the cycle number, and the separation efficiency is gradually reduced due to the gradual accumulation of some sediments in the chromatographic column, so that the purification effect of the chromatographic column is gradually reduced after multiple cycles, and the purity and the yield of the product are gradually reduced. However, since the chromatographic stationary phase is very expensive, frequent replacement of the chromatographic stationary phase will undoubtedly increase the production cost considerably, and thus the chromatographic stationary phase needs to be regenerated to improve the recycling rate. The economy of large-scale production of high-purity polypeptides plays a critical role in the commercialization of polypeptide products and the promotion of existing therapies.
WO 00/61493 discloses a process for regenerating granular materials (clays, sands, silica, etc.) of various origin. It is a 5-step process comprising: the material is contacted with a) an organic material extraction, b) an oxidizing agent, followed by c) an acid solution, d) the material is heated and e) the material is recovered. Therefore, the method for regenerating the chromatographic stationary phase by using the acidic solution is very complex, and the regenerated solution needs to be contacted with the stationary phase outside a chromatographic column, so that the method is not suitable for large-scale industrial application due to complex operation.
CN200480009293.4 discloses regenerating a chromatographic stationary phase with relatively pure formic acid (formic acid solution with water content less than 1%) as a regeneration solution. However, pure formic acid is a strong volatile acid and has extremely strong corrosiveness, so that the pure formic acid has corrosion risk and safety risk of production equipment.
Of course, regeneration of chromatographic stationary phases with caustic solutions, such as 0.1 molar sodium hydroxide (Videliediahl, "twelve years devoted to silica-based HPLC purification of peptides", Tides 2000, 5/10/2000, Las Vegas, USA) has also been investigated. The regeneration method can prolong the service life of the silicon dioxide for purifying the insulin to 100-600 cycles. However, silica materials are unstable when exposed to caustic conditions, and in particular, substituted silica materials may not be suitable for regeneration by caustic solutions.
Because of the defects of the existing chromatographic stationary phase regeneration method and the expensive price of the chromatographic stationary phase, in order to reduce the production cost and the operation difficulty of large-scale industrial production of high-purity polypeptide, it is necessary to develop a chromatographic stationary phase regeneration method which is simple and safe in operation, does not damage the chromatographic column stationary phase and is suitable for industrial application.
Disclosure of Invention
An object of the present invention is to provide a method for regenerating a chromatographic stationary phase suitable for industrial applications, which recovers the properties of the chromatographic stationary phase by contacting the chromatographic stationary phase with a regenerating solution, and which is simple, safe, economical and effective to operate.
The present invention provides a process for regenerating a chromatographic stationary phase wherein said chromatographic stationary phase is contacted with a regeneration solution comprising at least one weak base, an organic solvent and less than 70% (v/v) water.
The weak base is an important condition in the regeneration method, and the regeneration purpose can not be achieved under the condition of no weak base. In one embodiment of the present invention, the weak base includes ammonium compounds such as ammonia, carbonate, DIEA, etc., preferably ammonia.
In one embodiment of the invention, the weak base is present in a concentration of 0.1-2% (v/v), preferably 0.2-1.5%, more preferably 0.5-1.5%, more preferably 0.8-1.2%, more preferably 1%.
The organic solvent with a certain proportion is an important parameter in the regeneration method, the residue remained in the chromatographic column after elution is mostly a substance with stronger hydrophobicity, and the organic solvent with a certain proportion in the regeneration solution can enhance the solubility of the residue and reduce the residue remained in the chromatographic column.
In one embodiment of the present invention, the organic solvent includes acetonitrile, ethanol, methanol, isopropanol, etc., preferably acetonitrile. In another embodiment, the organic solvent concentration is from 30% to 70% (v/v), preferably from 35% to 65%, more preferably from 40% to 60%, more preferably from 45% to 55%, most preferably 50%.
In another embodiment, the water content in the regeneration liquid is less than 70% (v/v), more preferably less than 65%, 60%, 55% or 50%.
In another preferred embodiment, the weak base is ammonia and the organic solvent is acetonitrile. In another embodiment, the weak base is 0.1-2% (v/v) aqueous ammonia and the organic solvent is 30% -70% (v/v) acetonitrile.
In another embodiment, the regeneration liquid has a pH in the range of 9 to 12, preferably 9.5 to 11.5, more preferably 10 to 11, and most preferably a pH of 11.
In another embodiment, the present invention provides a process for regenerating a chromatographic stationary phase wherein said chromatographic stationary phase is contacted with a regeneration solution, wherein said regeneration solution comprises 0.1-2% (v/v) aqueous ammonia, 30-70% (v/v) acetonitrile and less than 70% (v/v) water, and the pH of the solution is in the range of 9-12.
In another preferred embodiment, the present invention provides a process for regenerating a chromatographic stationary phase wherein said chromatographic stationary phase is contacted with a regeneration solution comprising 0.2-1.5% (v/v) aqueous ammonia, 35% -65% (v/v) acetonitrile and less than 65% (v/v) water, the pH of the solution being in the range of 9.5-11.5.
In another preferred embodiment, the present invention provides a process for regenerating a chromatographic stationary phase wherein said chromatographic stationary phase is contacted with a regeneration solution comprising 0.5-1.5% (v/v) aqueous ammonia, 40% -60% (v/v) acetonitrile and less than 60% (v/v) water, the pH of the solution being in the range of 10-11.
In another preferred embodiment, the present invention provides a process for regenerating a chromatographic stationary phase wherein said chromatographic stationary phase is contacted with a regeneration solution comprising 0.8-1.2% (v/v) aqueous ammonia, 45% -55% (v/v) acetonitrile and less than 55% (v/v) water, the pH of the solution being in the range of 10-11.
In another preferred embodiment, the present invention provides a process for regenerating a chromatographic stationary phase wherein said chromatographic stationary phase is contacted with a regeneration solution comprising 1% (v/v) aqueous ammonia, 50(v/v) acetonitrile and less than 50% (v/v) water, the pH of the solution being in the range of 10.
In yet another embodiment, the chromatographic stationary phase is contacted with the regeneration solution, preferably within a chromatographic column. In this way, the loss of capacity due to the down time associated with the regeneration step is minimized. Therefore, the operation of regenerating the chromatographic stationary phase can be carried out without re-packing the column.
In another embodiment, the chromatographic stationary phase is fluidized during said regeneration.
In another embodiment, the chromatographic eluent or equilibration buffer is replaced with a water soluble organic solvent prior to contacting the chromatographic stationary phase with the regeneration solution. Preferably, the water-soluble organic solvent is also present in the chromatographic eluent or equilibration buffer. More preferably, the water-soluble organic solvent is also present in the regeneration solution.
In another embodiment of the present invention, the contacting of the chromatographic stationary phase with the regenerating liquid means that after eluting the product through the chromatographic column in a conventional chromatographic protocol, the regenerating liquid is removed for the purpose of recovering the performance of the chromatographic column, and the volume of the regenerating liquid is 0.5 to 2 column volumes.
In another embodiment, the chromatographic stationary phase is an RP-HPLC matrix. For RPThe chromatographic stationary phases of HPLC are mechanically very rigid substances, which may be silica or substituted silicas, such as C4, C6, C8, C10, C12, C16, C18, C30 or phenyl silica. Suitable silica materials for use as chromatographic stationary phases are spherical particles having a narrow pore size distribution and a particle size in the range of from 3 μm to 100 μm, the pore size being used in
Figure BDA0002350243790000061
For pressure stable polymeric materials, the pore size is
Figure BDA0002350243790000062
Or higher.
In another embodiment, the chromatographic stationary phase for RP-HPLC is a pressure stable substituted or unsubstituted polymeric material that is Source30Q or XAD 1180. Production scale chromatography columns typically have a diameter of 15-100cm, and such systems may have dynamic axial compression. For the production of small amounts of polypeptide, the production column may have a diameter of, for example, 15cm, 20cm or 25 cm. For the production of large amounts of polypeptide, the production column may have a diameter of, for example, 40cm, 60cm, 80cm or more.
In one embodiment of the method for regenerating a chromatographic stationary phase, the chromatographic stationary phase is contacted with the regeneration solution for at least 1 second, preferably at least 1 minute, more preferably at least 5 minutes, such as from 1 minute to 24 hours, from 1 minute to 5 hours, from 1 minute to 2 hours, from 1 minute to 60 minutes, from 5 minutes to 30 minutes.
In another embodiment of the method of regenerating a chromatographic stationary phase, the contacting of the chromatographic stationary phase with the regeneration solution is conducted at a temperature in the range of about 5 ℃ to 60 ℃, such as 10 ℃ to 50 ℃, such as 15 ℃ to 45 ℃, or 18 ℃ to 30 ℃.
In another embodiment of the method for regenerating a chromatographic stationary phase, the useful life of said chromatographic stationary phase is at least 500 chromatographic cycles, preferably at least 700 chromatographic cycles, more preferably at least 1000 chromatographic cycles, most preferably at least 2000 chromatographic cycles.
In another embodiment of the method of regenerating a chromatographic stationary phase, said method is applied to said chromatographic stationary phase for each chromatographic cycle, at least once every 2 chromatographic cycles, at least once every 5 chromatographic cycles, at least once every 10 chromatographic cycles, at least once every 20 chromatographic cycles, at least once every 50 chromatographic cycles, or at least once every 100 chromatographic cycles.
In another embodiment of the present invention, the number of regeneration operations performed on the chromatographic stationary phase is at least 25 times, at least 50 times, at least 100 times, at least 200 times, at least 400 times, or at least 1000 times.
It is another object of the present invention to provide a method for producing a therapeutic polypeptide or a precursor thereof, said method comprising at least one chromatographic treatment step in which a chromatographic stationary phase is regenerated by the above regeneration method.
Accordingly, the present invention provides a process for the production of a therapeutic polypeptide or a precursor thereof comprising at least one chromatographic treatment step wherein a chromatographic stationary phase is regenerated by a process for regenerating a chromatographic stationary phase wherein said chromatographic stationary phase is contacted with a regeneration solution comprising at least one weak base, an organic solvent and less than 70% (v/v) water at a temperature in the range of from 5 ℃ to 60 ℃; wherein the therapeutic polypeptide is selected from the group consisting of acylated GLP-1 or an acylated GLP-1 analog.
In one embodiment of the present invention said acylated GLP-1 or acylated GLP-1 analogs can in particular be referred to the various types of acylated GLP-1 analogs listed in CN97198413.1 and CN 200680006674.6. The acylation is defined as the attachment of a lipophilic substituent to the N-terminal or C-terminal amino acid residue of the GLP-1 polypeptide or analog thereof. The lipophilic substituents may be referred to in particular those listed in chinese patents CN97198413.1 and CN 200680006674.6. The lipophilic substituent attached to the GLP-1 moiety preferably contains 4 to 40 carbon atoms, especially 8 to 25 carbon atoms. The lipophilic substituent may be attached to the amino group of the GLP-1 moiety by formation of an amide bond between the carboxy group of the lipophilic substituent and the amino group of the amino acid residue to which it is attached. Or alternatively, the lipophilic substituent may be attached to the amino acid residue by formation of an amide bond between the amino group of the lipophilic substituent and the carboxyl group of the amino acid residue. Alternatively, the lipophilic substituent may be attached to the GLP-1 moiety via an ester bond. Alternatively, the lipophilic substituent is linked to the GLP-1 moiety by forming an amide bond with an amino group of the GLP-1 moiety via a carboxyl group of a spacer group. Examples of suitable spacer groups are dipeptides of succinic acid, Lys, Glu, Asp or Gly-Lys.
More preferably, the GLP-1 polypeptide or analog thereof to be acylated may be selected from GLP-1(7-37), GLP-1(8-37), GLP-1(9-37), GLP-1(10-37), GLP-1(11-37), GLP-1(12-37), GLP-1(13-37), GLP-1(14-37), GLP-1(15-37) or GLP-1 polypeptide analogs, including: arg34-GLP-1(7-37)，Arg26-GLP-1(7-37)，Lys36-GLP-1(7-37)，Arg26,34Lys38-GLP-1(7-38)，Arg26,34Lys39-GLP-1(7-39)，Arg26,34Lys40-GLP-1(7-40)，Arg26Lys36-GLP-1(7-37)，Arg34Lys36-GLP-1(7-37)，Arg26Lys39-GLP-1(7-39)，Arg34Lys40-GLP-1(7-40)，Arg26,34Lys36,39-GLP-1(7-39)，Arg26,34Lys36,40-GLP-1(7-40)，Gly8Arg26-GLP-1(7-37)，Gly8Arg34-GLP-1(7-37)，Gly8Lys36-GLP-1(7-37)，Gly8Arg26, 34Lys36-GLP-1(7-37)，Gly8Arg26,34Lys39-GLP-1(7-39)，Gly8Arg26,34Lys40-GLP-1(7-40)，Gly8Arg26Lys36-GLP-1(7-37)，Gly8Arg34Lys36-GLP-1(7-37)，Gly8Arg26Lys39-GLP-1(7-39)，Gly8Arg34Lys40-GLP-1(7-40)，Gly8Arg26,34Lys36,39-GLP-1(7-39)，Gly8Arg26,34Lys36,40-GLP-1(7-40)，Arg26,34Lys38-GLP-1(7-38)，Arg26,34Lys39-GLP-1(7-39)，Arg26,34Lys40-GLP-1(7-40)，Arg26,34Lys41-GLP-1(7-41)，Arg26,34Lys42-GLP-1(7-42)，Arg26,34Lys43-GLP-1(7-43)，Arg26,34Lys44-GLP-1(7-44)，Arg26,34Lys45-GLP-1(7-45)，Arg26,34Lys38-GLP-1(1-38)，Arg26, 34Lys39-GLP-1(1-39)，Arg26,34Lys40-GLP-1(1-40)，Arg26,34Lys41-GLP-1(1-41)，Arg26, 34Lys42-GLP-1(1-42)，Arg26,34Lys43-GLP-1(1-43)，Arg26,34Lys44-GLP-1(1-44)，Arg26, 34Lys45-GLP-1(1-45)，Arg26,34Lys38-GLP-1(2-38)，Arg26,34Lys39-GLP-1(2-39)，Arg26, 34Lys40-GLP-1(2-40)，Arg26,34Lys41-GLP-1(2-41)，Arg26,34Lys42-GLP-1(2-42)，Arg26, 34Lys43-GLP-1(2-43)，Arg26,34Lys44-GLP-1(2-44)，Arg26,34Lys45-GLP-1(2-45)，Arg26, 34Lys38-GLP-1(3-38)，Arg26,34Lys39-GLP-1(3-39)，Arg26,34Lys40-GLP-1(3-40)，Arg26, 34Lys41-GLP-1(3-41)，Arg26,34Lys42-GLP-1(3-42)，Arg26,34Lys43-GLP-1(3-43)，Arg26, 34Lys44-GLP-1(3-44)，Arg26,34Lys45-GLP-1(3-45)，Arg26,34Lys38-GLP-1(4-38)，Arg26, 34Lys39-GLP-1(4-39)，Arg26,34Lys40-GLP-1(4-40)，Arg26,34Lys41-GLP-1(4-41)，Arg26, 34Lys42-GLP-1(4-42)，Arg26,34Lys43-GLP-1(4-43)，Arg26,34Lys44-GLP-1(4-44)，Arg26, 34Lys45-GLP-1(4-45)，Arg26,34Lys38-GLP-1(5-38)，Arg26,34Lys39-GLP-1(5-39)，Arg26, 34Lys40-GLP-1(5-40)，Arg26,34Lys41-GLP-1(5-41)，Arg26,34Lys42-GLP-1(5-42)，Arg26, 34Lys43-GLP-1(5-43)，Arg26,34Lys44-GLP-1(5-44)，Arg26,34Lys45-GLP-1(5-45)，Arg26, 34Lys38-GLP-1(6-38)，Arg26,34Lys39-GLP-1(6-39)，Arg26,34Lys40-GLP-1(6-40)，Arg26, 34Lys41-GLP-1(6-41)，Arg26,34Lys42-GLP-1(6-42)，Arg26,34Lys43-GLP-1(6-43)，Arg26, 34Lys44-GLP-1(6-44)，Arg26,34Lys45-GLP-1(6-45)，Arg26Lys38-GLP-1(1-38)，Arg34Lys38-GLP-1(1-38)，Arg26,34Lys36,38-GLP-1(1-38)，Arg26Lys38-GLP-1(7-38)，Arg34Lys38-GLP-1(7-38)，Arg26,34Lys36,38-GLP-1(7-38)，Aib8,Arg34-GLP-1(7-37)，Aib8,22,Arg34-GLP-1(7-37)，Arg26,34Lys38-GLP-1(7-38)，Arg26Lys39-GLP-1(1-39)，Arg34Lys39-GLP-1(1-39)，Arg26, 34Lys36,39-GLP-1(1-39)，Arg26Lys39-GLP-1(7-39)，Arg34Lys39GLP-1(7-39) or Arg26,34Lys36 ,39-GLP-1(7-39)。
More preferably, the acylated GLP-1 or acylated GLP-1 analogue is selected from the group consisting of Liraglutide (Liraglutide) or somaglutide (Semaglutide).
CN200580038571.3 has reported that liraglutide drug formulation solutions tend to fibrillate when maintained over long periods of time and require treatment by a heating method that can improve the stability of liraglutide formulations by heating liraglutide solutions having a pH in the range of 8.0-10.0 to 50 ℃ and 80 ℃ for 3 minutes to 120 minutes. However, the inventors of the present application have unexpectedly found through a large number of experiments that an acylated GLP-1 or an acylated GLP-1 analogue such as liraglutide or somaglutide is prone to fibrillar aggregation at low pH conditions during production and purification, and in particular to the formation of high polymers under acidic conditions, which polymers deposit on the chromatographic stationary phase during purification and which, over a long period of time, deteriorate the purification performance of the chromatographic stationary phase. However, for unmodified GLP-1 polypeptides or analogs thereof, the formation of high polymers is not significant, possibly due to the influence of lipophilic substituents attached to the GLP-1 moiety.
For such contaminants deposited on the chromatographic stationary phase, CN200480009293.4 has disclosed regenerating the chromatographic stationary phase using relatively pure formic acid (formic acid solution with a water content of less than 1%) as a regeneration solution. However, pure formic acid belongs to strong volatile acid and has extremely strong corrosivity, and the pure formic acid has corrosion risk and safety risk of production equipment. Another approach is to regenerate the chromatographic stationary phase with an alkaline solution, but silica materials are unstable when exposed to caustic conditions, particularly substituted silica materials that may not be suitable for regeneration by strongly alkaline solutions.
The inventor of the present application finds, unexpectedly, that the high polymer pollutants are mainly formed in an acidic condition, and the high polymer pollutants can be destroyed under a weak base condition, so that the hydrophobicity of the pollutants is reduced, and the regeneration of the high polymer pollutants and a chromatographic stationary phase under a weak base condition is facilitated. This finding is clearly unexpected, since the inventors have experimentally demonstrated that the regeneration process under weak base conditions does not destroy the chromatographic stationary phase and can restore the chromatographic stationary phase to the pre-contaminated performance state, which is clearly a deviation from the prior art recognized knowledge that basic conditions would destroy the structure of the chromatographic stationary phase material.
In another embodiment of the invention, the invention provides a method for producing a therapeutic polypeptide or a precursor thereof, wherein the specific process parameters for regenerating the chromatographic stationary phase are as described above.
As described above, the present invention provides the following technical solutions:
1. a process for regenerating a chromatographic stationary phase wherein said chromatographic stationary phase is contacted with a regeneration solution comprising at least one weak base, an organic solvent and less than 70% (v/v) water.
2. The method according to claim 1, characterized in that: the weak base comprises ammonium compounds such as ammonia water, carbonate, DIEA and the like.
3. The method according to claim 2, characterized in that: the weak base is ammonia water.
4. The method according to claim 2, characterized in that: the weak base concentration is 0.1-2% (v/v), 0.2-1.5% (v/v), 0.5-1.5% (v/v), 0.8-1.2% (v/v) or 1% (v/v).
5. The method according to claim 1, characterized in that: the organic solvent comprises acetonitrile, ethanol, methanol or isopropanol.
6. The method according to claim 5, characterized in that: the organic solvent is acetonitrile.
7. The method according to claim 5, characterized in that: the concentration of the organic solvent is 30-70% (v/v), 35-65% (v/v), 40-60% (v/v), 45-55% (v/v), or 50% (v/v).
8. The method according to claim 1, characterized in that: the water content of the regeneration liquid is less than 65% (v/v), 60% (v/v), 55% (v/v) or 50% (v/v).
9. The method according to claim 1, characterized in that: the weak base in the regeneration liquid is ammonia water, and the organic solvent is acetonitrile.
10. The method according to claim 9, characterized in that: the weak base in the regeneration liquid is 0.1-2% (v/v) ammonia water, and the organic solvent is 30-70% (v/v) acetonitrile.
11. The method according to claim 1, characterized in that: the pH range of the regeneration liquid is 9-12, 9.5-11.5, 10-11 or pH is 11.
12. A process for regenerating a chromatographic stationary phase wherein said chromatographic stationary phase is contacted with a regeneration solution wherein said regeneration solution comprises 0.1-2% (v/v) aqueous ammonia, 30-70% (v/v) acetonitrile and less than 70% (v/v) water, and the pH of the solution is in the range of 9-12.
13. The method according to claim 12, wherein the regeneration solution comprises 0.2-1.5% (v/v) ammonia, 35-65% (v/v) acetonitrile and less than 65% (v/v) water, and the pH of the solution is in the range of 9.5-11.5.
14. The method of claim 13, wherein the regeneration solution comprises 0.5-1.5% (v/v) ammonia, 40-60% (v/v) acetonitrile and less than 60% (v/v) water, and the pH of the solution is in the range of 10-11.
15. The method of claim 14, wherein the regeneration solution comprises 0.8-1.2% (v/v) ammonia, 45% -55% (v/v) acetonitrile and less than 55% (v/v) water, and the pH of the solution is in the range of 10-11.
16. The method of claim 15, wherein the regeneration solution comprises 1% (v/v) ammonia, 50(v/v) acetonitrile and less than 50% (v/v) water, and the pH of the solution is in the range of 10.
17. The method according to any one of claims 1 to 16, wherein the contacting of the chromatographic stationary phase with the regenerating liquid means that after elution of the product through the chromatographic column in a conventional chromatographic protocol, the regenerating liquid is removed for the purpose of recovering the performance of the chromatographic column, and the volume of the regenerating liquid is 0.5 to 2 column volumes.
18. The process according to any of claims 1 to 16, wherein the chromatographic stationary phase is an RP-HPLC matrix.
19. The process according to claim 18, characterized in that the chromatographic stationary phase for RP-HPLC is silica or substituted silica, such as C4, C6, C8, C10, C12, C16, C18, C30 or phenyl silica.
20. The process according to claim 19, characterized in that the chromatographic stationary phases Source30Q or XAD1180 are used for RP-HPLC.
21. The method according to any of claims 1-16, characterized in that the chromatographic stationary phase is contacted with the regeneration solution for at least 1 second, at least 1 minute, at least 5 minutes, 1 minute-24 hours, 1 minute-5 hours, 1 minute-2 hours, 1 minute-60 minutes or 5 minutes-30 minutes.
22. The process according to any of claims 1 to 16, characterized in that the contacting of the chromatographic stationary phase with the regeneration solution is carried out at a temperature in the range of about 5 ℃ to 60 ℃, 10 ℃ to 50 ℃, 15 ℃ to 45 ℃ or 18 ℃ to 30 ℃.
23. The process according to any of claims 1-16, wherein the lifetime of the chromatographic stationary phase is at least 500 chromatographic cycles, at least 700 chromatographic cycles, at least 1000 chromatographic cycles or at least 2000 chromatographic cycles.
24. The method according to any of claims 1-16, wherein the method is applied to the chromatographic stationary phase for each chromatographic cycle, at least once every 2 chromatographic cycles, at least once every 5 chromatographic cycles, at least once every 10 chromatographic cycles, at least once every 20 chromatographic cycles, at least once every 50 chromatographic cycles, or at least once every 100 chromatographic cycles.
25. The process according to any of claims 1 to 16, wherein the number of regeneration operations performed on the chromatographic stationary phase is at least 25, at least 50, at least 100, at least 200, at least 400 or at least 1000.
26. A process for the production of a therapeutic polypeptide or a precursor thereof comprising at least one chromatographic treatment step wherein a chromatographic stationary phase is regenerated by a process for regenerating a chromatographic stationary phase wherein said chromatographic stationary phase is contacted with a regeneration solution comprising at least one weak base, an organic solvent and less than 70% (v/v) water at a temperature in the range of 5 ℃ to 60 ℃; wherein the therapeutic polypeptide is selected from the group consisting of acylated GLP-1 or an acylated GLP-1 analog.
27. The method of claim 26, wherein the acylation is defined as the attachment of a lipophilic substituent to the N-terminal or C-terminal amino acid residue of the GLP-1 polypeptide or analog thereof.
28. The method of claim 27, wherein the lipophilic substituent comprises 4 to 40 carbon atoms.
29. The method of claim 28, wherein the lipophilic substituent comprises from 8 to 25 carbon atoms.
30. The method of claim 27, wherein the lipophilic substituent is attached to the amino group of the GLP-1 moiety by forming an amide bond between the carboxy group of the lipophilic substituent and the amino group of the amino acid residue to which it is attached; or linked to the amino acid residue by forming an amide bond between the amino group thereof and the carboxyl group of the amino acid residue; or to a GLP-1 moiety via an ester bond; or linked to the GLP-1 moiety by an amide bond formed by a carboxy group of a spacer group and an amino group of the GLP-1 moiety, suitable spacers are dipeptides of succinic acid, Lys, Glu, Asp or Gly-Lys.
31. The method according to claim 26, wherein the GLP-1 polypeptide or analog thereof used for acylation is selected from the group consisting of GLP-1(7-37), GLP-1(8-37), GLP-1(9-37), GLP-1(10-37), GLP-1(11-37), GLP-1(12-37), GLP-1(13-37), GLP-1(14-37), GLP-1(15-37) and analogs of GLP-1 polypeptide, comprising: arg34-GLP-1(7-37)，Arg26-GLP-1(7-37)，Lys36-GLP-1(7-37)，Arg26,34Lys38-GLP-1(7-38)，Arg26, 34Lys39-GLP-1(7-39)，Arg26,34Lys40-GLP-1(7-40)，Arg26Lys36-GLP-1(7-37)，Arg34Lys36-GLP-1(7-37)，Arg26Lys39-GLP-1(7-39)，Arg34Lys40-GLP-1(7-40)，Arg26,34Lys36,39-GLP-1(7-39)，Arg26,34Lys36,40-GLP-1(7-40)，Gly8Arg26-GLP-1(7-37)，Gly8Arg34-GLP-1(7-37)，Gly8Lys36-GLP-1(7-37)，Gly8Arg26,34Lys36-GLP-1(7-37)，Gly8Arg26,34Lys39-GLP-1(7-39)，Gly8Arg26,34Lys40-GLP-1(7-40)，Gly8Arg26Lys36-GLP-1(7-37)，Gly8Arg34Lys36-GLP-1(7-37)，Gly8Arg26Lys39-GLP-1(7-39)，Gly8Arg34Lys40-GLP-1(7-40)，Gly8Arg26,34Lys36,39-GLP-1(7-39)，Gly8Arg26,34Lys36,40-GLP-1(7-40)，Arg26,34Lys38-GLP-1(7-38)，Arg26,34Lys39-GLP-1(7-39)，Arg26,34Lys40-GLP-1(7-40)，Arg26,34Lys41-GLP-1(7-41)，Arg26,34Lys42-GLP-1(7-42)，Arg26,34Lys43-GLP-1(7-43)，Arg26,34Lys44-GLP-1(7-44)，Arg26,34Lys45-GLP-1(7-45)，Arg26,34Lys38-GLP-1(1-38)，Arg26,34Lys39-GLP-1(1-39)，Arg26,34Lys40-GLP-1(1-40)，Arg26, 34Lys41-GLP-1(1-41)，Arg26,34Lys42-GLP-1(1-42)，Arg26,34Lys43-GLP-1(1-43)，Arg26, 34Lys44-GLP-1(1-44)，Arg26,34Lys45-GLP-1(1-45)，Arg26,34Lys38-GLP-1(2-38)，Arg26, 34Lys39-GLP-1(2-39)，Arg26,34Lys40-GLP-1(2-40)，Arg26,34Lys41-GLP-1(2-41)，Arg26, 34Lys42-GLP-1(2-42)，Arg26,34Lys43-GLP-1(2-43)，Arg26,34Lys44-GLP-1(2-44)，Arg26, 34Lys45-GLP-1(2-45)，Arg26,34Lys38-GLP-1(3-38)，Arg26,34Lys39-GLP-1(3-39)，Arg26, 34Lys40-GLP-1(3-40)，Arg26,34Lys41-GLP-1(3-41)，Arg26,34Lys42-GLP-1(3-42)，Arg26, 34Lys43-GLP-1(3-43)，Arg26,34Lys44-GLP-1(3-44)，Arg26,34Lys45-GLP-1(3-45)，Arg26, 34Lys38-GLP-1(4-38)，Arg26,34Lys39-GLP-1(4-39)，Arg26,34Lys40-GLP-1(4-40)，Arg26, 34Lys41-GLP-1(4-41)，Arg26,34Lys42-GLP-1(4-42)，Arg26,34Lys43-GLP-1(4-43)，Arg26, 34Lys44-GLP-1(4-44)，Arg26,34Lys45-GLP-1(4-45)，Arg26,34Lys38-GLP-1(5-38)，Arg26, 34Lys39-GLP-1(5-39)，Arg26,34Lys40-GLP-1(5-40)，Arg26,34Lys41-GLP-1(5-41)，Arg26, 34Lys42-GLP-1(5-42)，Arg26,34Lys43-GLP-1(5-43)，Arg26,34Lys44-GLP-1(5-44)，Arg26, 34Lys45-GLP-1(5-45)，Arg26,34Lys38-GLP-1(6-38)，Arg26,34Lys39-GLP-1(6-39)，Arg26, 34Lys40-GLP-1(6-40)，Arg26,34Lys41-GLP-1(6-41)，Arg26,34Lys42-GLP-1(6-42)，Arg26, 34Lys43-GLP-1(6-43)，Arg26,34Lys44-GLP-1(6-44)，Arg26,34Lys45-GLP-1(6-45)，Arg26Lys38-GLP-1(1-38)，Arg34Lys38-GLP-1(1-38)，Arg26,34Lys36,38-GLP-1(1-38)，Arg26Lys38-GLP-1(7-38)，Arg34Lys38-GLP-1(7-38)，Arg26,34Lys36,38-GLP-1(7-38)，Aib8,Arg34-GLP-1(7-37)，Aib8,22,Arg34-GLP-1(7-37)，Arg26,34Lys38-GLP-1(7-38)，Arg26Lys39-GLP-1(1-39)，Arg34Lys39-GLP-1(1-39)，Arg26,34Lys36,39-GLP-1(1-39)，Arg26Lys39-GLP-1(7-39)，Arg34Lys39GLP-1(7-39) or Arg26,34Lys36,39-GLP-1(7-39)。
32. The method of claim 26, wherein the acylated GLP-1 or acylated GLP-1 analog is selected from the group consisting of Liraglutide (Liraglutide) and somaglutide (Semagutide).
33. The process of claim 26, wherein the chromatographic stationary phase is contacted with a regeneration solution comprising at least one weak base, an organic solvent and less than 70% (v/v) water under regeneration conditions according to any of claims 1-25.
The method for regenerating the chromatographic stationary phase realizes the regeneration of the chromatographic stationary phase in the chromatographic purification cycle, so that the next purification cycle can be carried out after the chromatographic column is regenerated, and a plurality of purification cycles can be continuously carried out. Meanwhile, the regenerated chromatographic column can also be restored to the performance state before pollution, so that the purity and yield of the produced product are stable. The regeneration method is simple and safe to operate, has a good regeneration effect, does not damage a chromatographic stationary phase, and is suitable for industrial large-scale production of high-purity polypeptide.
Drawings
FIG. 1 shows the new and contaminated chromatographic columns
FIG. 2 shows the washing patterns of two regeneration liquids
FIG. 3 shows the test pattern of the regenerated and new chromatographic column
Detailed Description
The present invention will be described in further detail with reference to examples and drawings.
Materials:
liraglutide (Arg)34Lys26Crude products of (N-epsilon- (gamma-Glu (N- α -hexadecanoyl))) -GLP-1) are provided by Hangzhou nine-source genetic engineering company;
acetonitrile purchased from MERCK, lot No. I635730216; ammonia purchased from j.t.baker, lot No. J29028; the specification of the reverse phase silica gel column is 100-10-C84.6 multiplied by 250mm provided by kromasil corporation;
the instrument comprises the following steps: agilent HPLC 1100; UV-2550 UV-vis spectrophotometer;
column type: kromasil C8
Figure BDA0002350243790000201
4.6×250mm；
Example 1 chromatography purification separation test of Liraglutide injected into New and contaminated chromatography columns
The contaminated chromatographic column refers to a new chromatographic column which is subjected to multi-cycle purification and is not regenerated by the method.
The implementation process is as follows: equilibration buffer (0.1% TFA and 20% acetonitrile in water by volume) reverse phase equilibration chromatography column 1 column volume solution; injecting a crude liraglutide into an autosampler; equilibration buffer (0.1% TFA and 20% acetonitrile in water by volume) equilibrated to one column volume of solution; the eluate was eluted with an aqueous solution containing 0.1% TFA and acetonitrile. Where acetonitrile was eluted with a 20 column volume gradient from 35% up to 50% ratio.
As can be seen from fig. 1, the new and old reversed phase chromatography columns have a great difference in chromatographic peak shape. The new reversed phase chromatographic column has narrow and high peak shape and the old chromatographic column has short peak shape.
Example 2 reverse phase chromatography packing test of different component regeneration liquid cleaning contamination
The implementation process is as follows: an aqueous ammonia regenerant A, B, C, D (see Table 1) was prepared in different proportions, and four equal portions of the contaminated reverse phase chromatography packing material of example 1 were added to the regenerant A, B, C, D. Shaking on a shaker for 30min, collecting supernatant, and detecting absorption value with ultraviolet spectrophotometer. The higher the absorption value, the more the contaminants in the reversed phase chromatography packing migrate into the regenerant, and the better the cleaning effect of the regenerant. The results are shown in Table 1.
TABLE 1 cleaning effect of ammonia water in different proportions
Regeneration liquid Absorption number
A: 0.1% ammonia water, 50% acetonitrile and 49.9% water 2.230
B: 0.5% ammonia water, 50% acetonitrile and 49.5% water 2.839
C: 1% ammonia water, 50% acetonitrile and 49% water 3.053
D: 2% ammonia water, 50% acetonitrile and 48% water 3.148
The control solution selected by each group of sample detection is the regeneration solution of the group so as to ensure the reliability of detection data.
The result shows that the ammonia water is increased from 0.1% to 1%, the change of the absorption value is quick, and the cleaning effect is obviously improved; increasing to 2% again, the absorption value changes slowly and the effect increase is not significant.
EXAMPLE 3 determination of regeneration fluid composition
Washing the polluted reversed phase chromatographic column in the embodiment 1 by using the regeneration solution C in the embodiment 2, and leveling a base line when a peak is completely generated; then flushing with a D regeneration solution until the base line is leveled; and then carrying out a chromatographic purification separation test on the injection liraglutide. The washing result of the cleaning solution is shown in figure 2.
A peak is washed out by the regenerated solution C, which shows that a large amount of pollutants are washed out, and a base line is stable when the regenerated solution D is washed out, which shows that almost no pollutants are washed out, and shows that 1% of ammonia water can fully regenerate the polluted reversed phase chromatographic column.
Example 4 sample liraglutide after regeneration of contaminated reverse phase chromatography column for chromatographic purification separation test
The implementation process is as follows: after the contaminated reverse phase chromatography column of example 1 was directly regenerated with the regeneration solution of example 3, the crude liraglutide was injected into the autosampler, the equilibrium buffer equilibrated one column volume of solution, and the sample was gradient eluted with the eluent. The test result is shown in figure 3, and the peak becomes narrow and high after the polluted chromatographic column is regenerated, and the performance state is recovered to the performance state before the polluted chromatographic column is polluted.
Meanwhile, the regeneration of the chromatographic stationary phase is completed in the chromatographic purification cycle, so that the next purification cycle can be performed after the chromatographic column is regenerated, and a plurality of purification cycles can be continuously performed.

Claims (10)

1. A process for regenerating a chromatographic stationary phase wherein said chromatographic stationary phase is contacted with a regeneration solution comprising at least one weak base, an organic solvent and less than 70% (v/v) water.
2. The method of claim 1, wherein: the weak base comprises ammonium compounds such as ammonia water, carbonate, DIEA and the like.
3. The method of claim 2, wherein: the weak base concentration is 0.1-2% (v/v), 0.2-1.5% (v/v), 0.5-1.5% (v/v), 0.8-1.2% (v/v) or 1% (v/v).
4. The method of claim 1, wherein: the organic solvent comprises acetonitrile, ethanol, methanol or isopropanol.
5. The method of claim 4, wherein: the concentration of the organic solvent is 30-70% (v/v), 35-65% (v/v), 40-60% (v/v), 45-55% (v/v), or 50% (v/v).
6. The method of claim 1, wherein: the pH range of the regeneration liquid is 9-12, 9.5-11.5, 10-11 or pH is 11.
7. The method of claim 1, wherein contacting the chromatographic stationary phase with the regeneration solution is performed at a temperature in a range of about 5 ℃ to 60 ℃, 10 ℃ to 50 ℃, 15 ℃ to 45 ℃, or 18 ℃ to 30 ℃.
8. A process for the production of a therapeutic polypeptide or a precursor thereof comprising at least one chromatographic treatment step wherein a chromatographic stationary phase is regenerated by a process for regenerating a chromatographic stationary phase wherein said chromatographic stationary phase is contacted with a regeneration solution comprising at least one weak base, an organic solvent and less than 70% (v/v) water at a temperature in the range of 5 ℃ to 60 ℃; wherein the therapeutic polypeptide is selected from the group consisting of acylated GLP-1 or an acylated GLP-1 analog.
9. The method according to claim 8, wherein the acylated GLP-1 or acylated GLP-1 analogue is selected from Liraglutide (Liraglutide) or somaglutide (Semagutide).
10. The process according to claim 8, wherein the chromatographic stationary phase is contacted with a regeneration solution comprising at least one weak base, an organic solvent and less than 70% (v/v) water, under regeneration conditions according to the process of any one of claims 1 to 7.
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