Process Development And Evaluation For Recombinant Trypsin Based On QbD Conception

Trypsin?EC 3.4.21.4?,which belongs to the family of serine protease,has the ability of cutting carboxyl terminal of alkaline amino acids specifically,such as lysine and arginine.Trypsin has been widely used in pharmaceutical industry.Trypsin is easily extracted from the pancreas of animals?pigs,cow,etc.?,but the purity is low.Animal originated trypsin contains a large number of other enzymes?such as chymotrypsin?,so there is a risk of unknown virus and exogenous factor contamination.Due to the increasingly stringent regulation,the application of extracted trypsin from animal is limited while recombinant trypsin is used widely.This paper described a process development of the recombinant trypsin.The activity and purity of recombinant trypsin described in this paper is very high,and the recombinant trypsin can also be used in production of other products in commercial scale.Firstly,the upstream processes were developed with QbD strategy,including the processes of shaker,seed fermentor and fermentor.The process parameters determined were listed as follows:the inoculation amount of the shake flask was 0.5%,the temperature was28³2?,culture period of the shake flask was not more than 24 h,OD₆₀₀ was 19³2.The inoculation amount of seed fermentor was 0.5%¹.0%,culture temperature was 28³2?,dissolved oxygen was not more than 40%,the incubation period was 15²4 h,OD₆₀₀ was18³1.The inoculation amount of fermentor was 2.5⁷.5%,urea was used as nitrogen source,induction temperature was 25²8?,the pH iswas controlled between 4.0 and 5.0,the fermentation period was 90¹10 hours.Secondly,the downstream purification process was developed with method of QbD including:capture of trypsinogen,activation,ultrafiltration,and freeze-drying.A stable downstream production process was obtained.Capacity of chromatography for trypsinogen capture was 57 g/L gel,both of the flow rates of loading and elution were 0.08⁰.25 CV/min,elution conditions included that:pH was 3.0⁴.0,Nacl concentration was 40⁶0 mmol/L and solution elution volume was 3CV.It took 4⁵ h for activation under the condition of Ca²⁺40⁸0 mmol/L and pH 7.5⁸.0.The ultrafiltration was completed with 8K ultrafiltration membrane in 2 h under the largest transmembrane pressure allowed by the system.For freeze-drying,the pre-freeze-dried temperature was-20?,freeze-drying for 3 h,the main freeze-dried temperature was 8?,the final freeze-dried temperature was 25?,freeze-drying for 24 h.Then could botain the final product that the quality standard.Thirdly,the production data and application examples of trypsin in other projects indicate that the development process developed in the paper could be used in amplify production and the obtained product had good product quality.The expression level of upstream was more than 7.5 g/L,total yield of downstream was more than 15%,the product purity was 73.5%or higher,enzyme activity was 3856 IU/mg or more.This paper aims to expound a new process development of recombinant trypsin,using QbD method recommended by FDA.A stable process which could be enlarged was obtained.The above results provided the research foundation for the industrialization of trypsin and reference for other projects.