Fig 1.
(A) Left panel: Flows diagram for the commercial Prostak™ devices (red line for feed-retentate and blue line for permeate). Right panel: exploded view of small scale Prostak™ prototype (2 stak-0.063 m2). (B) Critical Permeate Flux Study (CPF) with the small scale Prostak™ prototype Tangential Flow Filtration system was run at the three cross flux settings of 4.5 L/min, 3 L/min and 1.5 L/min, with resulting critical permeate fluxes of, respectively, 37 LMH (L/m2/h), 28 LMH and 19 LMH. (C) Concentration step of the TFF clarification procedure with the critical data provided. (D) Diafiltration step of the TFF clarification procedure with the critical parameters provided.
Table 1.
Overview of insulin production and purification process from Pichia pastoris fermentation culture.
Fig 2.
(A) Toyopearl Giga cap S-650 M cation-exchange chromatography of IP. IP was eluted from the column with the mobile phase C: 20 mmol L-1 NaOAc, pH 4.0, EtOH 50%, NaCl 0.5 mol L-1. RP-HPLC profiles of insulin species from each step of the human insulin production process (panels B-F). (B) RP-HPLC profile of the insulin precursor after cation exchange chromatography. IP has experimental molecular mass 7043 Da (calculated average mass is 7042.04) and retention time 18.0 minutes. (C) RP-HPLC profile of insulin species after the transpeptidation reaction. Insulin ester-H-Thr(tBu)-OtBu is eluted at retention time 25.9 minutes (experimental and calculated average mass 5919.42 Da), insulin species cleaved at B29 without threonine ester are eluted at retention time 21.2 minutes (experimental and calculated average mass 5706.37 Da) and insulin species cleaved at B22 are eluted at retention time 19.6 minutes (experimental and calculated monoisotopic mass 4862.24 Da). (D) RP-HPLC profile of insulin ester-H-Thr(tBu)-OtBu after the reversed-phase chromatography purification. (E) RP-HPLC profile of deprotection reaction mixture where human insulin has a retention time 21.0 minutes (experimental and calculated average mass 5807.58 Da). (F) RP-HPLC profile of purified human insulin (after the last reversed-phase chromatography). The purity of the human insulin was at least 98%.
Fig 3.
(A) Detector traces (Evaporative Light Scattering Detector) from RP-HPLC separation of H-Thr(tBu)-OtBu standard and flow-through to first protein peak fraction from post transpeptidation purification. (B) Detector traces (Evaporative Light Scattering Detector) from RP-HPLC separation of the recovered H-Thr(tBu)-OtBu from transpeptidation reaction of IP. Retention time of H-Thr(tBu)-OtBu is 12.36 min and the purity is 99%. (C) RP-HPLC profile of insulin species after transpeptidation reaction with H-Thr(tBu)-OtBu. (D) RP-HPLC profile of insulin species after transpeptidation reaction with extracted H-Thr(tBu)-OtBu. No difference in efficiency of the transpeptidation reaction was observed.