A sequential coupling and purification method for preparing PEG-modified proteins

Abstract

This disclosure provides a continuous coupling and purification method for preparing PEG-modified proteins. The method utilizes a system integrating continuous reaction and purification modules. The target protein solution and the modifier solution are continuously pumped into a highly efficient mixing continuous reaction module in a specific ratio. The reaction is carried out under controlled temperature, pH, and retention time conditions. After completion, the reaction is quenched online and continuously purified. The high mixing efficiency of the continuous coupling process ensures reaction homogeneity, reduces batch-to-batch variability, improves yield, and reduces impurity levels. This disclosed process overcomes the shortcomings of traditional batch methods, enabling the efficient and controllable preparation of protein-modified drugs with well-defined modification sites, uniform modification degree, and high retention of biological activity. Product quality is significantly improved, and the process is easily scaled up for industrial production.

Description

Technical Field

[0001] This disclosure belongs to the interdisciplinary field of biopharmaceutical and chemical engineering, specifically relating to a method for the continuous coupling and purification of protein-modified drugs, and its application in the preparation of highly uniform and highly active modified protein drugs. Background Technology

[0002] Protein modification is a biotechnology that covalently links bioactive macromolecules (such as colony-stimulating factors, antibodies, and peptides) with functional molecules. Protein modification is often used to improve the pharmacokinetic properties of proteins (such as prolonging half-life and reducing immunogenicity) or to endow them with targeted killing capabilities. In the 1970s, to reduce the immunogenicity of protein drugs, researchers first proposed the concept of PEGylated proteins (Davis, 1977) and developed the first two PEGylated drugs, Adagen™ (1990) and Oncaspar™ (1994). Subsequent studies found that PEGylated protein drugs not only have reduced immunogenicity but also exhibit many advantages such as enhanced activity, prolonged onset of action, reduced toxicity, and passive targeting. Subsequently, PEGylated protein technology developed rapidly and extended to PEGylated peptides, PEGylated nucleic acids, and PEGylated liposomes. Currently, dozens of PEGylated protein drugs (or PEGylated peptide drugs or PEGylated nucleic acid drugs) have been approved for marketing for the treatment or prevention of leukemia, immune system diseases, tumors, viral infections, etc.

[0003] Traditional PEG-modified protein coupling reactions are mostly carried out in batch reactors (such as stirred tanks and reaction vessels), which have drawbacks such as long coupling reaction time, poor reaction uniformity, difficulty in process control, poor process reproducibility, low production efficiency, high raw material waste rate, and high quality risk. Therefore, developing a continuous protein coupling process that can achieve short preparation time, precise process control, high product uniformity, high process stability, recyclable raw materials, and easy scale-up is a technical problem that urgently needs to be solved in this field. Summary of the Invention

[0004] To overcome the shortcomings of existing technologies, this disclosure provides a continuous coupling and purification method with high efficiency, low loss, and controllable quality, and its application.

[0005] In a first aspect, this disclosure provides a continuous coupling and purification system for protein modification, characterized in that the system comprises at least one feed module, at least one continuous reaction (coupling) module, at least one continuous quenching and loading module, and at least one continuous purification module; wherein the feed module, continuous reaction module, continuous quenching and loading module, and continuous purification module are connected in series in the system via connectors or surge cells;

[0006] The feeding module is used to input the target protein to be modified, the modifier, and the catalyst into the continuous reaction module;

[0007] The continuous reaction module is used to continuously mix the liquid feed input from the feed module and perform a coupling reaction;

[0008] The continuous quenching and loading module is used to add a quencher (or diluent) to the coupling solution to terminate the reaction, and simultaneously deliver the coupling solution with the added quencher (or diluent) to the continuous purification module to complete the loading step; the coupling solution may also be delivered directly or after dilution to the continuous purification module without quenching to complete the loading step.

[0009] The continuous purification module, connected to the continuous quenching and loading module, is used to purify the modified protein solution, preferably recovering the unmodified target protein raw material.

[0010] In some embodiments, the catalyst is selected from reducing agents, oxidizing agents, enzymes, etc.

[0011] In some embodiments, the system further includes a return delivery module that delivers unmodified protein feedstock recovered from the continuous purification module back to the feed module.

[0012] In some embodiments, the continuous reaction module is selected from one or more of a microchannel reactor, a tubular reactor, or a packed bed reactor. Preferably, the reactor is equipped with a static mixing element, a serpentine channel structure, or a mixing valve system via a pump, with the mixture entering the pipeline after mixing.

[0013] In some implementations, the modules are transported between each other via pumps or buffer devices.

[0014] In some embodiments, the continuous purification module is selected from one or more of a chromatography system, a tangential flow filtration system, a filtration system, a salting-out or precipitation device.

[0015] In some implementations, the system further includes a monitoring module for real-time monitoring of each module.

[0016] In some embodiments, the system further includes at least one pump, at least one mixer, and at least one connector (or surge tank); the pump is used to transfer the solution from one module to another; the mixer is used to homogenize the solution; the connector (or surge tank) is used to buffer flow rate fluctuations between different systems, avoid drastic fluctuations in cross-system composition, and serve to homogenize and decouple the systems. The connector (or surge tank) is a key engineering component that ensures stable series connection between the continuous coupling system and the chromatography system, and supports the efficient and stable operation of the multi-column system.

[0017] In some embodiments, the continuous purification module is a continuous chromatography module including two or more columns, including two chromatography columns (including but not limited to ion exchange chromatography columns, affinity chromatography columns, and size exclusion chromatography columns) or more chromatography columns, which can continuously purify continuously coupled samples by using the method of alternating cyclic chromatography of two or more columns.

[0018] In some embodiments, before entering the continuous reaction module, there may be a module for online activation of the modifier (including chemical catalyst activation, enzyme catalytic activation, temperature-controlled activation, etc.) or a module for pretreatment of the protein (such as solution system replacement, reduction of disulfide bonds, etc.) to activate or enhance the reactivity and achieve precise control of the reaction process.

[0019] In some embodiments, the continuous reaction system is made of biocompatible materials, such as PEEK, 316L stainless steel, platinum silicone tubing, glass, or specific polymers, to reduce protein adsorption and ensure the retention of the activity of the modified small molecules.

[0020] In a second aspect, this disclosure provides a protein modification method, the method comprising coupling purification using the system of this disclosure, specifically including the following steps:

[0021] Step a) Input the target protein solution, the modifier solution, and / or the catalyst solution into the continuous reaction module;

[0022] Step b) In the continuous reaction module, the input solution is continuously and uniformly mixed and a coupling reaction is carried out;

[0023] Step c) After the reaction is complete, the coupling solution is continuously delivered to the continuous quenching and loading module, so that the coupling solution is mixed with the quencher (or diluent) and simultaneously input into the continuous purification module to complete the loading procedure; the coupling solution may also be input into the continuous purification module directly or after dilution without quenching reaction to complete the loading procedure.

[0024] Step d) Purify the coupling solution, harvest the target protein, and recover the unmodified target protein raw material.

[0025] In some implementations, step e) is also included, which involves feeding the recovered unmodified target protein raw material back to the feed module.

[0026] In some embodiments, the concentration of the target protein solution to be modified is about 1 mg / mL to 10 mg / mL; the concentration of the modifier solution is about 3 mg / mL to about 50 mg / mL; the concentration of the catalyst in the mixed solution is about 1 mM to about 100 mM; the buffer system for the reaction is a phosphate, acetate, or citrate, for example, sodium phosphate, potassium phosphate, sodium acetate, potassium acetate, sodium citrate, or potassium citrate; and the solvent used is water, a non-proton inert solvent, a proton solvent, or an inert solvent.

[0027] In some embodiments, the catalyst is selected from reducing agents, oxidizing agents, enzymes, etc.

[0028] In some embodiments, the reaction conditions include: a temperature of about 0 °C to about 37 °C, preferably 22 °C to 28 °C; a pH value of about 4.5 to about 6.5, preferably 4.8 to 5.2; and a residence time of 0.5 hours to about 6 hours, preferably 3 to 5 hours.

[0029] In some embodiments, the target protein solution to be modified is fed into the system via a feed pump A at a constant flow rate; preferably, the constant flow rate is from about 0.5 mL / min to 100 mL / min; more preferably, the constant flow rate is from about 0.5 mL / min to 20 mL / min; more preferably, the constant flow rate is from about 0.5 mL / min to 1 mL / min.

[0030] In some embodiments, the modifier solution is fed into the system at a constant flow rate via a B feed pump; preferably, the constant flow rate is from about 0.5 mL / min to 100 mL / min; more preferably, the constant flow rate is from about 0.5 mL / min to 20 mL / min; more preferably, the constant flow rate is from about 0.5 mL / min to 1 mL / min.

[0031] In some embodiments, the catalyst is fed into the system via a C-feed pump at a constant flow rate, preferably from about 0.01 mL / min to 10 mL / min; more preferably, the constant flow rate is from about 0.01 mL / min to 2.0 mL / min; more preferably, the constant flow rate is from about 0.01 mL / min to 0.1 mL / min.

[0032] In some implementations, the method specifically includes the following steps:

[0033] a) Introduce a solution of the target protein to be modified at a concentration of approximately 1.0 mg / ml to approximately 10 mg / ml into the system via feed pump A at a constant flow rate of approximately 0.5 mL / min to 1 mL / min. Introduce a solution of the modifier at a concentration of approximately 5 mg / ml to approximately 50 mg / ml into the system via feed pump B at a constant flow rate of approximately 0.5 mL / min to 1 mL / min. Introduce the catalyst via feed pump C at a constant flow rate of approximately 0.01 mL / min to 0.1 mL / min, so that the catalyst concentration in the final mixed solution is approximately 1 mM to 100 mM.

[0034] b) The feed liquids pumped in by pumps A, B and C are mixed in the mixer of the continuous reaction module, and then directly pumped into the reactor of the continuous reaction module for coupling reaction.

[0035] c) After the coupling reaction is complete, use pump D and pump E to mix the coupling solution and quenching solution to terminate the reaction and dilute the coupling solution. Simultaneously, pump the diluted coupling solution into the continuous purification module (continuous sample loading).

[0036] d) Separation and purification are performed in the continuous purification module to harvest the target protein. Preferably, the continuous purification module is a continuous chromatography module including two or more columns, including two chromatography columns (including but not limited to ion exchange chromatography columns, affinity chromatography columns, hydrophobic chromatography columns, reversed-phase chromatography columns, and size exclusion chromatography columns, etc.) or more chromatography columns. By using the method of alternating dual-column or multi-column chromatography, the continuously coupled samples can be continuously purified, the coupled target protein can be continuously separated and purified, and the unmodified target protein raw material can be recovered.

[0037] In some embodiments, the catalyst is selected from reducing agents, oxidizing agents, enzymes, etc.

[0038] In some implementations, the sequential chromatography purification steps are as follows:

[0039] i. System equilibration: Elute two column volumes using 20-80 mM phosphate buffer (chromatographic mobile phase A, pH 5.0) at a linear velocity of approximately 3-4 cm / min;

[0040] ii. Isocratic elution 1: Elute 4.5 column volumes with 50-100 mM phosphate buffer (chromatographic mobile phase B, pH 5.0) at a linear velocity of approximately 3-4 cm / min; this stage uses an automated chromatography program to capture the modified target protein;

[0041] iii. Isocratic elution 2: Continue eluting with 150-200 mM phosphate buffer (chromatographic mobile phase C, pH 5.0) for 4 column volumes; recover the unmodified target protein starting material;

[0042] iv. Column regeneration: Elute for 2 column volumes with 2M NaCl and 2 column volumes with 0.5M NaOH at a linear velocity of 3-4 cm / min;

[0043] v. System rebalancing and sample loading readiness: Use 50 mM phosphate buffer (chromatographic mobile phase A, pH 5.0) to elute 5 column volumes at a linear velocity of approximately 3 ~ 4 cm / min to restore the chromatography column to a usable state and complete this cycle (at this point, the chromatography column is ready to receive continuous coupling reaction solution for chromatographic sample loading at any time).

[0044] In some embodiments, the method further includes step e) feeding the recovered unmodified target protein raw material back to the feed module.

[0045] In some embodiments, the phosphate includes, but is not limited to, sodium phosphate, potassium phosphate, etc.

[0046] In some embodiments, the target protein to be modified includes, but is not limited to, antibodies, receptors, enzymes, peptides, cytokines, protein hormones, antibody-drug conjugates (ADCs), etc.

[0047] In some embodiments, the modifiers include, but are not limited to, PEG or activated PEG derivatives (such as mPEG-pALD, mPEG-SC, mPEG-SPA, mPEG-MAL, mPEG-NBZ, PEG-Diazonium, PEG-Thioester, mPEG-oxyamine, mPEG-keto-α-amine and their analogs), activated lipid derivatives (such as activated stearic acid, palmitic acid, myristic acid, eicosanoic acid, phospholipids, cholesterol, N-hydroxysuccinimide ester, phenyl isothiocyanate and their analogs), enzymes and their remodeling enzymes (sorting enzyme A, microbial transglutaminase, subtilisin, butterfly pea enzyme 1, lipoic acid protein ligase), cytotoxic drug payloads (such as MMAE, MMAF, DM1, DXd, SN38, etc.) and their linker-drugs (linker-payloads) and their derivatives, etc.

[0048] In some embodiments, the catalyst includes, but is not limited to, sodium cyanoborohydride, potassium cyanoborohydride, sodium borohydride, potassium borohydride, sodium periodate, potassium periodate, lithium periodate, pyridoxal phosphate and its analogues, sodium triacetoxyborohydride, pyridineborane, phenyl isothiocyanate, cuprous ions, vitamin C, tris(2-carboxyethyl)phosphine (TCEP), diphenylphosphinoacetic acid, 4-(diphenylphosphino)benzoic acid, 2-(diphenylphosphino)ethylamine, 3 1-(diphenylphosphino)propylamine, 3-(diphenylphosphine)propionic acid, 2-(diisopropylphosphino)ethylamine, 2-(diphenylphosphino)benzoic acid, (2-hydroxyphenyl)diphenylphosphine, 1,3,5-triaza-7-phosphotricyclic[3.3.1.13.7]decane, 2-[2-(diphenylphosphino)ethyl]pyridine, 3-(diphenylphosphino)benzenesulfonic acid, n-butyldi(1-adamantyl)phosphine, or any one or a combination of at least two of their salts.

[0049] In some embodiments, the protein to be modified, the modifying agent, and the catalyst are dissolved in a pharmaceutically acceptable buffer or organic solvent system. The buffer includes, but is not limited to, phosphates, Tris, acetates, citrates, histidines, and HEPES. The organic solvent is selected from polar aprotic solvents, ether solvents, haloalkanes solvents, ester solvents, or mixtures thereof. The organic solvent includes, but is not limited to, tetrahydrofuran (THF), N,N-dimethylformamide (DMF), N,N-dimethylacetamide (DMA), dimethyl sulfoxide (DMSO), or acetone, ethyl acetate, isopropanol, ethylene glycol, propylene glycol, glycerol, acetonitrile, methanol, and ethanol.

[0050] In some embodiments, coupling is carried out at a suitable system pH that is conducive to the reaction, typically in an aqueous buffer solution (or with an appropriate proportion of organic solvent) with a pH of 4.0 to 7.0.

[0051] In some embodiments, the reagents (or methods) used for online quenching include, but are not limited to, hydroxylamine, glycine, lysine, ethanolamine, Tris buffer, pH adjustment, oxidant, addition of metal ions, enzyme inhibitor, N-acetylcysteine, glutathione, cysteine, β-mercaptoethanol, dithiothreitol, EDTA or EGTA, azide or cyclooctyne, SDS, dilution, etc.

[0052] In some embodiments, the coupling reaction is carried out under strictly controlled conditions: the temperature of the pumped-in system is precisely controlled at 0°C to 40°C (preferably 25°C) via an external thermostat; the temperature of the reactor is precisely controlled at 10°C to 40°C (preferably 25°C or room temperature) via an external thermostat; the pH is maintained at 4.5 to 7.4 (preferably 6.0) via an online pH sensor and feedback system (or via buffer control); and the residence time is precisely controlled at 1 to 48 hours (preferably 4.5 hours) by adjusting the flow rate and the total flow volume (or number) of the reactors. This module design ensures that the reactants are in a constant, optimal reaction environment throughout the process, greatly improving reaction uniformity and reproducibility.

[0053] In some embodiments, the purification process employs continuous chromatography using dual or multiple columns, also performed in continuous mode. That is, after loading onto one column (column A), the elution program is immediately initiated, and the loading column is switched to another column (column B). The required number of columns is adjusted according to the actual situation. After elution from the column (e.g., column A) yields the target protein to be coupled, the process includes column regeneration and rebalancing, as well as a loading-ready procedure, allowing for the cyclical use of the columns.

[0054] In some implementations, the coupling solution is directly connected to the continuous chromatography purification module via a buffer device (such as a surge tank). Continuous chromatography employs an automated process encompassing sample loading, equilibration, elution, sample collection, protein recovery, and column regeneration, minimizing the impact of human factors on product quality during step-by-step operations. By directly connecting the continuous coupling solution in series with continuous chromatography, online synchronous quenching of the coupling solution and sample loading are achieved, eliminating numerous steps such as quenching, dilution, transfer, and storage required in batch production processes. This helps reduce equipment, material, and personnel costs, shorten process time, improve production efficiency, and ensure product quality and safety.

[0055] In some embodiments, before entering the continuous reaction module, there may be a module for online activation of the modifier (including chemical reducing agent activation, enzyme catalytic activation, temperature-controlled activation, etc.) or a module for pretreatment of the protein (such as solution system replacement, reduction of disulfide bonds, etc.) to activate or enhance the reactivity and achieve precise control of the reaction process.

[0056] In some embodiments, the reactor of the continuous reaction module is preferably a microchannel reactor, a tubular reactor, or a packed bed reactor. The reactor interior has a highly efficient mixing structure (such as serpentine channels or static mixing elements) to ensure uniform mixing of reactants at the molecular scale within milliseconds. High-efficiency material mixing can also be achieved by inserting a mixing module (such as an online mixer or a mixer integrated into the pump system) at the front end of the reactor.

[0057] In some implementations, the online quenching process is carried out in continuous mode. A quencher (such as an acid / alkaline buffer solution, glycine buffer solution, cysteine ​​buffer solution, diluent, etc.) is precisely added to the continuous coupling reaction solution via an E-feed pump to terminate the coupling reaction. The acid / alkaline buffer solution includes, but is not limited to, phosphate buffer solution, acetate buffer solution, citrate buffer solution, TRIS buffer solution, HIS buffer solution, diluent, etc. The quenching process of the coupling solution is also the purification and loading process. During the quenching stage (which may involve quenching reaction, dilution effect, small molecule dialysis, reducing agent removal from the system, temperature control, and other processes or principles), the coupling solution is simultaneously delivered to the continuous purification module for chromatographic loading. Simultaneous quenching and purification loading ensures rapid, uniform, and thorough termination of the reaction, prevents over-modification or side reactions, saves purification loading time, and simplifies the chromatographic method.

[0058] In some embodiments, the method is monitored in real time using process analysis techniques (PAT), including but not limited to online ultraviolet-visible spectroscopy (UV-Vis), online pH meter, online conductivity meter, online FTIR, online Raman spectroscopy, online HPLC, or online mass spectrometry, to monitor the reaction progress (conversion rate) and product quality in real time.

[0059] In a third aspect, this disclosure provides a method for modifying granulocyte colony-stimulating factor (G-CSF) with a PEG derivative monomethoxy polyethylene glycol propionaldehyde (mPEG-pALD), comprising the following steps:

[0060] a) Introduce a G-CSF solution of approximately 5.0 mg / ml to be modified into the system via feed pump A at a constant flow rate of approximately 0.75 mL / min. Introduce an mPEG-pALD solution of approximately 30 mg / ml into the system via feed pump B at a constant flow rate of approximately 0.72 mL / min. Introduce a NaBH3CN catalyst solution of approximately 1 M into the system via feed pump C at a constant flow rate of approximately 0.03 mL / min.

[0061] The G-CSF solution is prepared by replacing G-CSF protein in sodium phosphate buffer (pH 6.0) to a concentration of 5.0 mg / mL; the mPEG-pALD solution is prepared by dissolving mPEG-pALD in sodium phosphate buffer (pH 6.0) to a concentration of 30.0 mg / mL; and the NaBH3CN solution is prepared by dissolving NaBH3CN in DMSO to prepare a 1M stock solution.

[0062] b) The solutions pumped in by feed pumps A, B and C are mixed in the mixer of the continuous reaction module at room temperature. After mixing, the mixture is directly pumped into the reactor (reaction pipeline) of the continuous reaction module to continue the coupling reaction at 25±2 ℃ for 4.5 hours.

[0063] c) After the coupling reaction is complete, use feed pump D (to pump in the coupling solution) and feed pump E (to pump in the quenching solution) in combination at a flow rate of approximately 1.5 mL / min and a 1:1 ratio to mix the quencher and coupling solution to terminate the reaction. Simultaneously, load the mixture into the continuous purification module at a sample volume of approximately 17.3 g / L and load it into the chromatography column (chromatography column A). The total sample volume is approximately 701 mg. After the chromatographic loading is completed, start the online elution program and switch the loading column to the alternating chromatography column (chromatography column B) to continue continuous chromatographic loading.

[0064] The quenching solution is a sodium acetate solution with a pH of 3.0; both chromatography columns A and B are cation exchange chromatography columns, Capto... TM SP ImpRes, CV = 40.5 cm 3 .

[0065] d) For chromatography columns that have been loaded with samples (such as column A), start the following chromatography separation and purification procedure:

[0066] i. System equilibration: Wash two column volumes with approximately 50 mM sodium phosphate buffer (chromatographic mobile phase A, pH 5.0) at a linear velocity of approximately 3.7 cm / min;

[0067] ii. Isocratic elution 1: Elute 4.5 column volumes using approximately 80 mM sodium phosphate buffer (chromatographic mobile phase B, pH 5.0) at a linear velocity of approximately 3.7 cm / min; this stage uses an automated chromatography program. When the absorbance at A280 nm is >300 mAu, automated sample collection is initiated, and collection is stopped when the absorbance at A280 nm is ≤100 mAu, capturing the target protein mPEG-G-CSF;

[0068] iii. Isocratic elution 2: Continue eluting with approximately 200 mM sodium phosphate buffer (chromatographic mobile phase C, pH 5.0) for 4 column volumes; collect the chromatographic peak with an absorption intensity >30 mAu at A280 nm, and recover the unreacted target protein raw material G-CSF;

[0069] iv. Column regeneration: Elute 2 column volumes with approximately 2 M NaCl and 2 column volumes with approximately 0.5 M NaOH at a linear velocity of approximately 3.7 cm / min;

[0070] v. System rebalancing and sample loading readiness: Use approximately 50 mM sodium phosphate buffer (chromatographic mobile phase A, pH 5.0) to flush 5 column volumes at a linear velocity of approximately 3.7 cm / min to restore the column to a usable state and complete this cycle (at this point, the column is ready to receive continuous coupling reaction solution for chromatographic sample loading).

[0071] In some embodiments, the method further includes step e) feeding the recovered unmodified G-CSF back to the feed module.

[0072] In some implementations, the modified mPEG-G-CSF yield is over 60% and the purity is over 99%.

[0073] In some implementations, this method can recover unmodified G-CSF protein with a recovery rate of over 30%.

[0074] Beneficial effects

[0075] 1. Short reaction time: Traditional methods for preparing PEG-modified proteins require coupling reactions of more than 10 hours, while the method disclosed in this paper only requires coupling for 4.5 hours, greatly shortening the reaction time and improving the preparation efficiency. Furthermore, the reaction can be carried out at room temperature, improving operational convenience.

[0076] 2. High product uniformity: Efficient mass transfer and mixing in the continuous reactor, combined with precise residence time control, ensures uniformity of modification efficiency in the reactor, significantly increases the proportion of single-modification products, and reduces product heterogeneity.

[0077] 3. High process controllability: Key parameters such as temperature, pH, and residence time can be independently and precisely controlled, providing solid technical support for process optimization and quality control, and reducing batch-to-batch variations.

[0078] 4. Excellent protein activity retention: Rapid mixing and precise temperature control effectively shorten the exposure time of proteins under harsh reaction conditions. The continuous mixing method is gentler, and instantaneous quenching and simultaneous chromatographic loading can be achieved after the reaction, initiating the chromatographic purification process in a very short time. These characteristics of the continuous process minimize the risk of protein denaturation, aggregation, and inactivation.

[0079] 5. Easy to scale up and industrialize: Continuous processes follow the principle of "scale up by number". By adding parallel reaction modules or extending residence time, production capacity can be quickly scaled up. There is no scale-up effect problem of traditional batch reactions. Process transfer and large-scale production are simpler and less risky.

[0080] 6. Improve production efficiency and resource utilization: Continuous operation reduces equipment idle time and enables continuous large-scale production with small equipment; at the same time, precise chemical metering control reduces reagent waste, and the overall process is green, ecological, economical, and inherently safe.

[0081] 7. Cost savings: The method disclosed herein can recover unmodified target proteins with high purity and refeed them back into the feed module, reducing the waste of raw materials.

[0082] In summary, compared with traditional one-pot synthesis processes, the method disclosed herein exhibits numerous advantages, including higher capacity limits, milder reaction conditions, shorter and more refined processes, higher process stability, controllable product quality, and lower costs. The reaction time (taking mPEG-G-CSF as an example) is reduced from over 10 hours in traditional one-pot (batch) processes to 4-6 hours. Since continuous processes theoretically do not have a scale-up effect, capacity can be rapidly increased by adding continuous reactor modules or increasing flow rates and total pipeline volume, resulting in a very high capacity limit. Simultaneously, the coupling reaction can be performed efficiently at room temperature, which is more conducive to energy conservation and consumption reduction. Continuous coupling followed by continuous chromatography can directly obtain pure target proteins, reducing process steps and avoiding environmental exposure, which helps reduce costs and ensure product quality (strengthened aseptic assurance and reduced aggregate impurity levels). The continuous nature of the new process results in higher reaction homogeneity, ensuring product homogeneity and significantly reducing the risk of microbial exceedances introduced by environmental exposure and human operation, providing a solid material basis for the safety and efficacy of protein drugs. Attached Figure Description

[0083] Figure 1 This is a schematic diagram of the continuous coupling and purification system for protein modification disclosed herein.

[0084] Figure 2 The mPEG-G-CSF coupling reaction curve was prepared according to the process of Example 1 of this disclosure.

[0085] Figure 3 The conversion rate of the coupling reaction for preparing mPEG-G-CSF using the process of Example 1 of this disclosure.

[0086] Figure 4 The cation exchange chromatography pattern of mPEG-G-CSF prepared by the process of Example 1 of this disclosure is shown.

[0087] Figure 5 The IEC-HPLC purity analysis chromatogram of the cation exchange chromatography collection solution prepared by the process of Example 1 of this disclosure is shown.

[0088] Figure 6 The conversion rate of the coupling reaction for preparing mPEG-G-CSF using the process of Example 2 of this disclosure.

[0089] Figure 7 The cation exchange chromatography pattern of mPEG-G-CSF prepared by the process of Example 2 of this disclosure is shown.

[0090] Figure 8 The IEC-HPLC purity analysis chromatogram of the cation exchange chromatography collection solution prepared by the process of Example 2 of this disclosure is shown.

[0091] Figure 9 The conversion rate of the coupling reaction for preparing mPEG-G-CSF using the conventional one-pot process in Comparative Example 1 was determined.

[0092] Figure 10 The cation exchange chromatography pattern of mPEG-G-CSF purified using the conventional one-pot process in Comparative Example 1 is shown.

[0093] Figure 11 The IEC-HPLC chromatograms of the solution collected by manual cation exchange chromatography in Comparative Example 1 are shown. Detailed Implementation

[0094] The present disclosure will be further described in detail below with reference to specific embodiments and accompanying drawings. It should be emphasized that these embodiments are for illustrative purposes only and are not intended to limit the scope of the disclosure.

[0095] Unless otherwise specified, the technical details or conditions in the embodiments are understood to be in accordance with the techniques or conditions described in the literature or known in the art, or in accordance with the product instructions. The nomenclature and experimental procedures and techniques used in conjunction with the coupling reactions, organic synthesis, chromatographic purification, apparatus, analytical chemistry, or medicine described in this disclosure are known and commonly used in the art. Reagents or instruments whose manufacturers are not explicitly stated are considered to be conventional products that can be purchased through legitimate channels.

[0096] Terminology Definition

[0097] To better understand this disclosure, the relevant terms are defined and explained below.

[0098] Unless otherwise indicated, the terminology used in conjunction with this disclosure should be understood in the manner commonly understood by one of ordinary skill in the art. Throughout this application, unless the context otherwise requires, the terms "comprising," "including," and "containing" should be understood to include the stated steps or elements or groups of steps or elements, but do not exclude any other steps or elements or groups of steps or elements. … "Composition" refers to the phrase "composed of" and is limited to the phrase "made up of". … The elements that follow "composition".

[0099] Throughout this disclosure, the term "about" or "approximately" refers to a quantity, level, value, amount, percentage, frequency, size, weight, or length that varies relative to that quantity, level, value, amount, percentage, frequency, size, weight, or length by 50%, 40%, 30%, 20%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1%. In specific embodiments, the term "about" or "approximately" preceding a numerical value indicates a range of variation of that value plus or minus 20%, 10%, 5%, or 1%.

[0100] "Polyethylene glycol-modified protein drugs" or "PEGylated protein drugs" refer to modified products formed by covalently coupling polyethylene glycol to a protein through one or more suitable reactive linkers. "Polyethylene glycol-modified protein drugs" are typically in the form of "protein-linker-polyethylene glycol conjugates".

[0101] "Polyethylene glycol-modified antibody-drug conjugates" or "PEGylated ADCs" refer to modified products formed by covalently coupling antibody-drug conjugates with polyethylene glycol through one or more suitable reactive linkers. "Polyethylene glycol-modified antibody-drug conjugates" are typically in the form of "(antibody-linker-connector-drug)-linker-polyethylene glycol conjugate".

[0102] The "surge tank" described in this disclosure refers to a connecting device capable of smoothing fluctuations, decoupling units and homogeneous fluids, and enabling efficient, robust, and controllable end-to-end continuous production. It is far more than a simple storage tank (or a device with similar functions); rather, it is an active process coordination and stabilization unit, a key engineering component in modern continuous bioprocessing (CBP) and integrated continuous biomanufacturing (ICB). This definition emphasizes its function; any device capable of achieving surge functionality falls within the scope of the "surge tank" of this disclosure.

[0103] The term "catalyst" as used in this disclosure does not specifically refer to any particular chemical substance, but rather emphasizes its high activity and catalytic ability. The catalyst includes, but is not limited to, reducing agents (NaBH3CN, TCEP, cuprous ions, etc.), oxidizing agents (periodate, PLP, NCL, etc.), enzymes (sortase A, mTGAse, etc.). Those skilled in the art will understand that any substance capable of catalyzing a reaction falls within the scope of protection of this invention.

[0104] The "room temperature" mentioned in this invention refers to 20 ℃–30 ℃, preferably 25±2 ℃.

[0105] The “mixing temperature” mentioned in this disclosure refers to the temperature of the mixer or the temperature during the sample mixing stage.

[0106] The “reaction temperature” mentioned in this disclosure refers to the control temperature of the coupling reactor.

[0107] The “residence time” mentioned in this disclosure refers to the residence time of the coupling liquid in the reactor after mixing in the mixer, and can also be understood as the actual execution time of the coupling reaction.

[0108] Example 1: Continuous conjugation preparation of polyethylene glycol-modified granulocyte colony-stimulating factor (mPEG-G-CSF).

[0109] use Figure 1 The continuous coupling purification system shown is used for protein modification. Granulocyte colony-stimulating factor (G-CSF) is the target protein to be modified. Monomethoxy polyethylene glycol is used as the modifier and NaBH3CN is used as the catalyst to carry out the coupling reaction and purification to prepare monomethoxy polyethylene glycol modified granulocyte colony-stimulating factor (mPEG-G-CSF).

[0110] The specific preparation method is as follows:

[0111] 1) Solution preparation

[0112] a. Prepare a fresh buffer solution for G-CSF protein: Displace the G-CSF protein into sodium phosphate buffer (pH 6.0) at a concentration of 5.0 mg / mL, and set aside.

[0113] b. Prepare fresh modifier solution before use: Dissolve monomethoxy polyethylene glycol propionaldehyde (mPEG-pALD, 20 kDa) in sodium phosphate buffer (pH 6.0) to a concentration of 30.0 mg / mL and set aside.

[0114] c. Prepare a fresh catalyst stock solution: Dissolve NaBH3CN in DMSO to prepare a 1M stock solution for later use.

[0115] 2) System Configuration

[0116] A continuous coupling purification series system consisting of three plunger pumps, a mixer, one (or more) tubular reactors (0.125 inch inner diameter), a surge tank (or connector), a mixing quenching and loading unit, and a sample loading unit is used.

[0117] 3) Protein coupling step

[0118] a. Mixing and Coupling: The protein solution, modifier solution and catalyst stock solution were respectively placed in three mobile phase solvent bottles, pumped into a mixer through a three-pump system, and then directly connected to a continuous reactor for timed coupling reaction.

[0119] The process parameters are as follows:

[0120] Protein solution flow rate: 0.75 mL / min.

[0121] Flow rate of the modifier solution: 0.72 mL / min.

[0122] Catalyst solution flow rate: 0.03 mL / min.

[0123] Mixing temperature: room temperature.

[0124] Reaction temperature: 25±2 ℃ (controlled by a low-temperature constant reaction bath).

[0125] Duration of stay: 4.5 hours.

[0126] according to Figure 2 As shown in the reaction curve, under the parameter system of this embodiment, the main reaction can be completed in 4 hours of coupling. Extending the time did not show significant benefits. Therefore, 4.5 hours was selected as the reaction residence time to continue subsequent operations.

[0127] b. Quenching and sample loading

[0128] Quenching: After the coupling solution flows out, it is immediately mixed online with the diluent (i.e., quenching solution, sodium acetate solution, pH 3.0) at a ratio of 1:1 and a flow rate of 1.5 mL / min for quenching.

[0129] Chromatographic loading: The coupling solution and diluent are quenched online, and then chromatographic loading is performed (chromatographic column A, Capto). TM SPImpRes, CV = 40.5 cm 3 The cation exchange chromatography column (with a loading capacity of 17.3 g / L, total loading 701 mg) integrates continuous coupling and continuous chromatography in series. After loading, the online elution program is initiated, and the loading column is simultaneously switched to an alternating chromatography column (column B, Capto). TM SP ImpRes, CV = 40.5 cm 3 (Cation exchange chromatography column), continue continuous chromatography loading.

[0130] c. Purification and recovery

[0131] For the chromatography column (column A) that has already been loaded with samples, start the following chromatography program:

[0132] i. System equilibration: Wash two column volumes with 50 mM phosphate buffer (chromatographic mobile phase A, pH 5.0) at a linear velocity of approximately 3.7 cm / min;

[0133] ii. Isocratic elution 1: Elute 4.5 column volumes with 80 mM phosphate buffer (chromatographic mobile phase B, pH 5.0) at a linear velocity of 3.7 cm / min. An automated chromatography program was used in this stage. Automated sample collection was initiated when the absorbance at A280 nm was >300 mAu, and collection was stopped when the absorbance at A280 nm was ≤100 mAu. The target protein mPEG-G-CSF was purified and separated.

[0134] iii. Isocratic elution 2: Continue eluting with 200 mM phosphate buffer (chromatographic mobile phase C, pH 5.0) for 4 column volumes; collect the chromatographic peak with an absorption intensity >30 mAu at A280 nm, and recover the unreacted protein raw material G-CSF;

[0135] iv. Column regeneration: Elute 2 column volumes with 2M NaCl and 2 column volumes with 0.5M NaOH at a linear velocity of 3.7 cm / min;

[0136] v. System rebalancing and sample loading readiness: Use 50 mM phosphate buffer (chromatographic mobile phase A, pH 5.0) to elute 5 column volumes at a linear velocity of 3.7 cm / min to restore the chromatography column to a usable state and complete this cycle (at this point, the chromatography column is ready to receive continuous coupling reaction solution for chromatographic sample loading at any time).

[0137] 4) Results and Analysis: Coupled central control (IEC-HPLC analysis) showed that before purification by continuous cyclic alternating chromatography, the product of monomethoxy polyethylene glycol modified granulocyte colony-stimulating factor (mPEG-G-CSF) accounted for 78.3%, the content of impurities in the acid zone was 3.7%, and the remaining unmodified granulocyte colony-stimulating factor (G-CSF) was 18.0%. Figure 3 After continuous cyclic alternating chromatography purification, a total of 450.0 mg of captured mPEG-G-CSF was automatically collected, with a yield of 64.2%. Figure 4 ), purity 99.4% ( Figure 5 A total of 39.5 mg of G-CSF was recovered, with a recovery rate greater than 30% (the recovered pure G-CSF accounted for approximately 5.6% of the initial feed amount; the recovery rate here specifically refers to the percentage of recovered unreacted protein to the unreacted protein in the conjugated system).

[0138] Example 2: Study on the sequential conjugation preparation of monomethoxy polyethylene glycol modified granulocyte colony-stimulating factor (mPEG-G-CSF) under different conditions.

[0139] To validate the coupling method under different conditions such as target protein, modifier, catalyst concentration, flow rate, and residence time, the following experimental studies were also conducted.

[0140] 1) Solution preparation

[0141] a. Prepare a fresh buffer solution for G-CSF protein: Replace the G-CSF protein in a sodium phosphate buffer solution at pH 6.0, with a concentration of 2.0 mg / mL, and set aside.

[0142] b. Prepare fresh modifier solution before use: Dissolve monomethoxy polyethylene glycol propionaldehyde in sodium phosphate buffer at pH 6.0 to a concentration of 20.0 mg / mL, and set aside.

[0143] c. Catalyst stock solution: Dissolve NaBH3CN in DMSO to prepare a 2M stock solution, freeze at -20℃, and thaw at room temperature 2 hours before use.

[0144] 2) System Configuration

[0145] A continuous coupling purification series system consisting of three plunger pumps, a mixer, one (or more) tubular reactors (0.125 inch inner diameter), a surge tank (or connector), a mixing quenching and loading unit, and a sample loading unit is used.

[0146] 3) Protein coupling process

[0147] a. Mixing and Coupling: The protein solution, modifier solution and catalyst stock solution were respectively pumped into the mixer in appropriate proportions through a 3-pump system and directly connected to the continuous reactor. The system temperature was raised to room temperature and the coupling reaction was timed.

[0148] Process parameters:

[0149] Protein solution flow rate: 0.50 mL / min.

[0150] Flow rate of the modifier solution: 0.47 mL / min.

[0151] Catalyst stock solution flow rate: 0.03 mL / min.

[0152] Mixing temperature: 25 ℃ (controlled by a low-temperature constant reaction bath).

[0153] Reaction temperature: 25 ℃ (controlled by a low-temperature constant reaction bath).

[0154] Duration of stay: 20 hours.

[0155] b. Quenching and Sample Loading

[0156] Quenching: Immediately after the coupling solution flows out, it is mixed online with the diluent (quenching solution, sodium acetate solution, pH 3.5) at a flow rate of 1.5 mL / min for quenching.

[0157] Chromatographic loading: The coupling reaction solution and diluent were quenched online at a ratio of 1:1.5 and directly loaded onto the column (Column A, Capto). TM SP ImpRes, CV = 40.5 cm 3 The chromatographic column (cation exchange chromatography column) has a sample loading capacity of 21.6 g / L (total sample loading 876 mg), achieving tandem integration of continuous coupling and continuous chromatography. After sample loading, the online elution program is instantly initiated; and the chromatography column is simultaneously switched to an alternating chromatography column (column B, Capto). TM SP ImpRes, CV = 40.5 cm 3 (Cation exchange chromatography column), continue continuous chromatography loading.

[0158] c. Purification and recovery

[0159] The purification method is described in Example 1.

[0160] 4) Results and Analysis: The results of the coupled control system (using IEC-HPLC analysis) showed that the product of monomethoxy polyethylene glycol modified granulocyte colony-stimulating factor (mPEG-G-CSF) accounted for 90.2%, the content of impurities in the acid region was 3.1%, and the remaining granulocyte colony-stimulating factor (G-CSF) accounted for 6.8%. Figure 6 After chromatography purification, a total of 608 mg of mPEG-G-CSF was captured, with a yield of 69.4%. Figure 7 ), purity 99.5% ( Figure 8 A total of 20 mg of G-CSF was recovered, with a recovery rate of 33.6%.

[0161] Comparative Example 1: Traditional One-Pot Process

[0162] In a 250 mL reactor, 80 mL of 10 mg / mL G-CSF sodium phosphate buffer (50 mM, pH 6.0), 80 mL of 100 mg / mL mPEG-pALD sodium phosphate buffer (50 mM, pH 6.0), and 4 mL of 2 M sodium cyanoborohydride aqueous solution were added sequentially. The reaction was carried out at 35 °C for 10 hours at 50 rpm. After the reaction, the temperature was lowered to 25 °C, and 164 mL of quenching solution (50 mM sodium acetate buffer, pH 3.5) was added to the reaction system in one go to obtain the coupling reaction quenching solution. The entire coupling reaction quenching solution was directly loaded onto the sample for column chromatography (column A, Capto). TM After purification using SP ImpRes (cation exchange chromatography column), 389 mg of the target protein mPEG-G-CSF was manually collected.

[0163] Results: Coupled control (analyzed by IEC-HPLC) showed that the product content of monomethoxy polyethylene glycol modified granulocyte colony-stimulating factor (mPEG-G-CSF) was 68.9%, the content of impurities in the acid zone was 15.9%, and the remaining granulocyte colony-stimulating factor (G-CSF) was 15.2%. Figure 9 The overall yield of coupling chromatography was calculated to be 48.6%. Figure 10 ), purity 99.2% ( Figure 11 ).

[0164] In summary, the continuous process disclosed herein has the following advantages compared to the traditional one-pot process:

[0165] The reaction kinetics are improved. Studies show that, within the current parameter range, the coupling conversion efficiency is above 78.3%, the overall yield is above 64.2%, and the purity is not less than 99.4%. Compared with the traditional batch method (PEGylation efficiency approximately 23%~69%, yield around 48%, purity 99.2%), the new process significantly improves reaction efficiency while maintaining high yield, and achieves controllable reaction at room temperature (traditional methods typically require temperature control). Furthermore, the more refined continuous reaction conditions result in a higher proportion of single-modified products and lower impurity levels in the reaction system due to high mixing efficiency. The disclosed process also allows for efficient recovery of unmodified target protein raw materials, saving costs.

[0166] Automated chromatography programs make continuous processes more feasible. When preparing mPEG-G-CSF using the traditional one-pot method, the batch-produced coupling solution containing the target protein needs to be purified. Continuous production processes change this batch production logic. During the development of new processes, a continuous dual-column alternating cyclic chromatography method was developed specifically for the characteristics of continuous coupling processes. Through systematic optimization of this method, it is now perfectly adapted to continuous coupling processes, enabling continuous chromatographic purification of continuously prepared mPEG-G-CSF and continuous capture of the target protein. This compatible continuous process development provides strong technical support for the tandem integration of continuous coupling chromatographic purification processes.

[0167] Significant economic benefits, low quality risks, and environmental advantages. The new process significantly shortens reaction time, provides milder reaction conditions, and significantly reduces impurity levels. Superior process stability makes it more economically valuable for the production of expensive materials. Online monitoring and reaction control allow for appropriate adjustments to the coupling time, increasing yield and controlling costs over a wider range, unlocking economic potential. The new method simplifies experimental procedures, eliminating the need for post-processing of the coupling solution, saving significant manpower and resources, and further reducing costs and product contamination risks. This new method provides more possibilities for developing more economical, greener, and safer synthetic processes.

[0168] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for preparing polyethylene glycol-modified granulocyte colony-stimulating factor, characterized in that, The method includes preparation using a continuous coupling and purification system. The system includes at least one feeding module, at least one continuous reaction module, at least one continuous quenching and sample loading module, and at least one continuous purification module; the feeding module, continuous reaction module, continuous quenching and sample loading module, and continuous purification module are connected in series in the system via connectors or surge cells; The feeding module is used to input the target protein to be modified, the modifier, and the catalyst into the continuous reaction module; The continuous reaction module is used to continuously mix the liquid feed input from the feed module and perform a coupling reaction; The continuous quenching and loading module is used to add a quenching agent to the coupling solution to terminate the reaction and simultaneously transport the solution to the continuous purification module to complete the loading step. The continuous purification module is connected to the continuous quenching and loading module and is used to purify the modified protein solution. The method includes the following steps: a) A 5.0 mg / ml solution of the G-CSF to be modified is fed into the system via feed pump A at a constant flow rate of 0.75 mL / min; a 30 mg / ml mPEG-pALD solution is fed into the system via feed pump B at a constant flow rate of 0.72 mL / min; and a 1 M NaBH3CN catalyst solution is fed into the system via feed pump C at a constant flow rate of 0.03 mL / min. The method for preparing the NaBH3CN solution is as follows: dissolve NaBH3CN in DMSO to prepare a 1M stock solution; b) The solutions pumped in by feed pumps A, B and C are mixed at room temperature in the mixer of the continuous reaction module. After mixing, the mixture is directly pumped into the reactor of the continuous reaction module to continue the coupling reaction at 25±2℃ for 4.5 hours. c) After the coupling reaction is complete, use the D feed pump (for the coupling solution) and the E feed pump (for the quencher) to mix the quencher and coupling solution at a flow rate of 1.5 mL / min and a 1:1 ratio to terminate the reaction. Simultaneously, load the mixture into the continuous purification module at a loading rate of 17.3 g / L and then load it into column A. After the chromatographic loading is complete, start the online elution program and switch the loading column to the alternating chromatographic column B to continue continuous chromatographic loading. d) For the chromatography column that has been loaded with samples, start the following chromatography separation and purification procedure: i. System equilibration: Wash two column volumes with 50 mM sodium phosphate buffer, pH 5.0, at a linear velocity of 3.7 cm / min; ii. Isocratic elution 1: Elute 4.5 column volumes using 80 mM sodium phosphate buffer, pH 5.0, at a linear velocity of 3.7 cm / min. An automated chromatography program was used in this stage. Automated sample collection was initiated when the absorbance at A280 nm was >300 mAu, and collection was stopped when the absorbance at A280 nm was ≤100 mAu, capturing the target protein mPEG-G-CSF. iii. Isocratic elution 2: Continue eluting with 200 mM sodium phosphate buffer, pH 5.0, for 4 column volumes; collect the chromatographic peak with an absorption intensity >30 mAu at A280 nm, and recover the unreacted target protein raw material G-CSF; iv. Column regeneration: Elute 2 column volumes with 2M NaCl and 2 column volumes with 0.5M NaOH at a linear velocity of 3.7 cm / min; v. System rebalancing and sample loading: Use 50 mM sodium phosphate buffer, pH 5.0, to wash for 5 column volumes at a linear velocity of 3.7 cm / min to restore the chromatography column to a usable state and complete this cycle; Both column A and column B are cation chromatography columns; The quenching agent is a sodium acetate solution with a pH of 3.

0.

2. The method according to claim 1, characterized in that, The method further includes step e) feeding the recovered unmodified G-CSF back to the feed module.

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