Method suitable for recombinant collagen purification process development

Abstract

The invention discloses a method suitable for developing a recombinant collagen purification process, the method has the advantages of strong universality, high efficiency, convenience and strong guidance quality, the process developed by the method has high batch compatibility to feed liquid and good stability, the process obtained by a small-scale test can be stably transited to a pilot-scale test amplification process, and the method is suitable for large-scale production. The purification process development of different types of recombinant collagen can be met.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a method suitable for developing a purification process for recombinant collagen. Background Technology

[0002] Collagen, as a natural polymer, not only possesses excellent biocompatibility, degradability, and low immunogenicity, but also exhibits advantages such as high tensile strength and hemostatic function. Recombinant humanized collagen is derived from the original human collagen sequence. Highly water-soluble and bioactive portions are selected and recombined or tandemly repeated, with codon optimization to obtain the recombinant humanized collagen sequence. Large-scale production can be achieved by selecting suitable hosts and fermentation technologies. Increasing research confirms that recombinant humanized collagen has good water solubility and high bioactivity, outperforming natural human collagen, and has broad application prospects in biomedical materials, cosmetics, and health foods.

[0003] However, recombinant collagen is easily degraded by host proteases during fermentation, producing a series of collagen fragments of different molecular weights. These protein fragments usually have similar physical and chemical properties to the target protein, making them difficult to remove by simple affinity adsorption. This greatly increases the difficulty and cost of purification and limits its further industrial application.

[0004] Therefore, any recombinant collagen requires purification before use. Purification methods for each type of collagen need to be explored, and suitable purification conditions need to be determined. Currently, the process of exploring and establishing purification methods for recombinant collagen is extremely complex. Researchers can only refer to some directional guidelines; how to implement each specific step requires repeated exploration to determine.

[0005] It should be noted that the information disclosed in this background section is only for understanding the background technology of this application concept, and therefore, the above description is not considered to constitute prior art information. Summary of the Invention

[0006] To address the aforementioned problems, this invention aims to provide a universal and rapid method for developing collagen purification processes, thereby solving the issues of slow, time-consuming, labor-intensive, and inefficient development of existing collagen purification processes.

[0007] This invention provides a method for developing a collagen purification process, the method comprising the following steps: (1) Determine the filler based on collagen sequence information and isoelectric point; (2) Based on the protein characteristics, different NaCl concentrations of the first buffer were used for pH and conductivity bilinear gradient purification. The pH corresponding to when the target protein was completely eluted was found by the SDS-PAGE results. The pH of the first buffer was 3-8. (3) Provide a second buffer solution with a constant pH single-electro-linear gradient according to the pH determined above, wherein the pH of the second buffer solution is the pH selected in step (2); (4) Use the second buffer to perform constant pH single-wire linear gradient purification. After purification, find the conductivity values ​​Y1 and Y2 corresponding to when the impurity protein and the target protein are completely eluted according to the SDS-PAGE results, and calculate the corresponding salt concentrations of the third buffer C1≤Y1 and the fourth buffer C2≥Y2 for washing. (5) Prepare third and fourth buffer solutions with different NaCl concentrations according to the salt concentration determined in step (4), and purify the protein. The third buffer solution is used for washing impurities, and the fourth buffer solution is used for elution. The pH of the above buffer solutions is the pH determined in step (2). After purification, the purification process is determined according to the SDS-PAGE results. (6) If no obvious separation and purification effect is observed after steps (2) to (5), other packing materials need to be replaced and steps (2) to (5) are repeated until a suitable packing material is selected and subsequent steps are optimized.

[0008] Bilinear gradient purification based on pH and conductivity is a chromatography technique that combines pH gradient and conductivity regulation. By simultaneously adjusting the pH and conductivity of the buffer solution, it optimizes the separation of proteins (such as bispecific antibodies). The main principle of pH regulation is to utilize the differences in the charge state of proteins at different pH values. By continuously changing the pH, it adjusts the protein's interaction with the ion-exchange packing material, achieving highly selective separation. Conductivity reflects the ion concentration in the buffer solution and affects the electrostatic interaction between the protein and the packing material.

[0009] Correspondingly, constant pH single-electrode linear gradient purification is carried out at a constant pH.

[0010] Furthermore, the selection principle for the packing material in step (1) is as follows: If collagen carries a positive charge at physiological pH (pI>7.0), cationic fillers should be tried first; if collagen carries a negative charge at physiological pH (pI<7.0), anionic fillers should be tried first; if separation cannot be achieved after trying all ionic fillers, hydrophobic fillers should be tried.

[0011] The above selection is based on the stability of collagen under acidic conditions. Those skilled in the art can choose commercially available conventional fillers, including weakly cationic, strongly cationic, and multi-mode cationic fillers such as CM, SP / CD-S, and MMC fillers; weakly anionic, strongly anionic, and multi-mode anionic fillers such as DEAE Sepharose FF, QSepharose, and Capto adhere; and hydrophobic fillers such as phenyl and butyl fillers.

[0012] Furthermore, the elution volume in step (2) is 20~25 CV.

[0013] Furthermore, when using ionic packing material: the first buffer solution is prepared from the following components in the following concentration ratios: Buffer A1: 20 mM NaAC, 50 mM NaH2PO4, 50 mM Na2HPO4, 0 M NaCl, pH 3.0 Buffer B1: 20 mM NaAC, 50 mM NaH2PO4, 50 mM Na2HPO4, 1 M NaCl, pH 8.0.

[0014] The buffer solution provided in this application is a solution obtained by the inventors through repeated experiments. It requires no adjustment and can be used directly, making it quick and convenient.

[0015] Furthermore, when hydrophobic packing material is used, the first buffer solution is prepared by the following components in the following concentration ratio: Formula A R : 20mM NaAC, 50mM NaH2PO4, 50mM Na2HPO4, 2M (NH4)2SO4 / NaCl, pH3.0, Buffer B R : 20 mM NaAC, 50 mM NaH2PO4, 50 mM Na2HPO4, 0 M (NH4)2SO4 / NaCl, pH8.0.

[0016] Furthermore, the second buffer solution is prepared according to the following concentration ratio: Buffer A2: 20 mM NaAC, 50 mM NaH2PO4, 50 mM Na2HPO4, 0 M NaCl, adjusted to the selected pH; Buffer B2: 20 mM NaAC, 50 mM NaH2PO4, 50 mM Na2HPO4, 1 M NaCl, adjusted to the selected pH.

[0017] Further, the third buffer solution is prepared according to the following concentration ratio: 20 mM NaAC, 50 mM NaH2PO4, 50 mM Na2HPO4, C1M NaCl, adjusted to the selected pH. Further, C1 can be finely adjusted within the range of 0.05~0.1M.

[0018] Further, the fourth buffer solution is prepared according to the following concentration ratio: 20 mM NaAC, 50 mM NaH2PO4, 50 mM Na2HPO4, and C2M NaCl, wherein the concentration of NaCl is higher than the salt concentration corresponding to the Y2 conductivity. The elution salt concentration (C2) does not need to be optimized in principle. As long as the impurities are eluted cleanly, any salt concentration higher than the Y2 conductivity can be selected for elution. The lower the salt concentration, the longer the elution time required, which can be determined according to the actual situation.

[0019] Furthermore, the method also includes step (6) fine-tuning the third buffer solution, where the NaCl concentration is fine-tuned within the range of 0.05~0.1 M. After fine-tuning the washing concentration in step (6), the final pilot-scale process can generally be obtained. However, when converting the pilot-scale process to the pilot-scale process, due to changes in the height and diameter of the packing column, further fine-tuning is required.

[0020] Beneficial effects This invention patent provides a method for developing purification processes for recombinant collagen. This method has the advantage of high versatility, helping researchers quickly screen suitable chromatographic packing materials and buffer combinations for different collagen purification processes, and can meet the purification process development needs of different types of recombinant collagen. It also has the advantages of high efficiency, convenience, and strong guidance; after selecting the packing material, high-purity target protein can be obtained through 2-3 purification experiments. Based on the results of the first two steps, the gradient elution optimization experiment scheme can be roughly determined, greatly reducing the researchers' sense of uncertainty and helplessness when adjusting the process. Moreover, the process developed by this method has high batch compatibility and good stability, and the process obtained in the small-scale test can be stably transitioned to the pilot-scale amplification process. Attached Figure Description

[0021] Figure 1 Results of collagen CIMM-COL17-11 purification by bilinear gradient elution at pH 3.0~8.0 and 0~1 M pH conductivity. Figure 2 Results of collagen CIMM-COL17-11 purification by bilinear gradient elution at pH 3.0~6.0 and 0~1 M pH conductivity. Figure 3 Results of collagen CIMM-COL17-11 purification by 0-1M single-electrode linear gradient elution at pH 4.5. Figure 4 Collagen CIMM-COL17-11 pH 4.5 - Washing Optimization - 0.25M - 15CV Purification Results Figure 5 Collagen CIMM-COL17-11 pH 4.5 - Washing Optimization - 0.30 M - 15CV Purification Results Figure 6 Collagen CIMM-COL17-11 pH 4.5 - Washing Optimization - 0.35M - 15CV Purification Results Figure 7 Collagen CIMM-COL17-11 pH4.5-NaAC-PB-Maximum Loading Capacity Test Purification Results Figure 8 Collagen CIMM-COL17-11 pH 4.5 - Loading volume optimization - 40 mg / mL purification results Figure 9 Collagen CIMM-COL17-11 pH 4.5 - Loading volume optimization - 30 mg / mL purification results Figure 10 Collagen CIMM-COL17-11 Process Validation and Purification Results Figure 11 Results of CIMM-COL17-11 process for the amplification and purification of collagen Figure 12 Results of purification of collagen CIMM-COL3A1 by bilinear gradient elution at pH 3.0~8.0 and 0~1M. Figure 13 Results of purification of collagen CIMM-COL3A1 by pH 8.0, 0-1M single-electrode linear gradient elution. Figure 14 Results of purification of collagen CIMM-COL1A1-8-His6 by bilinear gradient elution at pH 3.0~8.0 and 0~1 M pH conductivity. Figure 15 Results of purification of collagen CIMM-COL1A1-8-His6 by pH 8.0, 0-1M single-electrode linear gradient elution. Figure 16 Results of purification of collagen CIMM-COL1A1-9-His6 by bilinear gradient elution at pH 3.0~8.0 and 0~1 M pH conductivity. Figure 17 Results of purification of collagen CIMM-COL1A1-9-His6 by 0-1M single-electrode linear gradient elution at pH 8.0. Figure 18Results of purification of collagen CIMM-COL1A1-9-His6 by bilinear gradient elution at pH 3.0~6.0 and 0~1M pH conductivity Figure 19 Results of purification of collagen CIMM-COL1A1-9-His using a single-electrode linear gradient elution at pH 5.0, 0–1 M. Figure 20 Results of purification of collagen CIMM-COL3A1-2 by bilinear gradient elution at pH 3.0~8.0 and 2~0 M pH. Figure 21 Results of collagen CIMM-COL3A1-2 purification by elution using a 2-0 M single-electrode linear gradient at pH 7.0. Detailed Implementation

[0022] The following specific examples illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention. It should be noted that the process equipment or apparatus not specifically specified in the following embodiments are all conventional equipment or apparatus in the art. Furthermore, it should be understood that one or more method steps mentioned in the present invention do not exclude the existence of other method steps before or after the combined steps, or the insertion of other method steps between these explicitly mentioned steps, unless otherwise stated; it should also be understood that the combined connection relationship between one or more devices / apparatus mentioned in the present invention does not exclude the existence of other devices / apparatus before or after the combined devices / apparatus, or the insertion of other devices / apparatus between these explicitly mentioned two devices / apparatus, unless otherwise stated. Moreover, unless otherwise stated, the numbering of each method step is only a convenient tool for identifying each method step, and not for limiting the order of the method steps or limiting the scope of the present invention. Changes or adjustments to their relative relationships, without substantially altering the technical content, should also be considered within the scope of the present invention.

[0023] Material: Collagen CIMM-COL17-11, amino acid sequence referenced from Chinese patent application number 2025107414665, "Recombinant Humanized Collagen and its Synthesis Method and Application". The amino acid sequence of collagen CIMM-COL3A1 is referenced in Chinese Patent Application No. 2024117088441, entitled "High Molecular Weight Recombinant Humanized Collagen and Its Synthesis Method and Application". The amino acid sequence of collagen CIMM-COL1A1-8 is referenced in Chinese Patent Application No. 2024117088441, entitled "Recombinant Humanized Collagen and Its Synthesis Method and Application". The amino acid sequence of collagen CIMM-COL1A1-9 is referenced in Chinese Patent Application No. 2024117088441, entitled "Recombinant Humanized Collagen and Its Synthesis Method and Application". The amino acid sequence of collagen CIMM-COL3A1-2 is as follows: 373 aa SEQ ID NO.1 PGGPGSDGKPGPPGSQGESGRPGPPGSGPRGQPGVMGFPGPKGNDGAPGKNGERGGPGGPGPQGPPGKNGETGPQGPPGPTGPGGDKGDTGPPGPQGLQGLPGTGGPPGENGKPGEPGPKGDAGAPGAPGGKGDAGAPGERGPPGLAGAPGLRGGAGPPGPEGGKGAAGPPGPPGAAGTPGLQGM PGERGLGSPGPKGDKGEPGGPGADGVPGKDGPGPPTGPIGPPGPAGQPGDKGEGGAPGLPGIAGPRGSPGERGETGPPGPAGFPGAPGQNGEPGGKGERGAPGEKGEGGPPGVAGPPGGSGPAGPPGPQGVKGERGSPGGPGAAGFPGARGLPGPPGSNGNPGPPGPSGSPGKDGPPGPAGNTGAP The nucleotide sequence is as follows: 1119 bp SEQ ID NO.2 Example 1: Fermentation of Pichia pastoris in a 5 L fermenter The yeast fermentation process can be implemented by those skilled in the art with reference to existing technologies. The following is the fermentation process flow of this application: Strike Pichia pastoris SMD1168H / pPIC9KM-CIMM-collagen at a concentration of 0.3 mg / mL. −1 On G418 YPD plates, incubate at 30°C for 72 h, then pick a single colony and inoculate it into 50 mL BMG medium and continue incubation until OD. 600 The OD reached 4.0–6.0, and after inoculation at a rate of 10%, it was transferred to 100 mL of BMG medium and cultured until the OD reached 8.0–12.0. The above secondary seed culture was then inoculated into a 5 L fermenter, and 4.35 mL·L⁻¹ of BMG medium was added simultaneously. −1 PTM 1 trace elements and 4.35 mL·L −1 Initiate fermentation with 0.02% biotin. When DO rapidly recovers to 50-70%, begin glycerol feeding at a rate of 10-20 mL / h. −1 ·L −1 The DO content was maintained at 25-30% by fine-tuning the glycerol feed rate. Feeding was stopped after 5-6 hours, and the mixture was "starved" for 30-60 minutes before methanol induction began. The methanol flow rate was 0.5-7.2 mL / h. −1 ·L −1 The induction temperature was 28℃, and samples were taken during the process to detect the wet weight of the bacterial cells and the protein concentration.

[0024] Seed culture medium BMG (1 L): 10 g glycerol, 2.3 g K2HPO4, 11.81 g KH2PO4, bring to a final volume of 900 mL, sterilize at 121℃ for 15 min, cool, and then add 100 mL 13.4% YNB and 2 mL 0.02% biotin. Fermentation medium BSM (1 L): 80 g glycerol, 26.7 mL H3PO4, 1.175 g CaSO4, 18.2 g K2SO4, 7.28 g MgSO4, 4.13 g KOH, dissolved in water and brought to a final volume of 2000 mL. After sterilization, add 4.35 mL PTM1. Fed growth medium: 50% (w / v) glycerol (containing 12 mL·L⁻¹) −1 PTM1) Fermentation induction medium: 100% methanol (containing 12 mL·L⁻¹) −1 PTM1).

[0025] PTM1 ingredients (1 L): CuSO4·5H2O 6.0 g, NaI 0.08 g, MnSO4·H2O 3.0 g, Na2MoO4·2H2O 0.2 g, H3BO3 0.02 g, CoCl2 0.5 g, ZnCl2 20.0 g, FeSO4·7H2O 65.0 g, H2SO4 5 mL·L −1 Biotin 0.2 g, filtered to sterilize, stored at 4℃ for later use.

[0026] Example 2: Process Development for Purification of Type XVII Collagen CIMM-COL17-11 Using MMC Columns (1) Determining buffer composition and initial purification conditions by pH and conductivity bilinear gradient elution The sequence composition of recombinant collagen determines its charge and isoelectric point, which affects its binding efficiency on different chromatographic columns. Therefore, the first step is to determine the initial purification buffer combination using two buffer combinations, Combination 1 and Combination 2. Generally, Combination 1 is preferred as it has a wide pH range (3.0 to 8.0) and a superimposed conductive gradient (0.3–1 M), which can achieve near-complete elution of collagen. However, if the elution rate is too fast and no significant purification effect is obtained, then Combination 2 should be tried. The specific purification steps are as follows: Combination 1: Buffer A: 20mM NaAC, 50mM NaH2PO4, 50mM Na2HPO4, 0M NaCl, pH 3.0 Buffer B: 20mM NaAC, 50mM NaH2PO4, 50mM Na2HPO4, 1M NaCl, pH 8.0 Buffer C: 0.5 M NaOH Combination 2: Buffer A: 20mM NaAC, 50mM NaH2PO4, 50mM Na2HPO4, 0M NaCl, pH 3.0 Buffer B: 20mM NaAC, 50mM NaH2PO4, 50mM Na2HPO4, 1M NaCl, pH6.0 Buffer C: 0.5 M NaOH Buffer was prepared according to Combination 1, and purification was performed using the Suzhou Saipu SCG-P100-V4 purification system. The column was an MMC column (4.7 mL pre-packed column, purchased from BorgLon (Zhejiang) Biotechnology Co., Ltd.). First, the column was equilibrated with 5 mL of CV Buffer A. After equilibration, the sample was loaded (total protein loading volume was 30 mg / mL of packing material) at a flow rate of 1 mL / min. After loading, the column was washed with Buffer A until the A was reached. 210 Once the concentration dropped below 100 mAU, a linear gradient of 0 to 100% Buffer B was set for elution, with an elution volume of 20 CV. A final 5 CV elution with Buffer B was performed, during which the eluent was collected. Protein purity was assessed using SDS-PAGE. After purification, CIP washing with 2 CV Buffer C was performed. The SDS-PAGE results are shown below. Figure 1 As shown, the target protein (theoretical molecular weight 8.2 kDa, migration factor 1.4, actually corresponding to a band of about 15 kDa) was completely eluted at pH 3.8~5.5, and no obvious separation from contaminating proteins (or degradation bands) was observed. Therefore, for CIMM-COL17-11, the elution power of buffer combination 1 is too strong, and buffer combination 2 can be used for a second purification attempt.

[0027] Buffer was prepared according to Combination 2, and purification was performed using the Suzhou Saipu SCG-P100-V4 purification system. The column was an MMC column (4.7 mL pre-packed column, purchased from Borglon (Zhejiang) Biotechnology Co., Ltd.). First, the column was equilibrated with 5 mL of CV Buffer A. After equilibration, the sample was loaded (total protein loading volume was 30 mg / mL of packing material) at a flow rate of 1 mL / min. After loading, the column was washed with Buffer A until the A was reached. 210 Once the concentration dropped below 100 mAU, a linear gradient of 0 to 100% Buffer B was set for elution, with an elution volume of 20 CV. A final 5 CV elution with Buffer B was performed, during which the eluent was collected. Protein purity was assessed using SDS-PAGE. After purification, CIP washing with 2 CV Buffer C was performed. The SDS-PAGE results are shown below. Figure 2 As shown, the target protein was completely eluted at pH 4.5. During purification, it was observed that it had a significant separation effect from impurity proteins (or degradation bands), with a protein purity of 99.7% (Table 1) and a recovery rate of 19.2%.

[0028] Although linear elution is important for initial process development, isocratic elution is generally preferred for process scale-up and stabilization. The latter has lower requirements for equipment precision and is more stable between batches. Therefore, we need to derive the conditions for further optimization based on the results of bilinear gradient elution and obtain a stable scale-up isocratic gradient elution process through step-by-step adjustments.

[0029] Based on the purification results of the previous step ( Figure 2 For the target protein CIMM-COL17-11, it can be completely eluted at pH 4.5, and the conductivity range of the eluted sample at pH 3.8~4.5 is 37.8~54.3 mS / cm. This means that if a constant pH of 4.5 is selected, a single conductive linear gradient (0~1 M NaCl) can also completely elute it.

[0030] (2) Determining initial isocratic elution conditions by constant pH single-electrode linear gradient elution Based on the pH determined in the previous step, proceed to the second purification step. The buffer formulation is shown in combination 3: Combination 3: Buffer A: 20mM NaAC, 50mM NaH2PO4, 50mM Na2HPO4, 0M NaCl, pH4.5 Buffer B: 20mM NaAC, 50mM NaH2PO4, 50mM Na2HPO4, 1M NaCl, pH4.5 Buffer C: 0.5 M NaOH Buffer was prepared according to combination 3, and purification was performed using the Suzhou Saipu SCG-P100-V4 purification system. The column was an MMC column (4.7 mL pre-packed column, purchased from Borglon (Zhejiang) Biotechnology Co., Ltd.). First, the column was equilibrated with 5 CV Buffer A. After equilibration, the sample was loaded (total protein loading volume was 30 mg / mL of packing material) at a flow rate of 1 mL / min. After loading, the column was washed with Buffer A until the A was reached. 210 Once the concentration dropped below 100 mAU, a linear gradient of 0 to 100% Buffer B was set for elution, with an elution volume of 20 CV. A final 5 CV elution with Buffer B was performed, during which the eluent was collected. Protein purity was assessed using SDS-PAGE. After purification, CIP washing with 2 CV Buffer C was performed. The SDS-PAGE results are shown below. Figure 3As shown in the figure, using a constant pH 4.5 single-wire linear gradient elution method, the target protein CIMM-COL17-11 achieved excellent separation from contaminating proteins (or degradation bands), with a protein purity of 99.5% and a recovery rate of 21.4% (Table 1). The elution conductivity range for the target protein was 33.3–72.8 mS / cm, meaning that contaminating proteins could be eluted with a conductivity below 33.3 mS / cm (corresponding to approximately 0.35 M NaCl), thus achieving separation from the target protein. Since the conductivity detected by the instrument is the conductivity of the buffer solution after mixing Buffer A and Buffer B, which differs slightly from the actual manually prepared buffer solution, optimization was required. Therefore, 0.25 M, 0.30 M, and 0.35 M NaCl were selected for elution of contaminating proteins, while 0.7 M NaCl (69.2 mS / cm) was selected for elution of the target protein. No optimization was required; any salt concentration buffer higher than 0.7 M could also be used for elution.

[0031] (3) Optimization of constant pH, conductivity and isocratic gradient elution to determine the final process Based on the washing range and elution conditions determined in the previous step, proceed to the third step of purification. The buffer formulation is shown in combinations 4-6: Combination 4: Buffer A: 20mM NaAC, 50mM NaH2PO4, 50mM Na2HPO4, 0M NaCl, pH4.5 Buffer B: 20mM NaAC, 50mM NaH2PO4, 50mM Na2HPO4, 0.25M NaCl, pH4.5 Buffer C: 20mM NaAC, 50mM NaH2PO4, 50mM Na2HPO4, 0.70M NaCl, pH4.5 Buffer D: 20mM NaAC, 50mM NaH2PO4, 50mM Na2HPO4, 1.0M NaCl, pH4.5 Buffer E: 0.5 M NaOH Combination 5: Buffer A: 20mM NaAC, 50mM NaH2PO4, 50mM Na2HPO4, 0M NaCl, pH4.5 Buffer B: 20mM NaAC, 50mM NaH2PO4, 50mM Na2HPO4, 0.30M NaCl, pH4.5 Buffer C: 20mM NaAC, 50mM NaH2PO4, 50mM Na2HPO4, 0.70M NaCl, pH4.5 Buffer D: 20mM NaAC, 50mM NaH2PO4, 50mM Na2HPO4, 1.0M NaCl, pH4.5 Buffer E: 0.5 M NaOH Combination 6: Buffer A: 20mM NaAC, 50mM NaH2PO4, 50mM Na2HPO4, 0M NaCl, pH4.5 Buffer B: 20mM NaAC, 50mM NaH2PO4, 50mM Na2HPO4, 0.35M NaCl, pH4.5 Buffer C: 20mM NaAC, 50mM NaH2PO4, 50mM Na2HPO4, 0.70M NaCl, pH4.5 Buffer D: 20mM NaAC, 50mM NaH2PO4, 50mM Na2HPO4, 1.0M NaCl, pH4.5 Buffer E: 0.5 M NaOH Buffer solutions were prepared according to combinations 4-6, and purification was performed using a Suzhou Saipu SCG-P100-V4 purification system. The column used was an MMC column (4.7 mL pre-packed column, purchased from BorgLon (Zhejiang) Biotechnology Co., Ltd.). The column was first equilibrated with 5V C Buffer A. After equilibration, the column was loaded with samples (total protein loading volume: 30 mg / mL packing material) at a flow rate of 1 mL / min. After loading, the column was washed with Buffer A until the A... 210 The concentration dropped below 100 mAU, followed by elution with Buffer B for 15 CV to remove impurities. Then, Buffer C was used for 5 CV to obtain the target protein, and finally, Buffer D was used for 5 CV. The eluent was collected during this process, and protein purity was determined by SDS-PAGE. After purification, CIP washing was performed with 2 CV of Buffer E. The SDS-PAGE results are shown below. Figures 4-6 As shown in the figure, the 0.25M NaCl in combination 4 has a weak cleaning ability, and a light-colored impurity band near the target band remains. Figure 4This resulted in a protein recovery rate of only 5.81% for proteins with a final purity of over 99% (Table 1). Buffer B 0.30 M NaCl in combination 5 had excellent cleaning ability, and the latter half of the cleaning process was already very clean. Figure 5 This resulted in a protein recovery rate of 30.4% with a final purity of over 99% (Table 1). However, the washing power of Buffer B 0.35M NaCl in Combination 6 was too strong, leading to significant loss of the target protein during the washing stage. Figure 6 This resulted in a protein recovery rate of 22.6% with a final purity of over 99% (Table 1). This result is consistent with the results of elution with a constant pH 4.5 single-wire linear gradient (recovery rate of 21.4%). In conclusion, the combined 5 buffer solution has the best purification effect.

[0032] Table 1 Comparison of purity and recovery rate in the CIMM-COL17-11 purification process development steps (4) Determine the maximum sample loading volume that is compatible with the final process. The isocratic gradient elution process was obtained in the above steps. To test the compatibility of this process with the sample loading amount, a maximum sample loading amount test was first performed: when the target band appeared in the flow passage, the sample loading amount was considered to be saturated. The specific purification steps are as follows: Combination 5: Buffer A: 20mM NaAC, 50mM NaH2PO4, 50mM Na2HPO4, 0M NaCl, pH4.5 Buffer B: 20mM NaAC, 50mM NaH2PO4, 50mM Na2HPO4, 0.30M NaCl, pH4.5 Buffer C: 20mM NaAC, 50mM NaH2PO4, 50mM Na2HPO4, 0.70M NaCl, pH4.5 Buffer D: 20mM NaAC, 50mM NaH2PO4, 50mM Na2HPO4, 1.0M NaCl, pH4.5 Buffer E: 0.5 M NaOH Buffer was prepared according to combination 5, and purification was performed using the Suzhou Saipu SCG-P100-V4 purification system. The column was an MMC column (4.7 mL pre-packed column, purchased from BorgLon (Zhejiang) Biotechnology Co., Ltd.). First, the column was equilibrated with 5 CV Buffer A. After equilibration, the sample was loaded (total protein loading volume was 90 mg / mL of packing material) at a flow rate of 1 mL / min. After loading, the column was washed with Buffer A until the A was reached. 210 The concentration dropped below 100 mAU, followed by elution with Buffer B for 15 CV to remove impurities. Then, Buffer C was used for 5 CV to obtain the target protein, and finally, Buffer D was used for 5 CV. The eluent was collected during this process, and protein purity was determined by SDS-PAGE. After purification, CIP washing was performed with 2 CV of Buffer E. The SDS-PAGE results are shown below. Figure 7 As shown, when the loading volume was increased to 90 mg / mL of packing material, the target protein began to appear in the flow through when the loading volume was 45 mL. At this point, the loading volume was 38 mg / mL of packing material, which was considered to have reached the maximum loading volume of the column.

[0033] Therefore, while maintaining the above purification process, the purification effect of total protein loading at 30 and 40 mg / mL packing material was further compared. The SDS-PAGE results are as follows: Figure 8 As shown in the gel images (40 mg / mL) and (30 mg / mL), their purification results are very consistent, and their purity and recovery rates are also basically the same. The former has a protein recovery rate of 29.8% with a purity of over 99%, while the latter has a protein recovery rate of 31.7% with a purity of over 99%. This means that increasing the loading amount to 40 mg / mL of packing material will not reduce the purification effect. Therefore, the maximum loading amount that this process can be compatible with is 40 mg / mL of packing material.

[0034] Table 2 Comparison of purity and recovery rates in the CIMM-COL17-11 purification process development steps (5) Final validation of purification process To verify the stability of the purification process, we prepared a new batch of fermentation broth and purified it according to the formulation of Combination 5. The specific steps were as follows: Buffer was prepared according to Combination 5, and purification was performed using a Suzhou Saipu SCG-P100-V4 purification system. The column was an MMC column (4.7 mL pre-packed column, purchased from BorgLon (Zhejiang) Biotechnology Co., Ltd.). First, the column was equilibrated with 5 CV Buffer A. After equilibration, the sample was loaded (total protein loading volume was 30 mg / mL of packing material) at a flow rate of 1 mL / min. After loading, the column was washed with Buffer A until the A... 210 The concentration dropped below 100 mAU, followed by elution with Buffer B for 15 CV to remove impurities. Then, Buffer C was used for 5 CV to obtain the target protein, and finally, Buffer D was used for 5 CV. The eluent was collected during this process, and protein purity was determined by SDS-PAGE. After purification, CIP washing was performed with 2 CV of Buffer E. The SDS-PAGE results are shown below. Figure 10 As shown in the figure, the results of SDS-PAGE are consistent with... Figure 5 , Figure 9 The results were very consistent, and the purity and recovery rate were also very consistent. The recovery rate of proteins with a purity of over 99% remained relatively stable at 30% (Table 3). Thus, the purification process of CIMM-COL17-11 protein was obtained.

[0035] Table 3. Comparison of the stability of CIMM-COL17-11 purification process Example 3: Scale-up of MMC column purification process for type XVII collagen (CIMM-COL17-11) In industrial applications, changes in column height and diameter necessitate adjustments and optimizations for scaling up from small-scale to pilot-scale processes. Therefore, to verify the scale-up stability of the above process, we performed a 400 mL column volume purification (both the column shell and packing material were purchased from BorgLone (Zhejiang) Biotechnology Co., Ltd., model BXK 50mm / 30cm, packing material was MMC, and the actual column height was 20cm). Buffer was prepared according to combination 5 above, and purification was performed as follows: First, the chromatography column was equilibrated with 5 CVBuffer A. After equilibration, sample was loaded (total protein loading volume was 30 mg / mL packing material) at a flow rate of 80 mL / min. After loading, the column was washed with Buffer A until the A... 210The concentration dropped below 100 mAU, followed by elution with Buffer B for 15 CV to remove impurities. Then, Buffer C was used for 5 CV to obtain the target protein, and finally, Buffer D was used for 5 CV. The eluent was collected during this process, and protein purity was determined by SDS-PAGE. After purification, CIP washing was performed with 2 CV of Buffer E. The SDS-PAGE results are shown below. Figure 11 As shown in the figure, the results of SDS-PAGE are consistent with... Figure 5 , 9 The results of 10 were very consistent, and the purity and recovery rate were also very consistent. The recovery rate of proteins with a purity of over 99% reached approximately 35.2% (Table 3), which was slightly higher than that of the small-scale process, further proving that the above process can be successfully scaled up.

[0036] Furthermore, to verify the stability of the scaled-up process, ten consecutive purification experiments were conducted on a 400 mL MMC column. The purification process, including loading, purification, collection, and analysis, was performed according to the methods described above. The final purification data for the ten experiments are shown in Table 4. As can be seen from the table, the protein SEC purity (the core indicator) obtained from these ten purifications remained at 99% or higher, with an average of 99.8% and a relative standard deviation of 0.29% (≤ 3%), indicating very small process fluctuations and satisfactory stability. The corresponding recoveries all reached 30% or higher, with an average of 32.8% and a relative standard deviation of 4.98% (≤ 5%), indicating minimal process fluctuations and satisfactory stability.

[0037] Table 4. Stability Study of CIMM-COL17-11 Scale-up Process Example 4: Process Development for MMC Column Purification of Type III Collagen CIMM-COL3A1 Based on the purification process development approach for CIMM-COL17-11 protein, for the purification process development of type III collagen CIMM-COL3A (theoretical molecular weight 91.6 kDa), we prioritized using combination 1 buffer for the first step of purification. We determined the buffer combination and initial purification conditions by pH and conductivity bilinear gradient elution. The specific steps are as follows: First, fermentation was carried out according to the process in Example 1. After fermentation, the fermentation supernatant was obtained by centrifugation and then set aside for later use.

[0038] Combination 1: Buffer A: 20mM NaAC, 50mM NaH2PO4, 50mM Na2HPO4, 0.3M NaCl, pH 3.0 Buffer B: 20mM NaAC, 50mM NaH2PO4, 50mM Na2HPO4, 1M NaCl, pH 8.0 Buffer C: 0.5 M NaOH Buffer was prepared according to Combination 1, and purification was performed using the Suzhou Saipu SCG-P100-V4 purification system. The column was an MMC column (4.7 mL pre-packed column, purchased from Borglon (Zhejiang) Biotechnology Co., Ltd.). First, the column was equilibrated with 5 CV Buffer A. After equilibration, the sample was loaded (total protein loading volume was 30 mg / mL of packing material) at a flow rate of 1 mL / min. After loading, the column was washed with Buffer A until the A value was reached. 210 Once the concentration dropped below 100 mAU, a linear gradient of 0 to 100% Buffer B was set for elution, with an elution volume of 20 CV. A final 5 CV elution with Buffer B was performed, during which the eluent was collected. Protein purity was assessed using SDS-PAGE. After purification, CIP washing with 2 CV Buffer C was performed. The SDS-PAGE results are shown below. Figure 12 As shown in the figure, for CIMM-COL3A1, a single target band can be clearly observed in the latter half of the elution. The SEC purity is 98.3% (Table 5), the recovery rate is 1.81%, and the pH range of the target band being eluted is 2.9~8.0. Based on the experience of the above method, for buffers where obvious separation effects can be observed, it is more appropriate to select the pH corresponding to the complete elution of the target protein (pH 8.0) as the constant pH single-electrode linear gradient elution optimization condition.

[0039] Combination 7: Buffer A: 20mM NaAC, 50mM NaH2PO4, 50mM Na2HPO4, 0M NaCl, pH8.0 Buffer B: 20mM NaAC, 50mM NaH2PO4, 50mM Na2HPO4, 1M NaCl, pH8.0 Buffer C: 0.5 M NaOH Buffer was prepared according to combination 7. Purification was performed using a Suzhou Saipu SCG-P100-V4 purification system. The column was an MMC column (4.7 mL pre-packed column, purchased from BorgLon (Zhejiang) Biotechnology Co., Ltd.). The column was first equilibrated with 5 CV Buffer A. After equilibration, the column was loaded with a total protein sample of 30 mg / mL of packing material at a flow rate of 1 mL / min. After loading, the column was washed with Buffer A until the A value was reached.210 Once the concentration dropped below 100 mAU, a linear gradient of 0 to 100% Buffer B was set for elution, with an elution volume of 20 CV. A final 5 CV elution with Buffer B was performed, during which the eluent was collected. Protein purity was assessed using SDS-PAGE. After purification, CIP washing with 2 CV Buffer C was performed. The SDS-PAGE results are shown below. Figure 13 As shown in the figure, the purification method using a constant pH 8.0 single-wire linear gradient can better separate the target protein CIMM-COL3A1 from other proteins. Compared with the first purification step, the yield of the target protein in this step is significantly improved, reaching 19.5% (Table 5), with an SEC purity of 99.7%. The conductivity range of the target protein being eluted is 16.4~57.0 mS / cm. Therefore, for the next step of isocratic gradient elution optimization, a salt concentration below 16.4 mS / cm (approximately 0.20 M NaCl) can be selected for elution optimization. For elution conditions, any salt concentration with a conductivity above 57.0 mS / cm can be selected.

[0040] Table 5 Comparison of CIMM-COL3A1 purification process development parameters Example 5: Development of a process for purifying type I collagen CIMM-COL1A1-8-His6 using MMC columns. To verify the adaptability of the above purification process to different collagen proteins, we selected two His6-tagged type I collagen proteins as examples to attempt to develop purification methods. This example uses CIMM-COL1A1-8-His6 (theoretical molecular weight 6.6 kDa) as an illustration, as follows: Combination 1: Buffer A: 20mM NaAC, 50mM NaH2PO4, 50mM Na2HPO4, 0.3M NaCl, pH 3.0 Buffer B: 20mM NaAC, 50mM NaH2PO4, 50mM Na2HPO4, 1M NaCl, pH 8.0 Buffer C: 0.5 M NaOH Buffer was prepared according to Combination 1, and purification was performed using the Suzhou Saipu SCG-P100-V4 purification system. The column was an MMC column (4.7 mL pre-packed column, purchased from Borglon (Zhejiang) Biotechnology Co., Ltd.). First, the column was equilibrated with 5 CV Buffer A. After equilibration, the sample was loaded (total protein loading volume was 30 mg / mL of packing material) at a flow rate of 1 mL / min. After loading, the column was washed with Buffer A until the A value was reached. 210 Once the concentration dropped below 100 mAU, a linear gradient of 0 to 100% Buffer B was set for elution, with an elution volume of 20 CV. A final 5 CV elution with Buffer B was performed, during which the eluent was collected. Protein purity was assessed using SDS-PAGE. After purification, CIP washing with 2 CV Buffer C was performed. The SDS-PAGE results are shown below. Figure 14 As shown in the figure, due to the small amount of contaminating protein, a single band of CIMM-COL1A1-8-His6 was obtained from the beginning of the purification stage onwards, achieving a rapid and good separation effect. The SEC purity was 97.1% (Table 6), and the yield was 19.2%. The target protein CIMM-COL1A1-8-His6 was completely eluted in the pH range of 4.7 to 7.6. Therefore, for the optimization of constant pH single-electro-linear gradient elution, we chose pH 8.0 (pH 7.6 rounded up) as its constant pH, corresponding to buffer combination 7.

[0041] Combination 7: Buffer A: 20mM NaAC, 50mM NaH2PO4, 50mM Na2HPO4, 0M NaCl, pH8.0 Buffer B: 20mM NaAC, 50mM NaH2PO4, 50mM Na2HPO4, 1M NaCl, pH8.0 Buffer C: 0.5 M NaOH Buffer was prepared according to combination 7. Purification was performed using a Suzhou Saipu SCG-P100-V4 purification system. The column was an MMC column (4.7 mL pre-packed column, purchased from BorgLon (Zhejiang) Biotechnology Co., Ltd.). The column was first equilibrated with 5 CV Buffer A. After equilibration, the column was loaded with a total protein sample of 30 mg / mL of packing material at a flow rate of 1 mL / min. After loading, the column was washed with Buffer A until the A value was reached. 210Once the concentration dropped below 100 mAU, a linear gradient of 0 to 100% Buffer B was set for elution, with an elution volume of 20 CV. A final 5 CV elution with Buffer B was performed, during which the eluent was collected. Protein purity was assessed using SDS-PAGE. After purification, CIP washing with 2 CV Buffer C was performed. The SDS-PAGE results are shown below. Figure 15 As shown in the figure, the purification method using a constant pH 8.0 single-wire linear gradient can achieve the separation of the target protein CIMM-COL1A1-8-His6 from other proteins. The SEC purity is 99.3% (Table 6), and the yield is 25.9%. The conductivity range in which the target protein is eluted in large quantities is 26.2~87.6 mS / cm. Therefore, for the next step of isocratic gradient elution optimization, a salt concentration below 26.2 mS / cm (about 0.30 M NaCl) can be selected for elution optimization. For elution conditions, any salt concentration with a conductivity above 87.6 mS / cm can be selected.

[0042] Table 6 Comparison of CIMM-COL1A1-8-His6 purification process development parameters Example 6: Development of a process for purifying type I collagen CIMM-COL1A1-9-His6 using MMC columns This example uses CIMM-COL1A1-9-His6 (theoretical molecular weight 6.7 kDa) as an illustration, as follows: Buffer was prepared according to combination 1, and purification was performed using a Suzhou Saipu SCG-P100-V4 purification system. The column was an MMC column (4.7 mL pre-packed column, purchased from Borglon (Zhejiang) Biotechnology Co., Ltd.). First, the column was equilibrated with 5 CV Buffer A. After equilibration, sample was loaded (total protein loading volume was 30 mg / mL of packing material) at a flow rate of 1 mL / min. After loading, the column was washed with Buffer A until A was reached. 210 Once the concentration dropped below 100 mAU, a linear gradient of 0 to 100% Buffer B was set for elution, with an elution volume of 20 CV. A final 5 CV elution with Buffer B was performed, during which the eluent was collected. Protein purity was assessed using SDS-PAGE. After purification, CIP washing was performed with 2 CV Buffer C. The SDS-PAGE results are shown below. Figure 16As shown in the figure, similar to CIMM-COL1A1-8-His6, CIMM-COL1A1-9-His6 quickly achieved complete separation of the target protein from impurities, achieving excellent purification results. The SEC purity was 99.2% (Table 7), and the yield was 9.01%. The pH range in which the target protein was eluted was 4.7~7.7. Therefore, the next step is to select a constant pH 8.0 single-wire linear gradient for purification optimization, corresponding to buffer combination 7.

[0043] Combination 7: Buffer A: 20mM NaAC, 50mM NaH2PO4, 50mM Na2HPO4, 0M NaCl, pH8.0 Buffer B: 20mM NaAC, 50mM NaH2PO4, 50mM Na2HPO4, 1M NaCl, pH8.0 Buffer C: 0.5 M NaOH Buffer was prepared according to combination 7. Purification was performed using a Suzhou Saipu SCG-P100-V4 purification system. The column was an MMC column (4.7 mL pre-packed column, purchased from BorgLon (Zhejiang) Biotechnology Co., Ltd.). The column was first equilibrated with 5 CV Buffer A. After equilibration, the column was loaded with a total protein sample of 30 mg / mL of packing material at a flow rate of 1 mL / min. After loading, the column was washed with Buffer A until the A value was reached. 210 Once the concentration dropped below 100 mAU, a linear gradient of 0 to 100% Buffer B was set for elution, with an elution volume of 20 CV. A final 5 CV elution with Buffer B was performed, during which the eluent was collected. Protein purity was assessed using SDS-PAGE. After purification, CIP washing with 2 CV Buffer C was performed. The SDS-PAGE results are shown below. Figure 17 As shown, the purification method using a constant pH 8.0 single-wire linear gradient can achieve rapid separation of the target protein CIMM-COL1A1-9-His6 from impurities. The SEC purity is 99.5% (Table 7), and the yield is 17.5%. The conductivity range of the target protein being eluted is 17.8~74.4 mS / cm. Therefore, for the next step of isocratic gradient elution optimization, a salt concentration below 17.8 mS / cm (approximately 0.20 M NaCl) can be selected for impurity elution optimization. For elution conditions, any salt concentration with a conductivity above 74.4 mS / cm can be selected.

[0044] Table 7 Comparison of CIMM-COL1A1-9-His6 purification process development parameters Example 7: Development of a CD-S column process for purifying type I collagen CIMM-COL1A1-9-His6 Examples 2-7 illustrate purification methods developed using MMC columns, covering three types (I, III, and XVII) of collagen with theoretical molecular weights ranging from 6.6 to 91 kDa. Both tagged and untagged proteins were included. To further verify that the purification method is independent of the packing material type, this example uses a strong cation exchange column, CD-S, as an example. Based on previous experimental experience, the binding of the same protein on CD-S is slightly weaker than on MMC. Therefore, for purification using this column, buffer combination 8 is recommended as the first choice. The specific purification method is as follows: Combination 8: Buffer A: 20mM NaAC, 50mM NaH2PO4, 50mM Na2HPO4, 0M NaCl, pH 3.0 Buffer B: 20mM NaAC, 50mM NaH2PO4, 50mM Na2HPO4, 1M NaCl, pH6.0 Buffer C: 0.5 M NaOH Buffer was prepared according to combination 8. Purification was performed using a Suzhou Saipu SCG-P100-V4 purification system. The column was a CD-S column (4.7 mL pre-packed, purchased from BorgLon (Zhejiang) Biotechnology Co., Ltd.). First, the column was equilibrated with 5 CV Buffer A. After equilibration, sample was loaded (total protein loading volume was 30 mg / mL of packing material) at a flow rate of 1 mL / min. After loading, the column was washed with Buffer A until the A... 210 Once the concentration dropped below 100 mAU, a linear gradient of 0 to 100% Buffer B was set for elution, with an elution volume of 20 CV. A final 5 CV elution with Buffer B was performed, during which the eluent was collected. Protein purity was assessed using SDS-PAGE. After purification, CIP washing with 2 CV Buffer C was performed. The SDS-PAGE results are shown below. Figure 18 As shown in the figure, CIMM-COL1A1-9-His6 achieved complete separation of the target protein from impurities in the latter part of the purification process. The SEC purity was 98.9% (Table 8), and the yield was 3.53%. The target protein was eluted in the pH range of 4.2 to 4.9. Based on the above method, the next step is to select a constant pH 5.0 single-electrode linear gradient for purification optimization, corresponding to buffer combination 9.

[0045] Combination 9: Buffer A: 20mM NaAC, 50mM NaH2PO4, 50mM Na2HPO4, 0M NaCl, pH5.0 Buffer B: 20mM NaAC, 50mM NaH2PO4, 50mM Na2HPO4, 1M NaCl, pH5.0 Buffer C: 0.5 M NaOH Buffer was prepared according to combination 9, and purification was performed using the Suzhou Saipu SCG-P100-V4 purification system. The column was a CD-S column (4.7 mL pre-packed column, purchased from BorgLon (Zhejiang) Biotechnology Co., Ltd.). First, the column was equilibrated with 5 CV Buffer A. After equilibration, sample was loaded (total protein loading volume was 30 mg / mL of packing material) at a flow rate of 1 mL / min. After loading, the column was washed with Buffer A until the A... 210 Once the concentration dropped below 100 mAU, a linear gradient of 0 to 100% Buffer B was set for elution, with an elution volume of 20 CV. A final 5 CV elution with Buffer B was performed, during which the eluent was collected. Protein purity was assessed using SDS-PAGE. After purification, CIP washing with 2 CV Buffer C was performed. The SDS-PAGE results are shown below. Figure 19 As shown, compared with the dual gradient method, the purification method using a constant pH 5.0 single-wire linear gradient can quickly separate the target protein CIMM-COL1A1-9-His6 from other proteins, and the yield of the target protein is significantly improved to 17.6%, with an SEC purity of 99.9% (Table 8). The conductivity range of the target protein being eluted is 46.2~86.6 mS / cm. Therefore, for the next step of isocratic gradient elution optimization, a salt concentration below 46.2 mS / cm (approximately 0.50 M NaCl) can be selected for elution optimization. For elution conditions, any salt concentration with a conductivity above 86.6 mS / cm can be selected.

[0046] Table 8 Comparison of purification process development parameters for CIMM-COL1A1-9-His6 Example 8: Process Development for Phenyl Column Purification of Type III Collagen CIMM-COL3A1-2 To verify the suitability of this method for developing purification processes using hydrophobic packing materials, this example uses type III collagen CIMM-COL3A1-2 (theoretical molecular weight 33.1 kDa) as an example to illustrate the development of a hydrophobic chromatography column purification process. Due to the special nature of the packing material, the conductivity needs to be adjusted before sample loading to meet binding requirements. Furthermore, the elution principle of the hydrophobic packing material is to gradually reduce the conductivity of the buffer solution to separate the target protein from the packing material. Therefore, the buffer combination may differ slightly from the system described above, but the basic purification principles remain the same. The specific purification steps are as follows: Combination 10: Buffer A: 20mM NaAC, 50mM NaH2PO4, 50mM Na2HPO4, 2M (NH4)2SO4, pH3.0 Buffer B: 20mM NaAC, 50mM NaH2PO4, 50mM Na2HPO4, 0M (NH4)2SO4, pH8.0 Buffer C: 0.5 M NaOH Buffer was prepared according to combination 10, and purification was performed using a Suzhou Saipu SCG-P100-V4 purification system. The column used was a Phenyl column (4.7 mL pre-packed column, purchased from Borglon (Zhejiang) Biotechnology Co., Ltd.). First, the column was equilibrated with 5 mL of CV Buffer A. After equilibration, sample was loaded (total protein loading volume was 30 mg / mL of packing material) at a flow rate of 1 mL / min. After loading, the column was washed with Buffer A until the A value was reached. 210 Once the concentration dropped below 100 mAU, a linear gradient of 0 to 100% Buffer B was set for elution, with an elution volume of 20 CV. A final 5 CV elution with Buffer B was performed, during which the eluent was collected. Protein purity was assessed using SDS-PAGE. After purification, CIP washing with 2 CV Buffer C was performed. The SDS-PAGE results are shown below. Figure 20 As shown in the figure, CIMM-COL3A1-2 achieved complete separation of the target protein from impurities in the final purification stage, with an SEC purity of 97.5% (Table 9) and a yield of 1.33%. The target protein was eluted within a pH range of 5.6–7.2. Based on the above method, the next step is to optimize purification using a single-electrode linear gradient with a constant pH of 7.0, corresponding to buffer combination 11. It should be noted that (NH4)2SO4 in combination 11 can be replaced with NaCl as needed.

[0047] Combination 11: Buffer A: 20mM NaAC, 50mM NaH2PO4, 50mM Na2HPO4, 2M(NH4)2SO4, pH 7.0 Buffer B: 20 ​​mM NaAC, 50 mM NaH2PO4, 50 mM Na2HPO4, 0 M (NH4)2SO4, pH 7.0 Buffer C: 0.5 M NaOH Buffer was prepared according to combination 11, and purification was performed using a Suzhou Saipu SCG-P100-V4 purification system. The column used was a Phenyl column (4.7 mL pre-packed column, purchased from Borglon (Zhejiang) Biotechnology Co., Ltd.). First, the column was equilibrated with 5 mL of CV Buffer A. After equilibration, sample was loaded (total protein loading volume was 30 mg / mL of packing material) at a flow rate of 1 mL / min. After loading, the column was washed with Buffer A until the A... 210 Once the concentration dropped below 100 mAU, a linear gradient of 0 to 100% Buffer B was set for elution, with an elution volume of 20 CV. A final 5 CV elution with Buffer B was performed, during which the eluent was collected. Protein purity was assessed using SDS-PAGE. After purification, CIP washing with 2 CV Buffer C was performed. The SDS-PAGE results are shown below. Figure 21 As shown in the figure, compared with the dual gradient, the purification effect of the single linear gradient with constant pH 7.0 was further improved. In the middle part of the purification, the target protein and the impurity protein were well separated. The SEC purity was 99.6% (Table 9), and the yield was significantly improved to 54%. The conductivity range of the target protein was eluted was 144~2.8 mS / cm. Therefore, for the next step of isocratic gradient elution optimization, a salt concentration higher than 144 mS / cm (about 1.5 M (NH4)2SO4) can be selected for impurity elution optimization. For elution conditions, any salt concentration with conductivity lower than 2.8 mS / cm can be selected.

[0048] Table 9 Comparison of CIMM-COL3A1-2 purification process development parameters In summary, the above embodiments tested the applicability of the recombinant collagen purification process development method disclosed in this application under different types of chromatographic packing materials, different types of collagen, and whether the protein contains tags, demonstrating its versatility and reproducibility. Furthermore, this method provides researchers with a reference outline for protein purification, enabling them to quickly obtain high-purity proteins and purification processes through simple and clear experimental design, reducing the sense of helplessness when purifying different types of proteins. Most importantly, the purification process obtained through this method exhibits good stability, high compatibility with feed solutions, and stable scale-up.

[0049] The above embodiments are for illustrating the implementation schemes disclosed in this invention and should not be construed as limiting the invention. Furthermore, various modifications listed herein, as well as variations in the methods and compositions of the invention, will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been specifically described in conjunction with various specific preferred embodiments, it should be understood that the invention should not be limited to these specific embodiments. In fact, various modifications as described above that are obvious to those skilled in the art to obtain the invention should be included within the scope of this invention.

Claims

1. A method for developing a collagen purification process, the method comprising the following steps: (1) Determine the filler based on collagen sequence information and isoelectric point; (2) Based on the protein characteristics, different NaCl concentrations of the first buffer were used for pH and conductivity bilinear gradient purification. The pH corresponding to when the target protein was completely eluted was found by the SDS-PAGE results. The pH of the first buffer was 3-8. (3) Provide a second buffer solution with a constant pH single-electro-linear gradient according to the pH determined above, wherein the pH of the second buffer solution is the pH selected in step (2); (4) Use the second buffer to perform constant pH single-wire linear gradient purification. After purification, find the conductivity values ​​Y1 and Y2 corresponding to when the impurity protein and the target protein are completely eluted according to the SDS-PAGE results, and calculate the corresponding salt concentrations of the third buffer C1≤Y1 and the fourth buffer C2≥Y2 for washing. (5) Prepare third and fourth buffer solutions with different NaCl concentrations according to the salt concentration determined in step (4), and purify the protein. The third buffer solution is used for washing impurities, and the fourth buffer solution is used for elution. The pH of the above buffer solutions is the pH determined in step (2). After purification, the purification process is determined according to the SDS-PAGE results. (6) If no obvious separation and purification effect is observed after steps (2) to (5), other packing materials need to be replaced and steps (2) to (5) are repeated until a suitable packing material is selected and subsequent steps are optimized.

2. The method according to claim 1, characterized in that, The selection principle for the packing in step (1) is as follows: If collagen carries a positive charge at physiological pH (pI > 7.0), cationic fillers should be tried first; if collagen carries a negative charge at physiological pH (pI < 7.0), anionic fillers should be tried first; if separation cannot be achieved after trying all ionic fillers, hydrophobic fillers should be tried.

3. The method according to claim 2, characterized in that, When ionic packing is used, the first buffer solution is prepared from the following components: Buffer A1: 20 mM NaAC, 50 mM NaH2PO4, 50 mM Na2HPO4, 0 M NaCl, pH 3.0 Buffer B1: 20 mM NaAC, 50 mM NaH2PO4, 50 mM Na2HPO4, 1 M NaCl, pH 8.

0.

4. The method according to claim 2, characterized in that, When hydrophobic packing is used, the first buffer solution is prepared from the following components: Buffer A R : 20mM NaAC, 50mM NaH2PO4, 50mM Na2HPO4, 2M (NH4)2SO4 / NaCl, pH3.0, Buffer B R : 20 mM NaAC, 50 mM NaH2PO4, 50 mM Na2HPO4, 0 M (NH4)2SO4 / NaCl, pH8.

0.

5. The method according to claim 1, characterized in that, The second buffer solution was prepared according to the following concentration ratio: Buffer A2: 20 mM NaAC, 50 mM NaH2PO4, 50 mM Na2HPO4, 0 M NaCl, adjusted to the selected pH; Buffer B2: 20 mM NaAC, 50 mM NaH2PO4, 50 mM Na2HPO4, 1 M NaCl, adjusted to the selected pH.

6. The method according to claim 1, characterized in that, The third buffer solution was prepared according to the following concentration ratio: 20 mM NaAC, 50 mM NaH2PO4, 50 mM Na2HPO4, ClM NaCl, and adjusted to the selected pH.

7. The method according to claim 1, characterized in that, The fourth buffer solution was prepared according to the following concentration ratio: 20 mM NaAC, 50 mM NaH2PO4, 50 mM Na2HPO4, C2M NaCl, and adjusted to the selected pH.

8. The method according to claim 1, characterized in that, The method further includes step (6) fine-tuning the third buffer solution, where the NaCl concentration is fine-tuned within the range of 0.05~0.1 M.

9. The method according to claim 1, characterized in that, The elution volume in steps (2) to (4) is 20 to 25 CV.

10. The method according to claim 1, characterized in that, The packing material is a weak cation, a strong cation, a composite multi-mode cation packing material, a weak anion, a strong anion, a composite multi-mode anion packing material, or a hydrophobic packing material.

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