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FEATURED CASE STUDY
Large Scale Production and Supply of Calpain (Sm-p80) Protein
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Background:

Schistosomiasis is a major neglected tropical disease of public health importance. It is caused due to penetration of the human skin by infectious larvae (cercariae) of Schistosomes, water-borne parasitic platyhelminths, which has infected ~252 million people worldwide. Host interactive schistosome protein, calpain (Sm-p80), plays an important role in the surface membrane renewal of schistosomes, which is employed by helminths to evade host immunity. At present, to our knowledge, Sm-p80 is the sole schistosome vaccine candidate that has been tested for its prophylactic and therapeutic efficacy in different vaccine formulations and approaches.

Problem/Rationale:

Most current vaccine approaches have focused on exploiting molecular and recombinant technology to identify possible protective antigens from parasites and deliver these purified extracts or recombinant constructs in various formulations. However, high throughput identification of antigen candidates and successful protective results will not be sufficient in the absence of reliable and consistent scale-up manufacturing process.
The client wanted to take the Sm-p80 molecule further to conduct various research studies and hence require material at larger scale. The objective of this project was to deliver the desired protein at larger scale.

Solution/Approach:

Based on discussions with the client, we developed a plan and workflow process to produce purified protein to conduct various studies (Figure 1-5). We developed a process at 5L scale to deliver the protein and then scale up the process to 50L (10X) for supply of protein for various studies.

Figure 1: Steps required to produce clinical grade material

Cell Banking System:

Figure 2: Cell Banking System – Process flow of Cell Banking system as per Q5D

Process Flow:

Figure 3: Process flow diagram for production of clinical grade material

Fermentation Parameters:

During the fermentation, we have optimized various parameters such as temperature, pH and agitator speed (Figure 4).

Figure 4: Fermentation Parameters – Set point V/S Actual (Temperature, pH & Agitator Speed)

Downstream Processing:

Ion exchange column (Q Sepharose XL) was used as the Protein Capture step to remove impurities. Protein captured onto Ion exchange column (Q Sepharose XL) was pooled and loaded onto Ni -NTA column to capture His tagged protein. During these steps, we screened wide range of buffer composition and additives for proper cushioning; developed mechanical setup for ultra-slow mixing resulting significantly less aggregation while doing buffer exchange.

Final Purified Protein: SDS Page

After the successful completion of the project, we supplied ~220mgs of protein in 3000 vials to the client along with certificate of analysis.

Figure 5: Purified Protein of Interest on 10% SDS Page Gel – M (Marker) & PP (Purified Protein)


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