Method of producing and purifying an active soluble sialyltransferase
By expressing in CHO cells and combining chromatographic purification technology, the problem of insufficient yield and purity of recombinant sialyltransferase polypeptides in the existing technology has been solved, and the production of highly active and high-purity ST6GalNAcI polypeptides has been achieved, which is suitable for glycosylation therapy. Proteins such as G-CSF.
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Embodiment 1
[0155] The purification process of ST6GalNAcI was started with a capture chromatography step using an anion exchange column. Subsequent purification steps include affinity chromatography, mixed mode chromatography, ultrafiltration / diafiltration, cation exchange chromatography and Final nanofiltration of membranes. Chromatographic steps are performed using a gradient chromatography system and run automatically. UV absorbance, conductivity, pH, flow rate and back pressure were recorded at each chromatography step using appropriate software.
[0156] The column types and resins used are shown in Table 1:
[0157]
[0158] First column: Anion exchange chromatography (Q-Sepharose FF)
[0159] Anion exchange chromatography with Q-Sepharose Fast Flow resin was used as the first chromatography step to capture ST6GalNAcI in the harvest and for volume reduction.
[0160] ST6GalNAcI was eluted by applying a salt step of 800 mM NaCl in 20 mM Tris HCl, pH 7.6. The eluate was collec...
Embodiment 2
[0181] First column: Anion exchange chromatography (Q-Sepharose FF)
[0182] This process step was used to capture cST6 from a clarified, sterile filtered bioreactor harvest by anion exchange chromatography. 1000 mL of supernatant from the bioreactor was diluted 1:3 in buffer A (20 mM Tris pH 7.6) and loaded onto an omni25 / 150Q Sepharose FF column. cST6 (0-1 M, 20 CV) was eluted by a linear NaCl gradient (0-1 M, 20 CV).
[0183] Second column: Affinity chromatography (CDP affinity matrix)
[0184] This affinity chromatography process step is an intermediate step that is important to increase the purity of cST6. The pool of fractions 9-11 from the Q Sepharose capture step was diluted 1:3 in buffer A (10 mM MES pH 6.8, 25% glycerol) and loaded onto an XK 10 / 55 CDP affinity column. To avoid excessive loading of the CDP column, the load was applied in two identical CDP runs. cST6 (0-1 M, 20 CV) was eluted by a linear NaCl gradient (0-1 M, 10 CV).
[0185] Third column: Anion ...
Embodiment 3
[0188] First column: Anion exchange chromatography (Q-Sepharose FF)
[0189] Anion exchange chromatography with Q-Sepharose Fast Flow resin was used as the first chromatography step to capture ST6GalNAcI in the harvest and for volume reduction. 300 mL of clarified and sterile filtered supernatant from the fed-batch culture bioreactor was purified.
[0190] Chromatographic conditions are summarized in Table 6:
[0191]
[0192] Second column: Intermediate Affinity Chromatography (Blue Sepharose 6FF)
[0193] This affinity chromatography step is an intermediate step that is important to increase the cST6 purity. The eluate of the capture chromatography step (29 mL) was diluted and purified by a Blue Sepharose chromatography step.
[0194] Chromatographic conditions are summarized in Table 7:
[0195] column
Omnifit10, bed height = 7.0cm, CV = 5.5mL
equilibration buffer A
25mM potassium phosphate buffer, pH7.5, 50mM NaCl
Elution buffer B
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