Extraction process of pancrelipase and evaluation thereof
The invention provided herein a pharmaceutical process used for an extraction of proteins from pancreatic sample and estimation of the extracted proteins. Moreover, the invention provides a use of suitable is selected from citrate-phosphate buffer and bicarbonate buffer capable to extract proteins from pancreatic sample. The invention further provides an analytical method to perform estimation of extracted proteins. This process provides an improved extraction method to quantify protein present in the pancreatic sample.
1 . A process for an extraction of a pancreatic protein from a pancreatic protein sample, the process comprising:
a. treating the pancreatic protein sample with a buffer;
b. dissolving the pancreatic protein sample in the buffer;
c. collecting the extracted pancreatic protein,
wherein the suitable buffer is selected from citrate-phosphate buffer and bicarbonate buffer;
wherein the pancreatic protein sample comprises a pharmaceutical composition comprising:
one or more enzymes selected from lipase, protease, and amylase; and
one or more enzymes selected from Triacylglycerol lipase, Co-lipase, CEL lipase, Phospholipase A2, Trypsin, Chymotrypsin, Elastase, Carboxypeptidase A1, Carboxypeptidase B, Kallikrien glandular, and Alpha amylase.
2 . The process according to claim 1 , wherein the extraction of the pancreatic protein is improved in comparison to an extraction process performed without using a suitable buffer selected from citrate-phosphate buffer and bicarbonate buffer.
3 . The process according to claim 1 , wherein:
the citrate-phosphate buffer concentration is selected from about 10 mM to about 200 mM; or
the bicarbonate buffer concentration is selected from about 10 mM to about 200 mM.
4 . The process according to claim 3 , wherein:
the citrate-phosphate buffer concentration is selected from about 100 mM; or
the bicarbonate buffer concentration is selected from about 100 mM.
5 . The process according to claim 1 , wherein the citrate phosphate buffer has a pH from about 6.0 to about 6.5.
6 . The process according to claim 1 , wherein of the bicarbonate buffer has a pH from about 9.5 to about 10.
7 . The process according to claim 1 , wherein dissolving the pancreatic protein sample in the buffer is performed by stirring or shaking.
8 . The process according to claim 7 , wherein stirring is performed at 200 rpm to 300 rpm.
9 . The process according to claim 8 , wherein stirring is performed for about 20 minutes to about 30 minutes.
10 . The process according to claim 1 , wherein the extracted pancreatic protein is further subjected to estimation of protein performed by using BCA, SDS-PAGE, CE-SDS, SE-HPLC, or RP-HPLC.
11 . The process according to claim 1 , wherein the extracted pancreatic protein is further subjected to quantification of protein performed by using SDS-PAGE, CE-SDS, SE-HPLC, or RP-HPLC.
12 . The process according to claim 1 , wherein the process provides more than about 80% yield of pancreatic protein.
13 . The process according to claim 1 , wherein the process provides more than about 90% yield of pancreatic protein.
14 . The process according to claim 1 , wherein the process provides more than about 98% yield of pancreatic protein.
15 . The process according to claim 1 , wherein the extracted pancreatic protein comprises a protein selected from amylase, lipase and protease.
16 . The process according to claim 1 , wherein the process provides a pharmaceutically acceptable pancreatic protein mixture comprising one or more enzymes selected from amylase, lipase and protease.
17 . The process according to claim 10 , wherein the extracted pancreatic protein is further subjected to estimation of protein based on molecular weight by using CE-SDS.
18 . The process according to claim 10 , wherein the extracted pancreatic protein is further subjected to estimation of purity profile by using RP-HPLC.
19 . The process according to claim 10 , wherein the extracted pancreatic protein is further subjected to estimation of protein based on molecular weight by using SDS-PAGE.
20 . The process according to claim 10 , wherein the extracted pancreatic protein is further subjected to estimation of purity profile by using SE-HPLC.