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OBJECTIVE To establish critical quality attributes analysis method for recombinant human collagen Ⅲ. METHODS The recombinant human collagen Ⅲ molecular weight (MW) distribution was determined by matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS) and size exclusion chromatography with multi-angle light scattering (SEC-MALS). Capillary electrophoresis-sodium dodecyl sulfate (CE-SDS) and size exclusion high performance liquid chromatography (SE-HPLC) were used for purity analysis. Ion exchange chromatography and capillary zone electrophoresis (CZE) were used to separate and analyze charge isomers. The “double” enzyme digestion peptide mapping method was used for identification and sequence coverage analysis. RESULTS The recombinant human collagen Ⅲ MW distribution measured by MALDI-TOF was 45.01×103. The MW of monomer recombinant human collagen Ⅲ determined by SEC-MALS was 45.17×103 (±0.226%). CE-SDS analysis showed that the purity of recombinant human collagen Ⅲ was 98.77% and that of other ingredients was 1.23%. The SE-HPLC purity was 98.07%, and that of other component was 1.93% (dimer). A total of 14 charge isomers were identified through strong cation exchange, and the acidic peak was 52.08%, the main peak was 26.22%, and the basic peak was 21.70%. Eight charge isomers were identified by CZE, including 52.10% acidic peak, 25.27% main peak, and 22.63% basic peak. Two identification methods for charge isomers have consistent distribution patterns. The “double” enzyme digestion peptide mapping method was used for identification and sequence coverage analysis, achieving 100% coverage of the recombinant human collagen Ⅲ sequence. CONCLUSION A series of critical quality attributes analysis methods for recombinant human collagen Ⅲ have been established. These methods provide a reference basis for the quality control of recombinant human collagen Ⅲ in China.
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| 科 Family | 属数 Number of genus | 种数 Number of species | 占总种数比例 Percentage of total species (%) | 属 Genus | 种数 Number of species | 占总种数比例 Percentage of total species (%) |
|---|---|---|---|---|---|---|
| 鹅膏菌科Amanitaceae | 2 | 11 | 5.26 | 鹅膏菌属 Amanita | 10 | 4.78 |
| 小菇科 Mycenaceae | 2 | 12 | 5.74 | 丝盖伞属 Inocybe | 5 | 2.39 |
| 多孔菌科 Polyporaceae | 8 | 14 | 6.70 | 蜡蘑属 Laccaria | 5 | 2.39 |
| 红菇科 Russulaceae | 3 | 23 | 11.00 | 小皮伞属 Marasmius | 6 | 2.87 |
| 小菇属 Mycena | 11 | 5.26 | ||||
| 光柄菇属 Pluteus | 5 | 2.39 | ||||
| 红菇属 Russula | 17 | 8.13 | ||||
| 栓菌属 Trametes | 5 | 2.39 |
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