Abstract: To establish a separation and purification process of recombinant humanized type Ⅱ collagen Ⅱ-23k, the type of chromatography column was first determined by comparing the perification performance of different ion-exchange columns. Then, the purification effects of clarification and filtration, concentration and buffer exchange, and ion exchange chromatography of the fermentation broth were investigated to establish the separation and purification process of Ⅱ-23k. The experimental results showed that compared to the Q strong anion, SP strong cation and CM weak cation exchange columns, the DEAE weak anion column performed the best. Using the DEAE column in flow-through mode, Ⅱ-23k yielded with a purity over 95% and a recovery rate greater than 90% at the buffer pH of 7.0. The clarification and filtration of the fermentation broth was carried out by tangential flow filtration (TFF) hollow fiber microfiltration, the concentration and buffer exchange were carried out by TFF flat membrane ultrafiltration, and the purification was carried out by ion exchange chromatography. Consequently, a separation and purification process was established, comprising the following steps: clarification and filtration, concentration and buffer exchange, DEAE flow-through chromatography, and freeze-drying. The purity of Ⅱ-23k obtained was about 95%, with no pigment residue. The average recovery rate of concentration and buffer exchange was about 75%, and the average recovery rate of DEAE column flow-through was about 78%. The overall process recovery reached nearly 60%. This study established a separation and purification process of recombinant humanized type Ⅱ collagen based on TFF and one-step column flow-through mode chromatography, which provides a reference for its large-scale production and industrial preparation.

Key words: recombinant humanized type Ⅱ collagen, separation and purification, ion exchange chromatography, flow-through mode, TFF