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Application of Ultrafiltration in Recombinant Human Insulin Production

2024.02.02 702


Introduction of Insulin

Insulin is a hormone secreted by pancreatic islet β cells in response to stimulation by endogenous or exogenous substances such as glucose, lactose, ribose, arginine and glucagon. It regulates the metabolism of carbohydrates, fats and protein by promoting the absorption of glucose from the blood into liver, fat and skeletal muscle cells, is considered to be the main anabolic hormone to lower blood sugar of the body. Insulin is an important medicine for diabetes treatment.



Figure 1 Introduction of Insulin

Insulin Therapeutics History and Progress

The development of insulin has gone through many iterations, starting with the earliest generation of animal-derived insulin, which was low-cost but did not have a stable glucose-lowering effect, and was also prone to immunogenicity, causing negative reactions such as allergies. With the development of technology, gradually began to use genetic engineering to achieve the development and production of insulin drugs, Along with the recombinant second-generation insulin, third-generation insulin has performed more outstandingly in reducing the risk of hypoglycemia.


Figure 2 Insulin Therapeutics History and Progress


Application of Ultrafiltration in Recombinant Human Insulin Production

Currently recombinant insulin production process is complex and involves multiple steps, through fermentation, proinsulin recovery collection, enzyme digestion, multi-step purification to lyophilization preparation and some others.

Multiple steps will require the use of ultrafiltration products for concentration and buffer exchange.

Here, some typical ultrafiltration application in human insulin production from E. coli is presented.

Ultrafiltration Application Points:

100 kD TFF Cassettes /Hollow Fiber – Removal of larger molecules from proinsulin refolding centrifugation supernatant
10 kD TFF Cassettes - Proinsulin concentration
30-50 kD- TFF Cassettes /Hollow Fiber- Removal of larger molecules from enzymatic cleavage insulin centrifugation supernatant
3 kD TFF Cassettes- Insulin concentration and diafiltration after multi-steps of chromatography


Case Studies

01 Case Studies of 100kD Hollow Fiber

Cobetter hollow fiber filter HFELA01000530P (Diameter 0.5mm, 100 kDa, EFA 236 cm2) is applied in proinsulin refolding centrifugation supernatant concentration and washing, permeate collection.


Process steps 35x Concentration, 20x Dialysis
Shearing rate (S-1) 6000
Average TMP (bar) 0.75
Average concentration permeate (LMH) 97.31
Average dialysis permeate (LMH) 109.8
Products recovery (%) 102.16
Water flux recovery (%) 83.57

Figure 3 Flux and TMP vs. Time



Conclusion: From above experimental cases, we can see that using of 100kD hollow fiber in the process of proinsulin macromolecule removal, with a good performance in throughput and recovery.

02 Case Studies of PES 10kD TFF Cassettes


Cobetter PES TFF Cassette UFELA0010010P (PES, 10kD, 0.11m2) is applied in proinsulin concentration and collection.


Process steps 15x Concentration
Feed Flux (LMM) 5
Average TMP (bar) 1.2
Average concentration permeate (LMH) 81.96
Products recovery (%) 98.59
Water flux recovery (%) 96.97

Figure 4 Flux Rate & TMP vs. Time

Conclusion: From above experimental cases, we can see that using of 10kD PES TFF cassettes in the process of proinsulin concentration, with a good performance in throughput and recovery.



03 Case Studies of RC 30kD TFF Cassettes

Cobetter RC TFF Cassette UFCLA0030010P (RC, 30kD, 0.11m2) is applied in removal of larger molecules from enzymatic cleavage insulin centrifugation supernatant, insulin collection.


Process steps 3x Concentration , 10x Dialysis
Feed Flux (LMM) 2.6
Average TMP (bar) 5-12
Average concentration permeate (LMH) 36.40 
Average dialysis permeate (LMH) 36.36
Products recovery (%) 91.40 
Water flux recovery (%) 97.32

Figure 5 TMP & Flux vs. Time

Conclusion: From above experimental cases, we can see that using of 30kD RC TFF cassettes in the process of enzymatic cleavage insulin concentration, with a good performance in throughput and recovery.

04 Case Studies of RC 3kD TFF Cassettes


Cobetter RC TFF Cassette UFCLA0003010P (RC, 3kD, 0.11m2) is applied in insulin concentration and diafiltration after multi-steps of chromatography. The following are repeated experimental data for the same TFF Cassettes.


Tests No. 1 2 3 4 5
Feed Flux (LMM) 3 3 3 3 3
Average TMP (bar) 2.46 2.53 2.53 2.53 2.53
Average concentration permeate (LMH) 14.86 16.22 15.91 15.91 15.35
Average dialysis permeate (LMH) 17.33 17.46 17.84 17.76 17.61
Products recovery (%) 101.67 96.98 100.3 103.72 99.13



Conclusion

From above experimental cases, we can see that using of 3kD RC TFF cassettes in the process insulin concentration and buffer change, with a good performance in throughput and recovery.


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