YY/T 1849-2022Recombinant collagen protein (English PDF)

3.1 Collagen

Collagen is a family of secretory proteins containing at least 20 genetically distinct types. It primarily functions as a structural support for the body, possessing a unique triple helix configuration composed of three polypeptide chains (called alpha chains) and exhibiting specific biological function.

3.2 Recombinant collagen protein

Recombinant DNA technology is used to genetically manipulate and/or modify the gene encoding the desired human collagen protein. The target gene is then introduced into a suitable host cell (bacteria, yeast, or other eukaryotic cells, etc.) using plasmids or viral vectors and expressed and translated into collagen (3.1) or a collagen-like (3.1) polypeptide. The collagen protein is then prepared through extraction and purification, etc.

4.1 General Rules

Due to differences in the type and sequence of recombinant gene fragments and variations in expression systems, the obtained products may not be exactly the same. When compared with natural collagen protein, there may be significant differences in amino acid composition, molecular weight, and/or structure, and sometimes they may be entirely new proteins. Therefore, comprehensive characterization (see Appendix A) and rigorous quality control are necessary.

The quality control of recombinant collagen protein is related to its molecular weight, structural characteristics, complexity of quality attributes, and production processes. The quality control system mainly includes raw and auxiliary material quality control, production process control, and final product testing, etc. A combination of finished product testing, process control, and process validation shall be used to ensure that all impurities have been eliminated or reduced to acceptable levels.

4.2 Quality Control of the Preparation Process

Recombinant collagen protein is a recombinant protein. Therefore, all requirements in "manufacturing" of the "General Principles of Recombinant DNA Protein Products for Human Use" in the Pharmacopoeia of the People's Republic of China, including. basic requirements, control of engineering cells (bacteria, yeast, or other eukaryotic cells, etc.) (expression vectors and host cells, cell bank systems, cell bank quality control, genetic stability of cell matrix), production process control (cell culture, extraction and purification, stock solution, semi- finished product, and finished product), and requirements for changes in the production processes shall be taken as a reference to establish a quality control system. Reasonable explanations must be provided for any inapplicable clauses or matters.

4.3 Quality Control of Recombinant Collagen Protein

Quality control of finished products of recombinant collagen protein includes evaluating known and/or potential product-related and process-related substances using reference standards and validated methods, and testing and analyzing product identification, biological function, purity, and impurities using appropriate methods.

Select one (or more) batch that has been proven sufficiently stable and suitable for clinical trials, or use a representative batch as a reference standard for various analyses of identification, physicochemical, and biological function, etc. Based on the characteristics of the recombinant DNA protein product, a comprehensive and in-depth characterization/characteristic analysis of the reference standard shall be performed. The establishment and preparation of the reference standard shall refer to the relevant requirements of the "Preparation and Standardization of National Standard Substances for Biological Products" in the Pharmacopoeia of the People's Republic of China.

Reference standards used for physicochemical determinations, for example, those for peptide mapping or isoelectric point determination, may be prepared directly from the stock solution and generally stored below -70 °C or under validated storage conditions. Based on the characteristics of the recombinant DNA protein product, the reference standard shall undergo necessary analytical identification, including protein content, isoelectric point, purity, N- terminal amino acid sequence, molecular weight, peptide mapping, fingerprint peptide library, disulfide bond analysis, and glycosylation analysis (yeast or other eukaryotic cell expression), etc.

5.1 General Rules

The harvested liquid, after extraction, purification, and aliquoting into intermediate storage containers, becomes the stock solution. The testing items for the stock solution depend on process validation, consistency confirmation, and the expected levels of product-related impurities and process-related impurities. Appropriate methods shall be used to test the quality of the stock solution, and when necessary, comparison with a reference standard shall be In accordance with the "Minimum Filling Volume Examination Method" in the Pharmacopoeia of the People's Republic of China, based on the different characteristics of the test sample (solution, gel, lyophilized sponge, or lyophilized powder, etc.) and filling volumes, determine the requirements for the allowable differences for the filling volume.

5.3 Identification

5.3.1 Peptide mapping

5.3.1.1 In accordance with Method 1, "Trypsin Cleavage-Reversed-Phase High-Performance Liquid Chromatography" of the "Peptide Mapping Examination Method" in the Pharmacopoeia of the People's Republic of China, conduct the determination. The peptide mapping shall be consistent with that of the reference standard, and the relative proportions (response intensities) of the main characteristic peptide fragments shall be substantially consistent with those of the reference standard.

NOTE. If Trypsin cannot hydrolyze, select another suitable protease.

5.3.1.2 In accordance with the "Mass Spectrometry Method" in the Pharmacopoeia of the People's Republic of China, conduct the determination. Appropriate mass spectrometry techniques shall be used to qualitatively determine the peptide segments corresponding to each chromatographic peak, and the sequence coverage shall meet the indicated range.

NOTE. If Trypsin cannot hydrolyze, select another suitable protease.

5.3.2 Terminal amino acid sequence

The N-terminal and/or C-terminal amino acid sequences shall be determined using an amino acid sequence analyzer or mass spectrometry. The result shall comply with theoretical predictions (this shall be performed at least once a year).

5.3.3 Molecular weight

5.3.3.1 The molecular weight shall be determined in accordance with the method conditions specified in YY/T 1805.2 and meet the indicated requirements.

5.3.3.2 When performing molecular weight analysis in accordance with the "High-performance Liquid Chromatography" in the Pharmacopoeia of the People's Republic of China, the peak retention time shall be consistent with that of the qualitative reference standard (recombinant collagen protein identified by time-of-flight mass spectrometry).

5.3.3.3 In accordance with the "Mass Spectrometry Method" in the Pharmacopoeia of the People's Republic of China, conduct the determination, and the molecular weight shall be consistent with the theoretical molecular weight.

Sterility

If the finished product of recombinant collagen protein is aseptically provided, the sterilization process shall be validated and routinely controlled in accordance with GB 18278.1, GB 18279.1 and GB 18280.1.

In accordance with the "Sterility Examination Method" in the Pharmacopoeia of the People's Republic of China, conduct the sterility testing, and the result shall be sterile.

5.9.3 Bacterial endotoxins

In accordance with the method specified in the "Bacterial Endotoxin Examination Method" in the Pharmacopoeia of the People's Republic of China, conduct the determination, which shall comply with the indicated limit.

NOTE 1.the bacterial endotoxin limit requirements for medical device products are related to factors, for example, their mode of contact with the human body. These factors should be considered when specifying the bacterial endotoxin limit for recombinant collagen protein as a raw material.

NOTE 2.the Pharmacopoeia of the People's Republic of China stipulates that for injectable preparations, the maximum acceptable endotoxin dose per kilogram of body weight per hour is 5 EU.

5.9.4 Microbiological limits

If the finished product of recombinant collagen protein is provided in a non-sterile manner, the total aerobic bacteria count per 1 g, 1 mL, or 10 cm² of the test sample must not exceed 10² CFU, the mold and yeast colony count must not exceed 10 CFU, and Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa must not be detected.

Testing method. use 0.9% sterile sodium chloride solution or pH 7.2 sterile phosphate buffer solution to prepare the test sample solution. In accordance with the methods "Microbiological Limit Examination for Non-Sterile Products. Microbial Count Method" and "Microbiological Limit Examination for Non-Sterile Products. Control Microorganism Examination Method" specified in the Pharmacopoeia of the People's Republic of China, conduct the testing.