Increased lifetime of RPC resins in insulin production by clean-up using WorkBeads 40S
Purification of recombinant insulin requires very high purity, often achieved by high-resolution reversed phase chromatography (RPC) based on silica. Impurities from the feed often cause fouling of the silica resins that is difficult to remove since the option of cleaning-in-place using sodium hydroxide is limited. These issues result in shortened lifetime of the RPC columns. We present here that an introduction of an ion exchange chromatography step before RPC removes the bulk of impurities from the feed which significantly will increase the lifetime of the RPC column.
By introducing a capture step, using WorkBeads™ 40S (a cation exchanger), a standard purification process involving a combination of two RPC steps with two different buffer systems (to obtain complementary selectivity) was improved.
WorkBeads 40S is an agarose based resin with sulfonate ligands which is stable in high concentration of sodium hydroxide thus allowing efficient cleaning-in-place. A feed containing 72.5% pure human insulin precursor (Met-Lys-Human insulin) was applied to the WorkBeads 40S column, followed by the two RPC steps. Each step was optimized to give a yield of 90% while maximizing the purity after each step. The amount of impurities in the applied feed on the first RPC column could be reduced from 27.5% to 12%. This improvement significantly reduces the fouling of the first RPC column and prolongs its lifetime. The final purity for the human insulin precursor was > 99%. A comparison was also done using Capto™ SP ImpRes cation exchange resin in the capture step which resulted in a less pure target protein compared to using WorkBeads 40S.