A bifunctional small molecule PEG-modified recombinant humanized collagen and its preparation method

Abstract

This invention discloses a bifunctional small-molecule PEG-modified recombinant humanized collagen and its preparation method. The method includes the following steps: preparing a borate buffer solution containing sodium cyanoborohydride, pH 7.5-8.5; dissolving lyophilized recombinant collagen powder in the buffer solution to obtain a substrate solution; adding crosslinking agent powder to the substrate solution according to a molar ratio of free lysine to crosslinking agent of 1:(1.5-4.0); the crosslinking agent is a bifunctional small-molecule PEG with PEG as the backbone and groups at both ends that can couple with the amino groups of collagen; reacting at 4°C in the dark for 2-5 hours. This invention not only overcomes the bottleneck of anti-enzymatic stability but also transforms recombinant humanized collagen into a multi-arm macromolecular crosslinking substrate with highly active groups on its surface, providing key chemical bonding sites for its subsequent targeted coupling or anchoring to polymer networks such as hydrogels.

Description

Technical Field

[0001] This invention belongs to the field of recombinant collagen modification technology, specifically relating to a bifunctional small molecule PEG-modified recombinant humanized collagen and its preparation method. Background Technology

[0002] Recombinant humanized collagen has important applications in tissue engineering and regenerative medicine due to its excellent biocompatibility and extremely low immunogenicity. Among them, recombinant type XVII collagen, as a key transmembrane hemispheric structural protein, plays an irreplaceable physiological role in anchoring epidermal basal cells and maintaining the stability of the epidermal stem cell microenvironment. However, in vitro recombinant expression of type XVII humanized collagen has a unique atypical spatial conformation, with a high density of active residues (especially the free ε-amino group of lysine) exposed on the surface of its polypeptide backbone. This physicochemical characteristic makes it highly susceptible to non-specific self-assembly and aggregation under microenvironmental stress in aqueous environments. Simultaneously, the exposed amino active sites are extremely sensitive to endogenous proteases in the in vivo microenvironment, resulting in a very short in vivo half-life, which severely restricts its engineering and clinical translation.

[0003] PEGylation, a classic strategy for chemically modifying biomolecules using polyethylene glycol (PEG), is to mask enzyme cleavage sites and increase hydrodynamic radius to prolong half-life. However, in the field of recombinant collagen modification, traditional PEGylation processes face insurmountable limitations: (1) Chemical inertness caused by monofunctional modification: Existing technologies generally use monofunctional methoxy polyethylene glycol activated ester (mPEG-NHS). This molecule has only one reactive end. After modification, the collagen surface is wrapped with an inert methoxy end. This modified protein is chemically inert and cannot be further integrated into other biomaterial matrices (such as gelatin or chitosan hydrogel containing free amino groups) through covalent bonds. It is very easy to burst release in the early stage of implantation and cannot achieve seamless integration with tissue engineering scaffolds.

[0004] (2) Loss of activity due to steric hindrance: Conventional modifications often use high molecular weight PEG, whose huge hydrodynamic radius will produce a strong "umbrella effect", which completely masks the key receptor binding domain of recombinant type XVII collagen, causing it to lose its inherent biological function.

[0005] Given the serious deficiencies of the existing technology, there is an urgent need in the field to develop a completely new precision modification process. Summary of the Invention

[0006] Technical problem solved: To address the above-mentioned technical problems, this invention provides a bifunctional small molecule PEG-modified recombinant humanized collagen and its preparation method. This not only breaks through the bottleneck of anti-enzymatic stability, but also transforms the recombinant humanized collagen into a multi-arm macromolecular crosslinking substrate with highly active groups on its surface, providing key chemical bonding sites for its subsequent targeted coupling or anchoring to polymer networks such as hydrogels.

[0007] Technical solution: A method for preparing bifunctional small molecule PEG-modified recombinant humanized collagen, comprising the following steps: S1. Preparation of microenvironment system: Prepare a borate buffer solution containing sodium cyanoborohydride, control the pH to 7.5~8.5, and pre-cool it for later use; S2. Preparation of macromolecular critical untangling substrate: Take recombinant collagen lyophilized powder, add borate buffer to make up to volume and dissolve to obtain substrate solution; S3. Addition of cross-linking agent: According to the molar ratio of free lysine in recombinant collagen to cross-linking agent of 1: (1.5~4.0), take the dry powder of cross-linking agent and add it to the substrate solution; the cross-linking agent is: a bifunctional small molecule PEG with PEG as the backbone and both ends connected with groups that can undergo coupling reaction with the amino groups of collagen; S4. Isothermal kinetic coupling reaction: The reaction was carried out at 4℃ in the dark for 2-5 hours to obtain the bifunctional small molecule PEG-modified recombinant humanized collagen.

[0008] Preferably, in S1, the concentration of sodium cyanoborohydride is 50~150 mmol / L.

[0009] Preferably, in step S2, the dissolution temperature is 0~8℃.

[0010] Preferably, in step S2, the concentration of the substrate solution is 1.0~5.0 mg / mL.

[0011] Preferably, in step S3, the molar ratio of free lysine to cross-linking agent in the recombinant collagen is 1:2.

[0012] Preferably, in step S3, the crosslinking agent is NHS-PEG-NHS.

[0013] Preferably, in step S3, the molecular weight of the crosslinking agent is 600~5000 Da.

[0014] Preferably, in step S4, the reaction is mechanically stirred at a speed of 200 rpm.

[0015] Preferably, the recombinant collagen is recombinant type XVII collagen.

[0016] The bifunctional small molecule PEG-modified recombinant humanized collagen was prepared by the above method.

[0017] Beneficial Effects: This invention constructs a specially designed reducing buffer microenvironment and applies extremely strict stoichiometric control (precise control of the molar ratio of free amino groups in collagen to bifunctional PEG) and low-temperature thermodynamic control at 4°C. This ensures that the highly reactive groups at one end of the bifunctional PEG molecule are covalently anchored to the collagen surface, while utilizing the principle of kinetic excess, the highly reactive groups at the other end are protected from non-specific cross-linking and remain intact on the outer side of the collagen hydration layer. This invention not only overcomes the bottleneck of anti-enzymatic stability but also transforms recombinant type XVII collagen into a multi-armed macromolecular cross-linking substrate rich in highly reactive groups, providing key chemical bonding sites for its subsequent targeted coupling or anchoring to polymer networks such as hydrogels. Attached Figure Description

[0018] Figure 1 The images show the electrophoresis results of Example 1 and Comparative Example 1, where ① is the protein before PEG modification and ② is the protein after PEG modification. Figure 2 The graph shows the relative molecular mass results for Example 1 and Comparative Example 1; Figure 3 The HPLC quantitative detection results of highly active NHS groups in Example 1 and Comparative Example 1 are shown in the figure. Figure 4 The graph shows the relative molecular mass results for Example 1 and Comparative Examples 2-5; Figure 5 The HPLC quantitative detection results of highly active NHS groups in Examples 1 and Comparative Examples 2-5 are shown in the figure. Detailed Implementation

[0019] The present invention will be further described below with reference to the accompanying drawings and specific embodiments. Example 1

[0020] A method for preparing bifunctional small molecule PEG-modified recombinant humanized collagen includes the following steps: S1. Preparation of the microenvironment system: Mixing parameters: The lower limit of pH control for borate buffer is 7.5, the upper limit is 8.5, and the optimal ratio is pH 8.0. The lower limit of final concentration of sodium cyanoborohydride (NaCNBH3) is 50 mmol / L, the upper limit is 150 mmol / L, and the optimal ratio is 100 mmol / L.

[0021] Detailed instructions: 1. Using an analytical balance, accurately weigh 6.18 g of boric acid (H3BO3) and 9.53 g of sodium tetraborate decahydrate (Na2B4O7·10H2O), place them in a 1000 mL beaker, and add 800 mL of ultrapure water.

[0022] 2. Add a magnetic stir bar and stir until completely dissolved. Insert the calibrated pH meter electrode into the solution and add 1 mol / L HCl or 1 mol / L NaOH solution dropwise using a dropper while stirring until the reading stabilizes at 8.00.

[0023] 3. Transfer to a 1000 mL volumetric flask, rinse the beaker three times with ultrapure water, add ultrapure water dropwise to bring the volume to the 1000 mL mark, and shake well by inverting 10 times.

[0024] 4. Weigh 6.28 g of sodium cyanoborohydride (NaCNBH3) powder and add it to the solution. Stir magnetically for 10 minutes until completely dissolved. Filter the solution using a 0.22 μm microporous membrane and pre-cool it at 4°C for later use. S2. Preparation of macromolecular critical untangling substrates: Mixing parameters: The lower limit of recombinant collagen protein concentration is 1.0 mg / mL, the upper limit is 5.0 mg / mL, and the optimal ratio is 3.0 mg / mL. The lower limit of the dissolution temperature is 0℃, the upper limit is 8℃, and the optimal value is 4℃.

[0025] Detailed instructions: 1. Accurately weigh 30.0 mg of recombinant type XVII humanized collagen lyophilized powder (with a calibrated Lys residue mass percentage of 4.9%) using a microanalytical balance and place it in a 25 mL beaker.

[0026] 2. Add 8.0 mL of the above buffer solution pre-cooled to 4°C, place in a 4°C cold room, and stir with a magnetic stirrer at 150 rpm for 30 minutes until the lyophilized powder is completely dissolved.

[0027] 3. Transfer to a 10 mL volumetric flask, rinse the beaker three times with the same buffer solution, add buffer solution to bring the volume to the 10 mL mark, invert and mix well to obtain a substrate solution of 3 mg / mL.

[0028] S3. Crosslinking agent addition: Mixing parameters: In the reaction system, the ratio of the molar amount of free lysine from collagen to the molar amount of bifunctional PEG has a lower limit of 1:1.5, an upper limit of 1:4.0, and an optimal ratio of 1:2.

[0029] Detailed instructions: 1. The total mass of recombinant collagen in the system was recorded as 30.0 mg. Based on the 4.9% Lys mass percentage, the total mass of Lys in the system was calculated to be 1.47 mg (i.e., 10.05 μmol).

[0030] 2. Based on the optimal ratio of Lys to bifunctional small molecule PEG of 1:2, the total number of PEG moles required is calculated to be 20.10 μmol.

[0031] 3. Calculate the corresponding absolute mass based on the molecular weight. Accurately weigh the required mass of bifunctional small molecule PEG powder using a micro-analytical balance with an accuracy of 0.01 mg, and add it directly to the prepared 10 mL substrate solution.

[0032] S4. Isothermal kinetic coupling reaction: Process conditions: The optimal reaction temperature is 4℃; the optimal mechanical stirring speed is 200 rpm; the lower limit of the reaction time is 2 hours, the upper limit is 5 hours, and the optimal value is 3.0 hours.

[0033] Detailed instructions: Seal the container tightly with sealing film and place it in a 4°C constant temperature water bath, wrapping it with aluminum foil to protect it from light. Add a magnetic stir bar, set the speed to 200 rpm, and stir continuously at a constant speed for a precise 3.0 hours. After the reaction is complete, remove the container to obtain a bifunctional small molecule PEG-modified recombinant humanized collagen stock solution.

[0034] Reaction effect detection: 1. Macromolecular mobility and molecular weight (including reagent preparation and electrophoretic detection): Electrophoresis reagent preparation: Prepare 100 mL of 30% Acr / Bis (29:1) stock solution (containing 29.0 g Acr and 1.0 g Bis); prepare separating gel buffer (pH 8.8) and stacking gel buffer (pH 6.8); prepare 10% SDS solution and freshly prepared 10% APS solution. Prepare 500 mL of Coomassie Brilliant Blue staining solution (containing 0.25 g R-250, methanol:acetic acid:water = 4:1:5) and 1000 mL of destaining solution (methanol:acetic acid:water = 4:1:5).

[0035] Glass Plate Assembly and Gel Preparation: Clean and assemble the glass plate assembly. Take a 15 mL centrifuge tube and sequentially add 1.6 mL ultrapure water, 2.0 mL Acr / Bis stock solution, 1.3 mL separating gel buffer, 0.05 mL 10% SDS, 50 μL 10% APS, and 5 μL LTEMED. Mix well and inject into the glass plate. Add 1.0 mL of ultrapure water for liquid sealing and polymerize for 30 minutes. Remove the sealing solution and inject the prepared 5.1% stacking gel (containing 0.33 mL stock solution, 0.25 mL stacking gel buffer, etc.). Insert a 10-well comb and polymerize for 30 minutes.

[0036] Denaturation and Quantitative Loading: After BCA quantification, pipette samples containing 50 μg of absolute protein (both pre- and post-modification) into EP tubes, add 5×SDS loading buffer, and bring the volume to 15 μL with water. Heat in a 100°C dry metal bath for precisely 5.0 minutes. After centrifugation, use a microsyringe to slowly dispense the denatured sample into the bottom of the well.

[0037] Electrophoresis and staining / destaining: Set a constant voltage of 100V and run for 20 minutes to allow the sample to enter the separating gel. Increase the voltage to 200V and turn on the circulating water cooling. Stop the electrophoresis when the bromophenol blue band is 1.5 cm from the bottom edge. Peel off the gel and mark the center of the indicator with a fine copper wire. Place the gel in the staining solution and shake on a shaker (50 rpm) for 3.0 hours. After rinsing with distilled water, add destaining solution and change it once per hour (for a total of 4 times) until the background is transparent.

[0038] Relative migration rate (Rm) determination: The vertical distance from the bottom of the sample well to the mark on the thin copper wire and to the center of the protein band was measured. Substituting into the formula Rm = sample migration distance / dye migration distance. Results: The Rm of the control group (before modification) was 60.2%; the Rm of the experimental group (after bifunctional PEG modification) decreased significantly to 57.6%, definitively verifying the effective alteration of the hydrodynamic radius of macromolecules by the crosslinking agent.

[0039] See electrophoresis results Figure 1 ① is the protein before PEG modification: the relative migration rate is 60.2%; ② is the protein after PEG modification: the relative migration rate is 57.6%.

[0040] Calculation of relative molecular mass: In SDS-PAGE gel electrophoresis, the relative migration rate (Rm) of a protein is strictly linearly negatively correlated with the logarithm of its relative molecular mass (logMW) within a certain range.

[0041] This invention uses pre-stained markers of standard proteins with known molecular weights (covering the range of 15 kDa to 130 kDa) as a reference. The migration distance of each standard protein is measured, its Rm value is calculated, and a standard curve is plotted with Rm as the abscissa and logMW as the ordinate to obtain a linear regression equation. Then, the Rm values ​​of the experimental group and the control group are substituted into this equation to accurately calculate the apparent relative molecular mass of the recombinant collagen before and after modification.

[0042] Standard curve equation: Based on experimental determination and linear fitting, the standard curve equation for this electrophoresis system is: log(MW) = -1.500 × Rm + 5.681 (R²=0.998). See [link to standard curve equation]. Figure 2 .

[0043] Normal group (unmodified protein): Rm = 0.602 was measured, and logMW = 4.778 was calculated by substituting into the equation, corresponding to an apparent molecular weight of 60.0 kDa.

[0044] PEG-NHS group (bifunctional PEG modified): Rm = 0.576 was measured, and logMW = 4.817 was calculated by substituting into the equation, corresponding to an apparent molecular weight of 65.6 kDa.

[0045] Conclusion: The apparent molecular weight increased by 5.6 kDa. Considering the molecular weight of the cross-linking agent used and the hydrodynamic expansion effect of the PEG hydration layer, this data confirms that the bifunctional PEG was successfully and moderately covalently anchored to the collagen surface without severe over-crosslinking.

[0046] 2. High NHS content in the product: Pyrolysis-High Performance Liquid Chromatography (HPLC) Combined Quantitative Method: In order to accurately quantify the content of NHS activated ester groups remaining on the surface of modified collagen, this invention adopts a specific chemical pyrolysis combined with HPLC detection scheme.

[0047] Principle: The purified PEGylated collagen product is extracted, and an excess of neutral hydroxylamine hydrochloride solution is added. Hydroxylamine hydrochloride can instantly cleave the unreacted NHS activated esters on the periphery of the product, releasing free N-hydroxysuccinimide (NHS) molecules.

[0048] Procedure: After 30 minutes of lysis reaction, remove large protein molecules by centrifugation using a 10 kDa ultrafiltration tube and collect the filtrate. Inject the filtrate into an HPLC system (equipped with a C18 reversed-phase column), and quantitatively detect the concentration of released free NHS at a UV detection wavelength of 260 nm (quantitatively using a pre-plotted pure NHS standard curve).

[0049] Activity retention rate calculation: High activity NHS retention rate (%) = (number of moles of free NHS released / number of moles of PEG bound to the protein) × 100%.

[0050] Experimental results: HPLC quantitative analysis showed that the experimental group prepared using the method of this invention at 4℃ and a 1:2 feed ratio exhibited a high retention rate of 82.5% for highly active NHS groups on its surface. This strongly demonstrates that the process of this invention successfully inhibits the hydrolysis of the crosslinking agent and internal self-crosslinking, perfectly realizing the design intent of "single-end anchoring and single-end free crosslinking".

[0051] In summary, this embodiment used homologous bifunctional small molecule PEG (molecular weight 2 kDa), with a collagen amino group to PEG molar ratio of 1:2, a reaction temperature of 4°C, and a buffer system of pH 8.0 borate buffer containing 100 mM NaCNBH3. The results showed that the product had an apparent molecular weight of 65.6 kDa, with a single and sharp electrophoretic band (no high molecular weight polymers); the lysis-HPLC method determined that its high-activity NHS retention rate was 82.5%. Subsequent secondary crosslinking and gelation ability was excellent. Comparative Example 1

[0052] This comparative example utilizes existing technology: based on conventional macromolecular monofunctional mPEG-NHS, indiscriminate PEGylation of protein substrates in phosphate-buffered saline (PBS), mainly including the following four stages: Substrate dispersion and buffer system construction stage: Recombinant collagen was directly dissolved in PBS solution at physiological pH. This system only provides acid-base buffering and does not introduce mild reducing agents such as NaCNBH3. It cannot maintain the reduction potential of the protein's spatial conformation under subsequent mechanical shear forces, which can easily lead to trace mismatches and aggregation of free groups within the protein molecule.

[0053] High molar ratio crosslinking agent feeding stage: The system introduces a single-functional macromolecule mPEG-NHS. Due to the low diffusion coefficient and huge steric hindrance of macromolecules in solution, in order to ensure sufficient modification conversion rate, existing technologies often adopt a crude ultra-high saturation feeding method (the molar ratio of PEG to amino is usually much higher than 5:1).

[0054] Room temperature random coupling reaction stage: In a room temperature environment of 20℃~25℃, the thermodynamic potential energy is relatively high. Three parallel reactions occur in this stage: (1) The activated ester group of mPEG-NHS undergoes disordered nucleophilic substitution with the amino group on the protein surface; (2) Due to the room temperature aqueous environment, a large amount of PEG-NHS activated ester undergoes spontaneous hydrolysis and deactivation, releasing acidic small molecules that cause local pH fluctuations; (3) Some collagen in a critically unstable state undergoes irreversible aggregation.

[0055] Separation, purification, and final product formation: After the reaction, the system exhibits a highly viscous and complex mixed state. Long-term dialysis or multiple ultrafiltration washes are necessary to remove extremely high concentrations of hydrolyzed PEG waste. The final modified product is only covered by a monofunctional inert methoxy group (-OCH3), completely losing its reactivity for further chemical cross-linking, and its large peripheral volume significantly hinders the spatial binding of the protein to the target cell receptor.

[0056] Regarding the problems observed in Comparative Example 1, Embodiment 1 of the present invention provides a targeted solution: 1. In Comparative Example 1, the use of monofunctional modification resulted in a chemically inert protein surface, making covalent integration with the tissue engineering matrix impossible and easily triggering burst release. In Example 1, the structure of the cross-linking agent molecular groups was fundamentally altered: Current technologies commonly employ monofunctional macromolecular activated esters (mPEG-NHS) to modify recombinant collagen. Essentially, this involves utilizing the single active group at the PEG chain end to bind to the protein. After modification, the protein surface is coated with a completely inert methoxy group (-OCH3). When this inert modified protein is applied in downstream tissue engineering (such as wound dressings and medical gel matrices), due to the lack of reactive chemical anchors, it can only exist in the matrix network in a physically blended form. In the complex in vivo fluid environment, this physical encapsulation easily leads to the rapid release and diffusion of collagen from the matrix (i.e., the "burst release effect"), making it impossible to achieve long-term targeted sustained release and continuous regulation of the tissue microenvironment.

[0057] This invention innovatively introduces "homologous bifunctional small molecule PEG succinimide ester (NHS-PEG-NHS)," a cross-linking agent with high activity at both ends, and precisely locks the molar ratio of free lysine from collagen to this bifunctional cross-linking agent at 1:2. Through this kinetic excess principle, one end of the bifunctional PEG (NHS) is covalently anchored to the protein backbone, while the other end (NHS), due to its spatial occupancy, avoids internal cross-linking, remaining intact and extending towards the aqueous phase. This method completely breaks through the inert barrier of traditional modifications, successfully transforming recombinant type XVII collagen into a "multi-arm macromolecular cross-linking substrate rich in highly active NHS groups," enabling it to undergo secondary covalent cross-linking with subsequent amino-containing matrices (such as methacrylamide gelatin, chitosan hydrogel, etc.), achieving a strong molecular-level bond with the matrix material and completely eliminating the shortcomings of burst release.

[0058] 2. In Comparative Example 1, the large molecular weight and ultra-high molar ratio triggered a strong "protective umbrella effect," leading to a severe loss of biological activity in the recombinant type XVII collagen. Example 1 carefully selected the molecular size and precisely locked the stoichiometric ratio: To compensate for the high hydrolysis rate of NHS groups at room temperature and pursue a long half-life, current technologies often employ high molecular weight PEG (e.g., 10kDa~20kDa) in extremely excessive amounts at a ratio of 5:1 to 10:1 or higher. Recombinant type XVII collagen, as a highly functional transmembrane hemidesmosome protein, has a surface densely packed with specific receptor-binding domains. The large hydrodynamic radius and high-density random modification of high molecular weight PEG form an extremely dense hydration shield around the protein (i.e., the "steric hindrance / umbrella effect"). While this blocks protease degradation, it also completely blocks the spatial recognition of collagen with target cell membrane receptors, resulting in a physiologically inactive "dead molecule" after modification.

[0059] This invention abandons traditional high-molecular-weight cross-linking agents and selects "bifunctional small-molecule PEG". Its shorter molecular chains provide adequate hydration protection without excessive shielding. The relatively short PEG chains construct a moderately hydrated layer on the protein surface. The thickness of this hydration layer is sufficient to block large-molecule proteases (such as collagenase) from approaching the backbone through steric hindrance, thereby significantly extending the half-life. However, this steric barrier is insufficient to prevent the specific recognition and binding of collagen functional domains to receptors such as integrins on cell membranes. Simultaneously, relying on a 1:2 limiting stoichiometric ratio, the modification density is precisely limited, ensuring that only single-chain coupling occurs at each lysine site, preventing the formation of multi-molecular cross-linking networks (cross-linking gelation). This achieves "point-to-point" modification of lysine residues on the collagen surface, rather than "network coating". This method effectively avoids the dense steric shielding (i.e., the "umbrella effect") produced by traditional large-volume, high-density PEGylation, significantly improving the protein's resistance to endogenous enzymatic degradation while perfectly preserving the core biorecognition activity of recombinant type XVII collagen.

[0060] 3. Comparative Example 1, with its room temperature and lack of reducing potential microenvironment, resulted in non-specific aggregation and extremely high hydrolytic loss of the crosslinking agent. Example 1, however, employed a specially designed redox microenvironment and extremely low-temperature thermodynamics for dual regulation. Existing techniques mostly involve disordered coupling reactions in conventional phosphate-buffered saline (PBS) buffer systems at room temperature (20℃~25℃). Under these conditions and with the shear force of mechanical stirring, recombinant type XVII collagen is highly susceptible to trace non-specific mismatches of intermolecular free groups (such as Schiff base crosslinking), leading to irreversible aggregation. Furthermore, NHS-activated esters have extremely short half-lives in aqueous phases at room temperature and are prone to spontaneous hydrolysis. This not only consumes large quantities of expensive crosslinking agents but also generates a large amount of acidic hydrolysis byproducts that severely disrupt the system's pH, resulting in extremely poor modification uniformity.

[0061] This invention addresses the thermodynamics and microenvironmental regulation of the reaction by constructing a borate buffer (pH 8.0) containing 100 mmol / L NaCNBH3 and applying strict low-temperature control at 4°C. The 4°C thermodynamic condition significantly raises the activation energy threshold of the NHS hydrolysis side reaction, greatly suppressing the spontaneous hydrolysis loss of expensive crosslinking agents in the aqueous phase, avoiding drastic pH fluctuations in the microenvironment, and returning reaction dominance to the nucleophilic substitution of amino groups. Simultaneously, the slightly alkaline NaCNBH3 acts as a mild reducing agent, precisely maintaining the redox dynamic balance of the system, effectively quenching free radicals, and completely preventing intermolecular mismatches and non-specific aggregation of recombinant proteins. This ensures high monomeric dispersibility and batch-to-batch uniformity of the modified products, significantly reducing the difficulty and cost of subsequent purification. Comparative Example 2

[0062] Compared to Example 1, this comparative example only changed the ratio of amino groups to PEG to 1:1.5 (due to insufficient PEG). The results showed that the apparent molecular weight of the product increased to 72.3 kDa, and numerous high-molecular-weight tails appeared in the electrophoresis pattern (indicating intermolecular cross-linking of two collagen molecules simultaneously at both ends of the bifunctional PEG). The retention rate of highly active NHS decreased to 51.3%, and the monomer dispersibility of the product decreased. Comparative Example 3

[0063] Compared to Example 1, this comparative example only changed the reaction temperature to the conventional room temperature of 25°C. The test results showed that the apparent molecular weight of the product was 64.1 kDa. Due to the rapid hydrolysis of the NHS-activated ester in the aqueous phase at room temperature, the retention rate of highly active NHS determined by pyrolysis-HPLC was only 15.2%. The product essentially lost its "secondary cross-linking" ability, verifying the absolute necessity of low-temperature control at 4°C. Comparative Example 4

[0064] This comparative example differs from Example 1 only in that the molecular weight of the crosslinking agent was changed to 5 kDa. The results showed that the product had an apparent molecular weight of 75.1 kDa and a single band on electrophoresis. The high-activity NHS retention rate was 78.4%. However, due to the increased hydrodynamic radius, cell receptor binding experiments showed that its biological activity was slightly lower than that of the product in Example 1 (2 kDa). This demonstrates that 2 kDa is the optimal solution for balancing activity and stability, while 5 kDa is an acceptable upper limit. Comparative Example 5

[0065] Compared to Example 1, this comparative example only replaced the buffer system with conventional PBS (pH 7.4) without a reducing agent. The results showed that the apparent molecular weight of the product was 65.0 kDa, but the electrophoresis pattern showed a high molecular weight cutoff band near the sample well. Due to the lack of the reducing protective potential of NaCNBH3, the protein underwent non-specific aggregation under mechanical shearing, resulting in significantly lower product yield and purity than in Example 1.

Claims

1. A method for preparing bifunctional small molecule PEG-modified recombinant humanized collagen, characterized in that, The steps include the following: S1. Preparation of microenvironment system: Prepare a borate buffer solution containing sodium cyanoborohydride, control the pH to 7.5~8.5, and pre-cool it for later use; S2. Preparation of macromolecular critical untangling substrate: Take recombinant collagen lyophilized powder, add borate buffer to make up to volume and dissolve to obtain substrate solution; S3. Addition of cross-linking agent: According to the molar ratio of free lysine in recombinant collagen to cross-linking agent of 1: (1.5~4.0), take the dry powder of cross-linking agent and add it to the substrate solution; the cross-linking agent is: a bifunctional small molecule PEG with PEG as the backbone and both ends connected with groups that can undergo coupling reaction with the amino groups of collagen; S4. Isothermal kinetic coupling reaction: The reaction was carried out at 4℃ in the dark for 2-5 hours to obtain the bifunctional small molecule PEG-modified recombinant humanized collagen.

2. The method for preparing a bifunctional small molecule PEG-modified recombinant humanized collagen according to claim 1, characterized in that, In S1, the concentration of sodium cyanoborohydride is 50~150 mmol / L.

3. The method for preparing a bifunctional small molecule PEG-modified recombinant humanized collagen according to claim 1, characterized in that, In S2, the dissolution temperature is 0~8℃.

4. The method for preparing a bifunctional small molecule PEG-modified recombinant humanized collagen according to claim 1, characterized in that, In S2, the concentration of the substrate solution is 1.0~5.0 mg / mL.

5. The method for preparing a bifunctional small molecule PEG-modified recombinant humanized collagen according to claim 1, characterized in that, In S3, the molar ratio of free lysine to cross-linking agent in recombinant collagen is 1:

2.

6. The method for preparing a bifunctional small molecule PEG-modified recombinant humanized collagen according to claim 1, characterized in that, In S3, the crosslinking agent is NHS-PEG-NHS.

7. The method for preparing a bifunctional small molecule PEG-modified recombinant humanized collagen according to claim 1, characterized in that, In S3, the molecular weight of the crosslinking agent is 600~5000 Da.

8. The method for preparing a bifunctional small molecule PEG-modified recombinant humanized collagen according to claim 1, characterized in that, In step S4, the reaction is mechanically stirred at a speed of 200 rpm.

9. The method for preparing a bifunctional small molecule PEG-modified recombinant humanized collagen according to claim 1, characterized in that, The recombinant collagen used is recombinant type XVII collagen.

10. Recombinant humanized collagen modified with bifunctional small molecule PEG, prepared by the method according to any one of claims 1 to 9.

Images