An improved method for separation and quantification of pancreatic enzymes by using ce-sds

Abstract

The present disclosure relates to a method for improving separation and quantification profile of pancreatic protein mixture comprising at least one enzyme amylase, protease, and lipase, trypsin, chymotrypsin, elastase and kallikrein glandular and variant thereof through capillary electrophoresis Sodium dodecyl sulfate (CE-SDS). Furthermore, the invention provides a stable pancreatic protein sample comprising substantially free of digested protein which makes the pancreatic sample more suitable for analytical characterization.

Description

[0001] TITLE: AN IMPROVED METHOD FOR SEPARATION AND QUANTIFICATION OF PANCREATIC ENZYMES BY USING CE-SDS

[0002] FIELD OF DISCLOSURE:

[0003] The present disclosure relates to a method for improving separation and quantification profile of pancreatic protein mixture comprising at least one enzyme amylase, protease, and lipase, trypsin, chymotrypsin, elastase and kallikrein glandular and variant thereof through capillary electrophoresis Sodium dodecyl sulfate (CE-SDS). More specifically present invention relates to a method that improves the separation efficiency and resolution of the proteins present in the pancreatic protein mixture selected from amylase, protease, and lipase, trypsin, chymotrypsin, elastase and kallikrein glandular by modulating gel matrix in capillary electrophoresis Sodium dodecyl sulfate (CE-SDS). Furthermore, the invention provides a stable pancreatic protein sample comprising substantially free of digested protein which makes the pancreatic sample more suitable for analytical characterization.

[0004] BACKGROUND OF INVENTION:

[0005] Pancreatic enzymes produced by the body are well known for the integral role they play in the digestion of the foods we eat. Pancreatic juice contains numerous enzymes, including amylase, lipase, protease, carboxyl ester lipase, and phospholipase, and the proenzymes trypsinogen, chymotrypsin, and pro-carboxypeptidase which are converted in the small intestine to their active forms, trypsin, chymotrypsin, and carboxypeptidase, respectively.

[0006] The one of the challenging tasks is to separate and quantify the proteins present in this pancreatic protein mixture. Ongoing efforts to improve the method for separation and quantification of the proteins leads to this invention to create a new and useful method. One such method is to separate and quantify proteins present in pancreatin is Capillary electrophoresis - sodium dodecyl sulfate (CE-SDS) which is known in the art to quantify protein fragments. However, pancreatic proteins are very fragile and susceptible to digestion that creates fragmentation which makes the CE-SDS analysis very challenging. Another problem lies with pancreatic proteins that it is complex mixture and many pancreatic proteins are closely associated due to their marginal different molecular weight which makes the CE-SDS profile complicated due to peak merging, poor resolution or separation that affect the quantification and analytical characterization to ensure the quality of the product.

[0007] The present invention successfully resolved all the problems and provides effective, improve CE- SDS method for the characterization or separation or quantification of pancreatic protein selected from protease, amylase, lipase, trypsin, chymotrypsin, elastase and kallikrein glandular and variant thereof. The invention provides a stable pancreatic protein sample comprising substantially free of digested protein which makes the pancreatic sample more suitable for analytical characterization. In present invention, while performing the CE-SDS to separate and quantify the proteins present in the pancreatic protein mixture, the gel matrix is modulated and maintain viscosity 75 cP and below resulting in improved resolution and the separation efficiency of the separated pancreatic proteins having weight not more than 65 kDa present in the pancreatic protein mixture.

[0008] SUMMARY OF INVENTION

[0009] The present invention provides a CE-SDS method that improves the separation of the proteins present in the sample of pancreatin protein mixture. The present invention also provides a CE-SDS method that improves the separation and / or quantification of the proteins present in the sample of pancreatin protein mixture.

[0010] In an embodiment, the present invention provides a CE-SDS method for improving the separation efficiency and resolution of the proteins present in the pancreatic protein mixture selected from protease, amylase, lipase, trypsin, chymotrypsin, elastase and kallikrein glandular and variant thereof by modulating gel matrix in capillary electrophoresis Sodium dodecyl sulfate (CE-SDS) wherein the gel matrix viscosity is 75cP and below.

[0011] In an embodiment, the present invention provides pancreatic protein mixture comprising two or more pancreatic protein selected from protease, amylase, lipase, trypsin, chymotrypsin, elastase and kallikrein glandular and variant thereof, wherein the pancreatic protein mixture does not contain pancreatic protein having molecular weight more than 65 KDa.

[0012] In an embodiment, the present invention provides a disclosure of pancreatic protein mixture treated with solvent to form a pre-treated pancreatic protein mixture suitable for analytical characterization and quantification through CE SDS technique, wherein, the suitable buffer is low pH phosphate buffer having pH below 7.

[0013] In an embodiment, the present invention provides a disclosure of pancreatic protein mixture treated with solvent and suitable buffer to form a stable pancreatic protein mixture suitable for analytical characterization and quantification through CE SDS technique, wherein, the suitable buffer is low pH phosphate buffer having pH below 7.

[0014] In an embodiment, the present invention provides a disclosure of stable pancreatic protein mixture treated with suitable buffer wherein, the suitable buffer is low pH phosphate buffer having pH below 7 wherein, the stable pancreatic protein mixture treated with low pH phosphate buffer has reduced amount of digested pancreatic proteins compared to the pancreatic protein mixture not treated with low pH phosphate buffer.

[0015] In an embodiment, the present invention provides a disclosure of a CE-SDS method for the separation of pancreatin protein presents in pancreatic protein mixture comprising; a. injecting the pancreatic protein mixture in CE-instrument, b. performing the CE-SDS; and, c. separating the at least more than two pancreatic proteins present in pancreatin protein mixture selected from protease, amylase, lipase, trypsin, chymotrypsin, elastase and kallikrein glandular; wherein, the CE-instrument comprises gel matrix having viscosity 75cP and below; wherein the gel matrix having viscosity 75cP and below improves the separation of the pancreatic protein mixture compared to pancreatic protein mixture separated through gel matrix having viscosity more than 75cP; wherein the separation of the pancreatic protein mixture improves resolution of the pancreatic proteins.

[0016] In an embodiment, the present invention provides a disclosure of a stable pancreatic protein mixture suitable for analytical characterization of pancreatin protein presents in pancreatic protein mixture comprising; a. treating the pancreatic protein mixture with suitable buffer to form stable pancreatic protein mixture; b. injecting the stable pancreatic protein mixture in CE-instrument, c. performing the CE-SDS; and, d. separating at least more than two pancreatic protein present in the pancreatin protein mixture selected from protease, amylase, lipase trypsin, chymotrypsin, elastase and kallikrein glandular; wherein, the suitable buffer is a low pH phosphate buffer having pH below 7; wherein, the stable pancreatic protein mixture has reduced amount of digested pancreatic proteins compared to the pancreatic protein mixture not treated with low pH phosphate buffer.

[0017] In another embodiment, of the present invention, the gel matrix in the CE-instrument is prepared by mixing SDS-MW gel with suitable modulator selected from water, SDS-MW buffer, and low pH phosphate buffer. In another aspect of the present invention, the gel matrix in the CE-instrument is prepared by mixing SDS-MW gel with low pH phosphate buffer wherein the gel matrix comprises SDS and low pH phosphate buffer in suitable ratio comprising SDS in higher amount than low pH phosphate buffer; wherein the gel matrix improves the separation of pancreatic protein selected from protease, amylase, lipase, trypsin, chymotrypsin, elastase and kallikrein glandular and variant thereof,.

[0018] In an embodiment, the gel matrix comprises SDS and suitable modulator in suitable ratio comprising SDS in higher amount than suitable modulator.

[0019] In another aspect of the present invention, the the gel matrix in the CE-instrument is prepared by mixing SDS-MW gel with low pH phosphate buffer wherein the gel matrix comprises SDS and low pH phosphate buffer in suitable ratio comprising SDS amount is at least two times or three times or four times higher amount than low pH phosphate buffer.

[0020] In another aspect of the present invention, the the gel matrix used in the CE-instrument is prepared by mixing SDS-MW gel with low pH phosphate buffer in a ratio of about 80:20, about 70:30 and about 40:60.

[0021] In an embodiment, the present invention provides a stable pancreatic protein mixture is injected in CE-instrument to separate two or more enzymes selected from protease, amylase, lipase trypsin, chymotrypsin, elastase and kallikrein glandular wherein CE-instrument comprising gel matrix has viscosity about 75cP or 75 cP and below suitable to separate pancreatic protein wherein, the gel matrix is prepared by mixing SDS-MW gel with low pH phosphate buffer in suitable ratio wherein, the suitable ratio comprising SDS in higher amount than low pH phosphate buffer.

[0022] In an embodiment, the present invention provides a CE-SDS method for the separation of pancreatin protein presents in pancreatic protein mixture comprising; a. preparing or modulating the gel matrix used CE instrument having viscosity about 75cP or 75 cP and below; b. injecting the pancreatic protein mixture in CE-instrument, c. performing the CE-SDS; and, d. separating at least more than two pancreatic protein presents in pancreatin protein mixture selected from protease, amylase, lipase trypsin, chymotrypsin, elastase and kallikrein glandular; wherein, the separation of the pancreatic protein mixture is improved by modulating gel matrix having viscosity 75cP and 75 cP and below compared to pancreatic protein mixture separated through gel matrix having viscosity more than 75cP; wherein the separation of the pancreatic protein mixture improves resolution of the pancreatic proteins.

[0023] In an embodiment, the present invention provides CE SDS method for the separation of pancreatin protein presents in pancreatic protein mixture comprising; a. injecting the pancreatic protein mixture in CE-instrument, b. performing the CE-SDS; and, c. separating the at least more than two pancreatic protein present in pancreatin protein mixture selected from protease, amylase, lipase trypsin, chymotrypsin, elastase and kallikrein glandular; wherein, the CE-instrument comprises gel matrix having viscosity 75cP and below; wherein, the gel matrix having viscosity 75cP and below improves the separation of the pancreatic protein mixture compared to pancreatic protein mixture separated through gel matrix having viscosity more than 75cP; wherein, the separation of the pancreatic protein mixture improves resolution of the pancreatic proteins.

[0024] In an embodiment, the invention provides a method for improving the separation of pancreatic proteins present in the pancreatic protein mixture comprising: a. pancreatic protein mixture comprising two or more pancreatic protein selected from protease, amylase, lipase, trypsin, chymotrypsin, elastase and kallikrein glandular and variant thereof, wherein the pancreatic protein mixture does not contain pancreatic protein having molecular weight more than 65 KDa; b. treating the pancreatic protein mixture with suitable solvent and buffer to form treated pancreatic protein mixture; c. injecting the treated protein mixture in CE-instrument, d. performing the CE-SDS; and, e. separating at least two enzymes present in pancreatin protein mixture; wherein, the CE-instrument comprising a gel matrix has viscosity 75cP and below; wherein, the gel matrix comprises SDS and suitable modulator in suitable ratio comprising SDS in higher amount than suitable modulator; wherein, the suitable modulator comprises a buffer having pH below 7 ; wherein, the gel matrix is suitable to separate at least two enzymes present in pancreatin protein mixture selected from protease, amylase, lipase trypsin, chymotrypsin, elastase and kallikrein glandular.

[0025] In an embodiment, the invention provides a method for improving the separation of pancreatic proteins present in the pancreatic protein mixture comprising: a. pancreatic protein mixture comprising pancreatic protein selected from protease, amylase, lipase, trypsin, chymotrypsin, elastase and kallikrein glandular and variant thereof, wherein the pancreatic protein mixture does not contain pancreatic protein having molecular weight more than 65 KDa; b. treating the pancreatic protein mixture with suitable solvent and buffer to form treated pancreatic protein mixture; c. injecting the treated protein mixture in CE-instrument, d. performing the CE-SDS; and, e. separating at least two enzymes present in pancreatin protein mixture; wherein, the CE-instrument comprising a gel matrix has viscosity 75cP and below; wherein, the gel matrix comprises SDS and low pH phosphate buffer in suitable ratio having SDS at least two time higher than low pH phosphate buffer. wherein, the low pH phosphate buffer having pH below 7 preferably about pH 6.5; wherein, the gel matrix is suitable to separate at least two enzymes present in pancreatin protein mixture selected from protease, amylase, lipase trypsin, chymotrypsin, elastase and kallikrein glandular.

[0026] In an embodiment, the invention provides a method for improving the separation of pancreatic proteins present in the pancreatic protein mixture comprising: a. pancreatic protein mixture comprising pancreatic protein selected from protease, amylase, lipase, trypsin, chymotrypsin, elastase and kallikrein glandular and variant thereof, wherein the pancreatic protein mixture does not contain pancreatic protein having molecular weight more than 65 KDa; b. treating the pancreatic protein mixture with suitable solvent and buffer to form treated pancreatic protein mixture; c. injecting the treated protein mixture in CE-instrument, d. performing the CE-SDS; and, e. separating at least two enzymes present in pancreatin protein mixture; wherein, the CE-instrument comprising a gel matrix has viscosity 75cP and below; wherein, the gel matrix comprises SDS and suitable modulator in suitable ratio comprising SDS in higher amount than suitable modulator; wherein, the suitable modulator comprises a buffer having pH below 7 ; wherein, the gel matrix is suitable to separate protease, amylase, lipase trypsin, chymotrypsin, elastase and kallikrein glandular present in pancreatin protein mixture.

[0027] In an embodiment the invention provides a method to improve resolution of pancreatic proteins selected from lipase trypsin, chymotrypsin, elastase and kallikrein glandular.

[0028] In an embodiment, the invention provides a method for improving the separation of pancreatic proteins present in the pancreatic protein mixture comprising: a. pancreatic protein mixture comprising pancreatic protein selected from protease, amylase, lipase, trypsin, chymotrypsin, elastase and kallikrein glandular and variant thereof, wherein the pancreatic protein mixture does not contain pancreatic protein having molecular weight more than 65 KDa; b. treating the pancreatic protein mixture with suitable solvent and buffer to form treated pancreatic protein mixture; c. injecting the treated protein mixture in CE-instrument, d. performing the CE-SDS; and, e. separating at least two enzymes present in pancreatin protein mixture; wherein, the CE-instrument comprising a gel matrix has viscosity 75cP and below; wherein, the gel matrix comprises SDS and low pH phosphate buffer in suitable ratio comprising SDS in higher amount than suitable modulator; wherein, the low pH phosphate buffer having pH below 7 preferably about pH 6.5; wherein, the gel matrix is suitable to separate at least two enzymes present in pancreatin protein mixture selected from protease, amylase, lipase trypsin, chymotrypsin, elastase and kallikrein glandular. wherein, the process provides an improved resolution of pancreatic proteins.

[0029] In an embodiment, the invention provides a method for improving the separation of pancreatic proteins present in the pancreatic protein mixture comprising: a. pancreatic protein mixture comprising pancreatic protein selected from protease, amylase, lipase, trypsin, chymotrypsin, elastase and kallikrein glandular and variant thereof, wherein the pancreatic protein mixture does not contain pancreatic protein having molecular weight more than 65 KDa; b. treating the pancreatic protein mixture with suitable solvent and buffer to form treated pancreatic protein mixture; c. injecting the treated protein mixture in CE-instrument, d. performing the CE-SDS; and, e. separating at least two enzymes present in pancreatin protein mixture; wherein, the CE-instrument comprising a gel matrix has viscosity 75cP and below; wherein, the gel matrix comprises SDS and low pH phosphate buffer in suitable ratio having 80:20; wherein, the low pH phosphate buffer having pH below 7 preferably about pH 6.5; wherein, the gel matrix is suitable to separate at least two enzymes present in pancreatin protein mixture selected from protease, amylase, lipase trypsin, chymotrypsin, elastase and kallikrein glandular. wherein, the process provides an improved resolution of pancreatic proteins selected from lipase trypsin, chymotrypsin, elastase and kallikrein glandular.

[0030] In another embodiment the invention further improved resolution of pancreatic protein present in pancreatic mixture selected from co-lipase, phospholipase A2, elastase, Trypsin, Kallikrein glandular, chymotrypsin, carboxypeptidase B, Carboxypeptidase A, Amylase and Carboxy ester lipase (CEL).

[0031] In an embodiment, the invention provides a method for improving the quantification of pancreatic proteins present in the pancreatic protein mixture comprising: a. pancreatic protein mixture comprising two or more pancreatic protein selected from protease, amylase, lipase, trypsin, chymotrypsin, elastase and kallikrein glandular and variant thereof, wherein the pancreatic protein mixture does not contain pancreatic protein having molecular weight more than 65 KDa; b. treating the pancreatic protein mixture with suitable solvent and buffer to form treated pancreatic protein mixture; c. injecting the treated protein mixture in CE-instrument, d. performing the CE-SDS; and, e. separating and detecting at least two enzymes present in pancreatin protein mixture; wherein, the CE-instrument comprising a gel matrix has viscosity below 75cP; wherein, the gel matrix comprises SDS and suitable modulator in suitable ratio comprising SDS in higher amount than suitable modulator; wherein, the suitable modulator comprises a buffer having pH below 7 ; wherein, the gel matrix is suitable to separate at least two enzymes present in pancreatin protein mixture selected from protease, amylase, lipase trypsin, chymotrypsin, elastase and kallikrein glandular.

[0032] In an embodiment, the invention provides a method for improving the separation of pancreatic proteins present in the pancreatic protein mixture comprising: a. pancreatic protein mixture comprising two or more pancreatic protein selected from protease, amylase, lipase, trypsin, chymotrypsin, elastase and kallikrein glandular colipase, phospholipase A2, elastase, Trypsin, Kallikrein glandular, chymotrypsin, carboxypeptidase B, Carboxypeptidase A, Amylase and Carboxy ester lipase (CEL) and variant thereof, wherein the pancreatic protein mixture does not contain pancreatic protein having molecular weight more than 65 KDa; b. treating the pancreatic protein mixture with suitable solvent and buffer to form treated pancreatic protein mixture; c. injecting the treated protein mixture in CE-instrument, d. performing the CE-SDS; and, e. separating at least two enzymes present in pancreatin protein mixture; wherein, the separation efficiency of the separated pancreatic enzymes is improved compared to pancreatic protein mixture separated through CE SDS without treating with suitable buffer and solvent; wherein, the buffer is low pH phosphate buffer comprising pH below 7.

[0033] In an embodiment, the invention provides a method for improving the quantification of pancreatic proteins present in the pancreatic protein mixture comprising: a. pancreatic protein mixture comprising two or more pancreatic protein selected from protease, amylase, lipase, trypsin, chymotrypsin, elastase and kallikrein glandular, colipase, phospholipase A2, elastase, Trypsin, Kallikrein glandular, chymotrypsin, carboxypeptidase B, Carboxypeptidase A, Amylase and Carboxy ester lipase (CEL) and variant thereof, wherein the pancreatic protein mixture does not contain pancreatic protein having molecular weight more than 65 KDa; b. treating the pancreatic protein mixture with solvent and low pH phosphate buffer to form treated pancreatic protein mixture; c. injecting the treated protein mixture in CE-instrument, d. performing the CE-SDS; and, e. separating and detecting at least two enzymes present in pancreatin protein mixture; wherein, the quantification of the separated pancreatic enzymes is improved compared to pancreatic protein mixture separated through CE SDS without treating with suitable buffer and solvent; wherein, the buffer is low pH phosphate buffer comprising pH below 7.

[0034] In an embodiment, the pancreatic protein mixture is pre-treated with organic solvent, wherein organic solvent is selected from carbon tetrachloride, trichloroethylene, Diethyl ether, Dichloroethane, chlorobenzene, cyclohexane, 1,2-dichloroethene, Dichloromethane, 1,2- Dichloromethane, N,N- Dimethylacetamide, 1,4- dioxane, 2- Ethoxyethanol, Ethylene glycol, Formamide, Hexane, Methanol, 2-Methoxyethanol, Methylbutylketone, pyridine, Sulfolane, Tetralin, Toluene, hydrochloric acid, NaOH, phosphate buffer, water, citrate phosphate buffer, and trifluoroacetic acid (TFA) in acetonitrile (ACN).

[0035] In an embodiment, pancreatic protein mixture is pre-treated with organic solvent in the concentration of about 0.5% trifluoroacetic acid (TFA) in 25% Acetonitrile (ACN), about 0.2% trifluoroacetic acid (TFA) in 25% Acetonitrile (ACN), about 0.1% trifluoroacetic acid (TFA) in 25% Acetonitrile (ACN).

[0036] In an embodiment the pancreatic protein treated with solvent improves the resolution of pancreatic protein or enzyme during CE-SDS.

[0037] In another embodiment, pre-treated pancreatic protein mixture is treated with buffer and mixed with suitable alkylating agent, wherein buffer is selected from but not limited to Sodium dodecyl sulfate- molecular weight sample buffer, low pH Sodium dodecyl sulfate- molecular weight sample buffer and low pH Phosphate SDS Sample buffer, citrate phosphate buffer, Ammonium phosphate buffer, ammonium formate buffer.

[0038] In an embodiment, the present disclosure is related to an improved method for separation of pancreatic proteins present in the pancreatic protein mixture comprising two or more enzymes comprising: a. pancreatic proteins comprising two or more enzymes selected from protease, amylase, lipase trypsin, chymotrypsin, elastase and kallikrein glandular, co-lipase, phospholipase A2, elastase, Trypsin, Kallikrein glandular, chymotrypsin, carboxypeptidase B, Carboxypeptidase A, Amylase and Carboxy ester lipase (CEL) wherein, the protein mixture does not contain protein having molecular weight more than 65kDa; b. treating the pancreatic protein with suitable solvent and buffer to form treated pancreatic protein mixture; a. injecting the treated protein mixture in CE-instrument comprises gel matrix having viscosity 75 cP and below; c. performing CE-SDS; and, d. separating the pancreatic enzymes; wherein, the separation of the pancreatic enzymes is improved by modulating gel matrix having viscosity 75 cP and below compared to pancreatic protein mixture separated through gel matrix having viscosity more than 75cP; wherein the separation of the pancreatic protein mixture improves resolution of the pancreatic proteins.

[0039] In an embodiment the invention provides a disclosure of the separation and / or resolution of one and more pancreatic enzymes selected from co-lipase, phospholipase A2, elastase, Trypsin, Kallikrein glandular, chymotrypsin, carboxypeptidase B, Carboxypeptidase A, Amylase and Carboxy ester lipase (CEL).

[0040] In an embodiment, the present disclosure is related to a method to improve the separation and resolution of pancreatic proteins present in the pancreatic protein mixture comprising: a. pancreatic protein mixture comprising two or more pancreatic enzymes selected from protease, amylase, lipase, trypsin, chymotrypsin, elastase and kallikrein glandular wherein the pancreatic protein mixture does not contain pancreatic protein having molecular weight more than 65 KDa; b. treating the pancreatic protein mixture with suitable buffer and solvent to form treated pancreatic protein mixture; c. injecting the treated protein mixture in CE-instrument; d. performing the CE-SDS; and, e. separating the one or more enzymes present in pancreatin protein mixture; wherein the separation improves resolution of one or more pancreatic enzyme as follow;

[0041] (i) lipase peak is obtained in chromatogram at about 25 minutes to about 42 minutes during CE-SDS at separation voltage between 6 KV to about 10 KV; (ii) protease peak is obtained in chromatogram at about 26 minutes to about 37 minutes during CE-SDS at separation voltage between 6 KV to about 10 KV;

[0042] (iii) amylase peak is obtained in chromatogram at about 32 minutes to about 42 minutes during CE-SDS at separation voltage between 6KV to about 10 KV;

[0043] (iv) trypsin peak is obtained in chromatogram at about 29 minutes to about 31 minutes during CE-SDS at separation voltage between 6KV to about 10 KV;

[0044] (v) elastase peak is obtained in chromatogram at about 29 minutes to about 31 minutes during CE-SDS at separation voltage between 6KV to about 10 KV;

[0045] (vi) chymotrypsin peak is obtained in chromatogram at about 29 minutes to about 31 minutes during CE-SDS at separation voltage between 6KV to about 10 KV;

[0046] (vii) kallikrein glandular peak is obtained in chromatogram at about 29 minutes to about 31 minutes during CE-SDS at separation voltage between 6KV to about 10 KV.

[0047] In an embodiment, the present disclosure is related to a method to improve the separation, detection of pancreatic proteins present, in the pancreatic protein mixture comprising: a. pancreatic protein mixture comprising two or more pancreatic enzymes selected from protease, amylase, lipase, trypsin, chymotrypsin, elastase and kallikrein glandular wherein the pancreatic protein mixture does not contain pancreatic protein having molecular weight more than 65 KDa; b. treating the pancreatic protein mixture with suitable buffer and solvent to form treated pancreatic protein mixture; c. injecting the treated protein mixture in CE-instrument; d. performing the CE-SDS; and, e. separating and detecting the one or more enzymes present in pancreatin protein mixture; wherein the separation improves resolution of one or more pancreatic enzyme as follow; i. lipase peak is obtained in chromatogram at about 25 minutes to about 42 minutes during CE- SDS at separation voltage between 6 KV to about 10 KV; ii. protease peak is obtained in chromatogram at about 26 minutes to about 37 minutes during CE- SDS at separation voltage between 6 KV to about 10 KV; iii. amylase peak is obtained in chromatogram at about 32 minutes to about 42 minutes during CE- SDS at separation voltage between 6KV to about 10 KV; iv. trypsin peak is obtained in chromatogram at about 29 minutes to about 31 minutes during CE- SDS at separation voltage between 6KV to about 10 KV; v. elastase peak is obtained in chromatogram at about 29 minutes to about 31 minutes during CE- SDS at separation voltage between 6KV to about 10 KV; vi. chymotrypsin peak is obtained in chromatogram at about 29 minutes to about 31 minutes during

[0048] CE-SDS at separation voltage between 6KV to about 10 KV; vii. kallikrein glandular peak is obtained in chromatogram at about 29 minutes to about 31 minutes during CE-SDS at separation voltage between 6KV to about 10 KV.

[0049] In an embodiment, the present disclosure is related to a method to improve the separation, detection of pancreatic proteins present, in the pancreatic protein mixture comprising: a. pancreatic protein mixture comprising two or more pancreatic enzymes selected from protease, amylase, lipase, trypsin, chymotrypsin, elastase and kallikrein glandular wherein the pancreatic protein mixture does not contain pancreatic protein having molecular weight more than 65 KDa; b. treating the pancreatic protein mixture with suitable buffer and solvent to form treated pancreatic protein mixture; c. injecting the treated protein mixture in CE-instrument; d. performing the CE-SDS; and, e. separating and / or detecting the one or more enzymes present in pancreatin protein mixture according to figure 5(a).

[0050] In an embodiment the invention discloses a CE SDS method provides major peaks for one or more pancreatic protein selected from protease, amylase, lipase, trypsin, chymotrypsin, elastase, kallikrein glandular, co-lipase, phospholipase A2 (PLA2), Carboxy ester lipase (CEL) and Carboxypeptidase B.

[0051] In an embodiment the invention discloses a CE SDS method provides at least major peaks for pancreatic protein selected from trypsin, chymotrypsin, elastase, kallikrein glandular.

[0052] DESCRIPTION OF THE FIGURES:

[0053] Figure 1: Shows the separation profile of pancreatic proteins through CE-SDS when pancreatic proteins are mixed in citrate phosphate buffer at pH 6.2 and pre-treated with SDS MW Sample buffer as described in the example 1.

[0054] Figure 2: Shows the separation profile of pancreatic proteins through CE-SDS when pancreatic proteins are mixed in different solvents and pre-treated with SDS MW Sample buffer as described in the example 2. Figure 3: Shows the separation profile of pancreatic proteins through CE-SDS when pancreatic proteins are mixed in different concentration of trifluoroacetic acid (TFA) in acetonitrile (ACN) and pre-treated with different buffers as described in the example 3.

[0055] Figure 4: Shows the comparative separation profile of pancreatic proteins as sample with Reference product through CE-SDS performed with modulated gel matrix as described in the example 4.

[0056] Figure 5 (a): Shows the separation profile of pancreatic proteins through CE-SDS wherein CE- instrument comprising a gel matrix is modulated comprising viscosity 75 cP and below as described in the example 5.

[0057] Figure 5 (b): Shows the separation profile of pancreatic proteins through CE-SDS wherein CE- instrument comprising commercially available gel matrix comprising viscosity above 75cP as described in the example 5.

[0058] DETAILED DESCRIPTION OF THE INVENTION:

[0059] In order that the present disclosure can be more readily understood, certain terms are first defined. As used in this application, except as otherwise expressly provided herein, each of the following terms shall have the meaning set forth below. Additional definitions are set forth throughout the application.

[0060] Unless the context clearly requires otherwise, throughout the invention, the words “comprise”, “comprising”, and the like are to be construed in an inclusive sense as opposed to an exclusive or exhaustive sense; that is to say, in the sense of “including, but not limited to”.

[0061] The term “about” as used herein is intended to refer to ranges of approximately 5% to 20% greater than or less than the referenced value. In certain circumstances, one skill in the art will recognize that, due to the nature of the referenced value, the term about can mean more or less than 5% to 20% deviation from that value.

[0062] The term “below” or “less than” are interchangeable herein for an example 75 cP and below should be interpreted less than 75cP. However, in certain embodiment gel viscosity below or less than 75cP includes 75 cP.

[0063] The term “pancrelipase samples” or “pancreatic sample” or “pancreatic protein mixture”, are interchangeable with each other and herein refers to pancreatic digestive enzymes which is purified or semi purified and further formulated in any pharmaceutical composition. In an embodiment, the pancrelipase sample is selected from granules, tablet, capsules, and powder. The “pancrelipase samples” or “pancreatic sample” or “pancreatic protein mixture”, comprises at least one or more enzyme selected from lipase, protease, amylase, and combination thereof. In an embodiment, the “pancrelipase samples” or “pancreatic sample” or “pancreatic protein mixture”, obtained from crude, partially purified, substantially purified and microbially synthesize. The pancreatic protein mixture is pre-treated with suitable solvent before treating the sample mixture with suitable buffer. In certain embodiment the pre-treated pancreatic protein sample is characterized by CE SDS. In an embodiment the pancreatic protein treated with solvent improves the resolution of pancreatic protein or enzyme during CE-SDS.

[0064] The term “organic solvent” refers to solvent selected from but not limited to carbon tetrachloride, trichloroethylene, Diethyl ether, Dichloroethane, chlorobenzene, cyclohexane, 1,2-dichloroethene, Dichloromethane, 1,2- Dichloromethane, N,N- Dimethylacetamide, 1,4- dioxane, 2- Ethoxyethanol, Ethylene glycol, Formamide, Hexane, Methanol, 2-Methoxyethanol, Methylbutylketone, pyridine, Sulfolane, Tetralin, Toluene, citrate phosphate buffer, and trifluoroacetic acid (TFA) in acetonitrile (ACN).

[0065] The term “pre-treated pancreatic protein sample” or “pre-treated pancreatic proteins” or “pretreated pancreatic protein mixture” are interchangeable with each other and herein refers to pancreatic protein mixture prepared by dissolving pancreatic protein mixture in an organic solvent.

[0066] The term “stable pancreatic protein mixture” is prepared by mixing the pancreatic protein mixture with suitable buffer. In an embodiment the stable pancreatic protein mixture optionally treated with solvent. The stability of the pancreatic protein mixture is achieved by treating with buffer. In an embodiment the treating sample with suitable buffer improves the stability of the sample by reducing proteolytic and / or autocatalytic activity in the pancreatic sample thereby reduces formation of digestive protein or enzyme.

[0067] The term “digestive protein” or “digestive pancreatin protein” or “digestive pancreatin enzyme” are interchangeable with each other and herein refers to truncated or degraded pancreatic protein.

[0068] The term “treated pancreatic protein solution” or “treated pancreatic protein sample” or “treated pancreatic protein mixture” are interchangeable with each other and herein refers to as the protein mixture prepared by mixing the pre-treated pancreatic protein mixture with suitable buffer.

[0069] The term “buffer” refers to buffer selected from but not limited to Sodium dodecyl sulfate- molecular weight sample buffer, Sodium dodecyl sulfate- molecular weight sample buffer and low pH Phosphate SDS Sample buffer, citrate phosphate buffer, Ammonium phosphate buffer, ammonium formate buffer. The term “low pH phosphate buffer” or “low pH phosphate SDS buffer” or “low pH phosphate SDS sample buffer” are interchangeable herein.

[0070] The “SDS-MW buffer” or “Sodium dodecyl sulfate- molecular weight sample buffer” herein is prepared by mixing 100 mM Tris-HCl, pH 9.0, in 1% SDS.

[0071] In an embodiment, the pancreatic sample contains “enzymes” or “pancreatic enzymes” or “pancreatic proteins” are interchangeable herein and are selected from protease, amylase, lipase, Triacylglycerol lipase, Co-lipase, carboxyl ester lipase (CEL lipase), Phospholipase A2, Trypsin, Chymotrypsin, Elastase, Carboxypeptidase Al or Carboxypeptidase A , Carboxypeptidase B, kallikrein glandular, and Alpha amylase are the prominent functionally important enzymes.

[0072] The term “variant” refers to a member of a set of highly similar or identical proteins that originate from a single gene or gene family and are the result of genetic differences. In an embodiment, the protein variant is at least 70% or about 75% or about 80% or about 85% or about 90% or about 91% or about 92% or about 93% or about 94% or about 95% or about 96% or about 97% or about 98% and about 99% identical or similar to protein of interest.

[0073] The term “amylase” refers to an enzyme that helps in the chemical process of digestion by hydrolysing starch into more available saccharide forms.

[0074] The term “lipase” refers to an enzyme that is secreted by the pancreas and is responsible for the hydrolysis of dietary fat molecules in the human digestive system. Lipases comprises of Triacylglycerol lipase, Co-lipase, Carboxy ester lipase, and phospholipase A2 (PLA 2).

[0075] The term “protease” refers to an enzyme that breaks down proteins into smaller polypeptides or amino acids. The protease comprises trypsin, chymotrypsin, elastase, kallikrein glandular, Carboxypeptidase Al, Carboxypeptidase B, and chymotrypsin.

[0076] The term “untreated pancreatic protein mixture” or “not treated pancreatic protein mixture” herein refers to the pancreatic protein mixture which is not treated with suitable solvent and buffer.

[0077] The “CE-instrument” or “capillary electrophoresis instrument” herein refers to a device that uses a capillary to separate and analyse charged compounds in small sample volumes. The Capillary electrophoresis (CE) is a technique that uses an electric field to separate proteins by charge and size in a capillary tube. The “analytical characterization” herein refers to the process of analysing the properties and biological functions of proteins and separating them for identification.

[0078] The “gel matrix” herein refers to a sieving polymer that separates proteins by molecular weight and hydrodynamic radius in capillary gel electrophoresis (CE-SDS). Herein the Gel Matrix is modulated by mixing SDS-MW gel with suitable modulator. In certain embodiment the CE SDS gel matrix provides major peaks for one or more pancreatic protein selected from protease, amylase, lipase, trypsin, chymotrypsin, elastase, kallikrein glandular, co-lipase, phospholipase A2 (PLA2), Carboxy ester lipase (CEL) and Carboxypeptidase B. In certain embodiment the CE SDS gel matrix provides major peaks at least for pancreatic protein selected from chymotrypsin, elastase, kallikrein glandular.

[0079] The term “insoluble particles” refers to cell debris remained during the preparation of pancreatic protein sample mixture.

[0080] The present invention provides a CE-SDS method that improves the separation of the proteins present in the sample of pancreatin protein mixture. The present invention also provides a CE-SDS method that improves the separation and / or quantification of the proteins present in the sample of pancreatin protein mixture.

[0081] In an embodiment, the present invention provides a CE-SDS method for improving the separation efficiency and resolution of the proteins present in the pancreatic protein mixture selected from protease, amylase, lipase, trypsin, chymotrypsin, elastase and kallikrein glandular and variant thereof by modulating gel matrix in capillary electrophoresis Sodium dodecyl sulfate (CE-SDS) wherein the gel matrix viscosity is 75 cP and below.

[0082] In an embodiment, the present invention provides pancreatic protein mixture comprising two or more pancreatic protein selected from protease, amylase, lipase, trypsin, chymotrypsin, elastase and kallikrein glandular and variant thereof, wherein the pancreatic protein mixture does not contain pancreatic protein having molecular weight more than 65 KDa.

[0083] In an embodiment, the present invention disclosure of pancreatic protein mixture treated with suitable buffer and solvent to form a stable pancreatic protein mixture suitable for analytical characterization and quantification, wherein, the suitable buffer is low pH phosphate buffer having pH below 7.

[0084] In an embodiment, the present invention disclosure of stable pancreatic protein mixture treated with suitable buffer wherein, the suitable buffer is low pH phosphate buffer having pH below 7 wherein, the stable pancreatic protein mixture treated with low pH phosphate buffer has reduced amount of digested pancreatic proteins compared to the pancreatic protein mixture not treated with low pH phosphate buffer. In an embodiment, the invention provides a method for improving the separation of pancreatic proteins present in the pancreatic protein mixture comprising: a. pancreatic protein mixture comprising pancreatic protein selected from protease, amylase, lipase, trypsin, chymotrypsin, elastase and kallikrein glandular and variant thereof, wherein the pancreatic protein mixture does not contain pancreatic protein having molecular weight more than 65 KDa; b. treating the pancreatic protein mixture with suitable solvent and buffer to form treated pancreatic protein mixture; c. injecting the treated protein mixture in CE-instrument, d. performing the CE-SDS; and, e. separating at least two enzymes present in pancreatin protein mixture; wherein, the CE-instrument comprising a gel matrix has viscosity 75 cP and below; wherein, the gel matrix comprises SDS and low pH phosphate buffer in suitable ratio having SDS at least two time higher than low pH phosphate buffer. wherein, the low pH phosphate buffer having pH below 7 preferably about pH 6.5; wherein, the gel matrix is suitable to separate at least two enzymes present in pancreatin protein mixture selected from protease, amylase, lipase trypsin, chymotrypsin, elastase and kallikrein glandular.

[0085] In an embodiment, the present invention provides a disclosure of a stable pancreatic protein mixture suitable for analytical characterization of pancreatin proteins presents in pancreatic protein mixture comprising; a. treating the pancreatic protein mixture with suitable buffer to form stable pancreatic protein mixture; b. injecting the stable pancreatic protein mixture in CE-instrument, c. performing the CE-SDS; and, d. separating at least more than two pancreatic protein present in the pancreatin protein mixture selected from protease, amylase, lipase trypsin, chymotrypsin, elastase and kallikrein glandular; wherein, the suitable buffer is a low pH phosphate buffer having pH below 7; wherein, the stable pancreatic protein mixture has reduced amount of digested pancreatic proteins compared to the pancreatic protein mixture not treated with low pH phosphate buffer. In another embodiment, of the present invention, the gel matrix in the CE-instrument is prepared by mixing SDS-MW gel with suitable modulator selected from water, SDS-MW buffer, and low pH phosphate buffer.

[0086] In another aspect of the present invention, the gel matrix in the CE-instrument is prepared by mixing SDS-MW gel with low pH phosphate buffer wherein the gel matrix comprises SDS and low pH phosphate buffer in suitable ratio comprising SDS in higher amount than low pH phosphate buffer; wherein the gel matrix improves the separation of pancreatic protein selected from protease, amylase, lipase, trypsin, chymotrypsin, elastase and kallikrein glandular and variant thereof.

[0087] In an embodiment, the gel matrix comprises SDS and suitable modulator in suitable ratio comprising SDS in higher amount than suitable modulator.

[0088] In another aspect of the present invention, the the gel matrix in the CE-instrument is prepared by mixing SDS-MW gel with low pH phosphate buffer wherein the gel matrix comprises SDS and low pH phosphate buffer in suitable ratio comprising SDS amount is at least two times or three times or four times higher amount than low pH phosphate buffer.

[0089] In another aspect of the present invention, the the gel matrix used in the CE-instrument is prepared by mixing SDS-MW gel with low pH phosphate buffer in a ratio of about 80:20, about 70:30 and about 40:60.

[0090] In an embodiment, the present invention provides a stable pancreatic protein mixture is injected in CE-instrument to separate two or more enzymes selected from protease, amylase, lipase trypsin, chymotrypsin, elastase and kallikrein glandular wherein CE-instrument comprising gel matrix has viscosity 75 cP and below suitable to separate pancreatic protein wherein, the gel matrix is prepared by mixing SDS-MW gel with low pH phosphate buffer in suitable ratio wherein, the suitable ratio comprising SDS in higher amount than low pH phosphate buffer

[0091] In an embodiment, the present invention provides an improved separation of pancreatic protein mixture using CE-SDS comprising modulated gel matrix having viscosity 75 cP and below suitable to separate pancreatic proteins selected from protease, amylase, lipase trypsin, chymotrypsin, elastase and kallikrein glandular wherein, the separation efficiency and resolution of the pancreatic protein mixture is improved by modulating gel matrix having viscosity 75 cP and below compared to pancreatic protein mixture separated through gel matrix having viscosity more than 75cP. In an embodiment, the present invention provides a CE-SDS method for the separation of pancreatin protein presents in pancreatic protein mixture comprising; a. preparing or modulating the gel matrix used CE instrument having viscosity 75 cP and below; b. injecting the pancreatic protein mixture in CE-instrument, c. performing the CE-SDS; and, d. separating at least more than two pancreatic protein presents in pancreatin protein mixture selected from protease, amylase, lipase trypsin, chymotrypsin, elastase and kallikrein glandular; wherein, the separation of the pancreatic protein mixture is improved by modulating gel matrix having viscosity 75 cP and below compared to pancreatic protein mixture separated through gel matrix having viscosity more than 75cP; wherein, the separation of the pancreatic protein mixture improves resolution of the pancreatic proteins.

[0092] In an embodiment, the present invention provides CE SDS method for the separation of pancreatin protein presents in pancreatic protein mixture comprising; a. injecting the pancreatic protein mixture in CE-instrument, b. performing the CE-SDS; and, c. separating the at least more than two pancreatic protein present in pancreatin protein mixture selected from protease, amylase, lipase trypsin, chymotrypsin, elastase and kallikrein glandular; wherein, the CE-instrument comprises gel matrix having viscosity 75 cP and below; wherein the gel matrix having viscosity 75 cP and below improves the separation of the pancreatic protein mixture compared to pancreatic protein mixture separated through gel matrix having viscosity more than 75cP; wherein, the separation of the pancreatic protein mixture improves resolution of the pancreatic proteins.

[0093] In an embodiment, the invention provides a method for improving the separation of pancreatic proteins present in the pancreatic protein mixture comprising: a. pancreatic protein mixture comprising two or more pancreatic protein selected from protease, amylase, lipase, trypsin, chymotrypsin, elastase and kallikrein glandular and variant thereof, wherein the pancreatic protein mixture does not contain pancreatic protein having molecular weight more than 65 KDa; b. treating the pancreatic protein mixture with suitable solvent and buffer to form treated pancreatic protein mixture; c. injecting the treated protein mixture in CE-instrument, d. performing the CE-SDS; and, e. separating at least two enzymes present in pancreatin protein mixture; wherein, the CE-instrument comprising a gel matrix has viscosity 75 cP and below; wherein, the gel matrix comprises SDS and suitable modulator in suitable ratio comprising SDS in higher amount than suitable modulator; wherein, the suitable modulator comprises a buffer having pH below 7 ; wherein, the gel matrix is suitable to separate at least two enzymes present in pancreatin protein mixture selected from protease, amylase, lipase trypsin, chymotrypsin, elastase and kallikrein glandular.

[0094] In an embodiment, the invention provides a method for improving the separation of pancreatic proteins present in the pancreatic protein mixture comprising: a. pancreatic protein mixture comprising pancreatic protein selected from protease, amylase, lipase, trypsin, chymotrypsin, elastase and kallikrein glandular and variant thereof, wherein the pancreatic protein mixture does not contain pancreatic protein having molecular weight more than 65 KDa; b. treating the pancreatic protein mixture with suitable solvent and buffer to form treated pancreatic protein mixture; c. injecting the treated protein mixture in CE-instrument, d. performing the CE-SDS; and, e. separating at least two enzymes present in pancreatin protein mixture; wherein, the CE-instrument comprising a gel matrix has viscosity 75 cP and below; wherein, the gel matrix comprises SDS and low pH phosphate buffer in suitable ratio comprising SDS in higher amount than suitable modulator; wherein, the low pH phosphate buffer having pH below 7 preferably about pH 6.5; wherein, the gel matrix is suitable to separate at least two enzymes present in pancreatin protein mixture selected from protease, amylase, lipase trypsin, chymotrypsin, elastase and kallikrein glandular. wherein, the process provides an improved resolution of pancreatic proteins. In an embodiment, the invention provides a method for improving the separation of pancreatic proteins present in the pancreatic protein mixture comprising: a. pancreatic protein mixture comprising pancreatic protein selected from protease, amylase, lipase, trypsin, chymotrypsin, elastase and kallikrein glandular and variant thereof, wherein the pancreatic protein mixture does not contain pancreatic protein having molecular weight more than 65 KDa; b. treating the pancreatic protein mixture with suitable solvent and buffer to form treated pancreatic protein mixture; c. injecting the treated protein mixture in CE-instrument, d. performing the CE-SDS; and, e. separating at least two enzymes present in pancreatin protein mixture; wherein, the CE-instrument comprising a gel matrix has viscosity 75 cP and below; wherein, the gel matrix comprises SDS and low pH phosphate buffer in suitable ratio having 80:20; wherein, the low pH phosphate buffer having pH below 7 preferably about pH 6.5; wherein, the gel matrix is suitable to separate at least two enzymes present in pancreatin protein mixture selected from protease, amylase, lipase trypsin, chymotrypsin, elastase and kallikrein glandular. wherein, the process provides an improved resolution of pancreatic proteins selected from lipase trypsin, chymotrypsin, elastase and kallikrein glandular.

[0095] In an embodiment, the invention provides a method for improving the quantification of pancreatic proteins present in the pancreatic protein mixture comprising: a. pancreatic protein mixture comprising two or more pancreatic protein selected from protease, amylase, lipase, trypsin, chymotrypsin, elastase and kallikrein glandular and variant thereof, wherein the pancreatic protein mixture does not contain pancreatic protein having molecular weight more than 65 KDa; b. treating the pancreatic protein mixture with suitable solvent and buffer to form treated pancreatic protein mixture; c. injecting the treated protein mixture in CE-instrument, d. performing the CE-SDS; and, e. detecting at least two enzymes present in pancreatin protein mixture; wherein, the CE-instrument comprising a gel matrix has viscosity 75 cP and below; wherein, the gel matrix comprises SDS and suitable modulator in suitable ratio comprising SDS in higher amount than suitable modulator; wherein, the suitable modulator comprises a buffer having pH below 7 ; wherein, the gel matrix is suitable to separate at least two enzymes present in pancreatin protein mixture selected from protease, amylase, lipase trypsin, chymotrypsin, elastase and kallikrein glandular.

[0096] In an embodiment, the invention provides a method for improving the separation and quantification of pancreatic proteins present in the pancreatic protein mixture comprising: a. pancreatic protein mixture comprising two or more pancreatic protein selected from protease, amylase, lipase, trypsin, chymotrypsin, elastase and kallikrein glandular and variant thereof, wherein the pancreatic protein mixture does not contain pancreatic protein having molecular weight more than 65 KDa; b. treating the pancreatic protein mixture with suitable solvent and buffer to form treated pancreatic protein mixture; c. injecting the treated protein mixture in CE-instrument, d. performing the CE-SDS; and, e. separating and detecting at least two enzymes present in pancreatin protein mixture; wherein, the CE-instrument comprising a gel matrix has viscosity 75 cP and below; wherein, the gel matrix comprises SDS and suitable modulator in suitable ratio comprising SDS in higher amount than suitable modulator; wherein, the suitable modulator comprises a buffer having pH below 7 ; wherein, the gel matrix is suitable to separate at least two enzymes present in pancreatin protein mixture selected from protease, amylase, lipase trypsin, chymotrypsin, elastase and kallikrein glandular.

[0097] In an embodiment, the invention provides a method for improving the separation and quantification of pancreatic proteins present in the pancreatic protein mixture comprising: a. pancreatic protein mixture comprising pancreatic protein selected from protease, amylase, lipase, trypsin, chymotrypsin, elastase and kallikrein glandular and variant thereof, wherein the pancreatic protein mixture does not contain pancreatic protein having molecular weight more than 65 KDa; b. treating the pancreatic protein mixture with suitable solvent and buffer to form treated pancreatic protein mixture; c. injecting the treated protein mixture in CE-instrument, d. performing the CE-SDS; and, e. separating and detecting at least two enzymes present in pancreatin protein mixture; wherein, the CE-instrument comprising a gel matrix has viscosity 75 cP and below; wherein, the gel matrix comprises SDS and low pH phosphate buffer in suitable ratio comprising SDS in higher amount than suitable modulator; wherein, the low pH phosphate buffer having pH below 7 preferably about pH 6.5; wherein, the gel matrix is suitable to separate at least two enzymes present in pancreatin protein mixture selected from protease, amylase, lipase trypsin, chymotrypsin, elastase and kallikrein glandular. wherein, the process provides an improved resolution of pancreatic proteins.

[0098] In an embodiment, the invention provides a method for improving the separation and quantification of pancreatic proteins present in the pancreatic protein mixture comprising: a. pancreatic protein mixture comprising pancreatic protein selected from protease, amylase, lipase, trypsin, chymotrypsin, elastase and kallikrein glandular and variant thereof, wherein the pancreatic protein mixture does not contain pancreatic protein having molecular weight more than 65 KDa; b. treating the pancreatic protein mixture with suitable solvent and buffer to form treated pancreatic protein mixture; c. injecting the treated protein mixture in CE-instrument, d. performing the CE-SDS; and, e. separating and detecting at least two enzymes present in pancreatin protein mixture; wherein, the CE-instrument comprising a gel matrix has viscosity 75 cP and below; wherein, the gel matrix comprises SDS and low pH phosphate buffer in suitable ratio having 80:20; wherein, the low pH phosphate buffer having pH below 7 preferably about pH 6.5; wherein, the gel matrix is suitable to separate at least two enzymes present in pancreatin protein mixture selected from protease, amylase, lipase trypsin, chymotrypsin, elastase and kallikrein glandular. wherein, the process provides an improved resolution of pancreatic proteins selected from lipase trypsin, chymotrypsin, elastase and kallikrein glandular.

[0099] In an embodiment the invention provides a disclosure of the separation and / or resolution of one and more pancreatic enzymes selected from co-lipase, phospholipase A2, elastase, Trypsin, Kallikrein glandular, chymotrypsin, carboxypeptidase B, Carboxypeptidase A, Amylase and Carboxy ester lipase (CEL).

[0100] In an embodiment, the invention provides a method for improving the separation of pancreatic proteins present in the pancreatic protein mixture comprising: a. pancreatic protein mixture comprising two or more pancreatic protein selected from protease, amylase, lipase, trypsin, chymotrypsin, elastase and kallikrein glandular and variant thereof, wherein the pancreatic protein mixture does not contain pancreatic protein having molecular weight more than 65 KDa; b. treating the pancreatic protein mixture with suitable solvent and buffer to form treated pancreatic protein mixture; c. injecting the treated protein mixture in CE-instrument, d. performing the CE-SDS; and, e. separating at least two enzymes present in pancreatin protein mixture; wherein, the separation efficiency of the separated pancreatic enzymes is improved compared to pancreatic protein mixture separated through CE SDS without treating with suitable solvent and buffer; wherein, the buffer is low pH phosphate buffer comprising pH below 7.

[0101] In an embodiment, the invention provides a method for improving the quantification of pancreatic proteins present in the pancreatic protein mixture comprising: a. pancreatic protein mixture comprising two or more pancreatic protein selected from protease, amylase, lipase, trypsin, chymotrypsin, elastase and kallikrein glandular and variant thereof, wherein the pancreatic protein mixture does not contain pancreatic protein having molecular weight more than 65 KDa; b. treating the pancreatic protein mixture with solvent and low pH phosphate buffer to form treated pancreatic protein mixture; c. injecting the treated protein mixture in CE-instrument, d. performing the CE-SDS; and, e. separating and detecting at least two enzymes present in pancreatin protein mixture; wherein, the quantification of the separated pancreatic enzymes is improved compared to pancreatic protein mixture separated through CE SDS without treating with suitable buffer and solvent; wherein, the buffer is low pH phosphate buffer comprising pH below 7.

[0102] In an embodiment, the invention provides a method for improving the separation and quantification of pancreatic proteins present in the pancreatic protein mixture comprising: a. pancreatic protein mixture comprising two or more pancreatic protein selected from protease, amylase, lipase, trypsin, chymotrypsin, elastase and kallikrein glandular and variant thereof, wherein the pancreatic protein mixture does not contain pancreatic protein having molecular weight more than 65 KDa; b. treating the pancreatic protein mixture with low pH phosphate buffer to form treated pancreatic protein mixture; c. injecting the treated protein mixture in CE-instrument, d. performing the CE-SDS; and, e. separating and detecting at least two enzymes present in pancreatin protein mixture; wherein, the separation efficiency of the separated pancreatic enzymes is improved compared to pancreatic protein mixture separated through CE SDS without treating with suitable buffer and solvent; wherein, the buffer is low pH phosphate buffer comprising pH below 7.

[0103] In one aspect of the present invention, the pancreatic protein mixture comprises proteins having weight not more than 65 kDa. In another embodiment, the protein mixture comprises proteins having weight, about 65 kDa, about 60 kDa, about 55 kDa, about 50 kDa, about 45 kDa, about 40 kDa, about 35 kDa, about 30 kDa, about 25 kDa, about 20 kDa, about 15 kDa, about 10 kDa, about 5 kDa. In an embodiment the pancreatic protein mixture comprises proteins having weight about 10 KDa to about 65 KDa.

[0104] In an embodiment, the pancreatic protein mixture is pre-treated with organic solvent, wherein organic solvent is selected from carbon tetrachloride, trichloroethylene, Diethyl ether, Dichloroethane, chlorobenzene, cyclohexane, 1,2-dichloroethene, Dichloromethane, 1,2- Dichloromethane, N,N- Dimethylacetamide, 1,4- dioxane, 2- Ethoxyethanol, Ethylene glycol, Formamide, Hexane, Methanol, 2-Methoxyethanol, Methylbutylketone, pyridine, Sulfolane, Tetralin, Toluene, hydrochloric acid, NaOH, phosphate buffer, water, citrate phosphate buffer, and trifluoroacetic acid (TFA) in acetonitrile (ACN). In an embodiment, pancreatic protein mixture is pre-treated with organic solvent in the concentration of about 0.5% trifluoroacetic acid (TFA) in 25% Acetonitrile (ACN), about 0.2% trifluoroacetic acid (TFA) in 25% Acetonitrile (ACN), about 0.1% trifluoroacetic acid (TFA) in 25% Acetonitrile (ACN). In preferred embodiment pre-treated pancreatic protein mixture is dissolved in 0.1% trifluoroacetic acid (TFA) in 25% Acetonitrile (ACN).

[0105] In another embodiment, pre-treated pancreatic protein mixture is treated with buffer and mixed with suitable alkylating agent, wherein buffer is selected from but not limited to Sodium dodecyl sulfate- molecular weight sample buffer, low pH Sodium dodecyl sulfate- molecular weight sample buffer and low pH Phosphate SDS Sample buffer, citrate phosphate buffer, Ammonium phosphate buffer, ammonium formate buffer.

[0106] In an embodiment pancreatic protein mixture is treated preferably in low pH Phosphate SDS Sample buffer.

[0107] In another embodiment, pre-treated pancreatic protein mixture is treated with buffer and mixed with suitable alkylating agent, wherein buffer is selected from but not limited to Sodium dodecyl sulfate- molecular weight sample buffer, low pH Sodium dodecyl sulfate- molecular weight sample buffer and low pH Phosphate SDS Sample buffer, citrate phosphate buffer, Ammonium phosphate buffer, ammonium formate buffer. In another embodiment pancreatic protein mixture is treated preferably in low pH Phosphate SDS Sample buffer.

[0108] In an embodiment, the low pH phosphate buffer is prepared by mixing suitable amount of phosphate in SDS at suitable pH. In an embodiment phosphate is mixed with SDS in molar concentration selected from about 35 mM, about 36 mM, about 37 mM, about 38 mM, about 39 mM, about 40 mM, about 41 mM, about 42 mM, about 43 mM, about 44 mM, about 45 mM. In another phosphate is mixed with SDS in molar concentration selected from about 37 mM, about 38 mM, about 39 mM, about 40 mM, about 41 mM, about 42 mM. In preferred embodiment, phosphate is mixed with SDS more preferably in molar concentration of about 40 mM in SDS at suitable pH.

[0109] In another embodiment, 40mM of phosphate is mixed with SDS at suitable pH. In yet another embodiment 40 mM phosphate is mixed with 1% SDS at suitable pH.

[0110] In an embodiment, 40 mM phosphate is mixed with 1% SDS at pH below 7. In another embodiment, 40 mM phosphate is mixed with 1% SDS at pH selected from about 5.5 pH, about 5.6, about 5.7, about 5.8, about 5.9, about 6, about 6.1, about 6.2, about 6.3, about 6.5, about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, about 7.0. In yet another embodiment 40 mM phosphate is mixed with 1% SDS at pH selected from about 5.9, about 6, about 6.1, about 6.2, about 6.3, about 6.5, about 6.5, about 6.6, about 6.7, about 6.8, about 6.9. In an embodiment the low pH phosphate buffer is prepared by mixing 40 mM phosphate with 1% SDS preferably at pH 6.5.

[0111] In an embodiment, the present invention provides a method to improve the separation efficiency of the proteins, wherein a stable sample is prepared comprising low pH phosphate buffer preventing the digestion of proteins present in the pancreatic protein mixture.

[0112] In one aspect of this invention, the pre-treated pancreatic protein mixture is further treated with low pH phosphate buffer and mixed in alkylating agent for suitable time at suitable temperature.

[0113] In an embodiment, the alkylating agent is Iodo acetamide.

[0114] In an embodiment, pre-treated pancreatic protein mixture is treated with low pH phosphate buffer in the concentration selected from about 45 pL, about 46 pL, about 47 pL, about 48 pL, about 49 pL, about 50 pL, about 51 pL, about 52 pL, about 53 pL, about 54 pL, about 55 pL. In an embodiment the pre-treated pancreatic protein mixture is treated with low pH phosphate buffer in the concentration 45 pL.

[0115] In another embodiment, low pH phosphate buffer concentration is selected from about 45 pL, about 46 pL, about 47 pL, about 48 pL, about 49 pL, about 50 pL, about 51 pL, about 52 pL, about 53 pL, about 54 pL, about 55 pL. In another embodiment the 45 pL pre-treated pancreatic protein mixture is treated with 50 pL low pH phosphate buffer.

[0116] In an embodiment, the alkylating agent is lodoacetamide. In another embodiment 45 pL pre-treated pancreatic protein mixture is treated with 50 pL low pH phosphate buffer and is mixed with 5 pL iodoacetamide for suitable time at suitable temperature.

[0117] In another embodiment, 45 pL pre-treated pancreatic protein mixture is allowed to react with 50 pL low pH phosphate buffer and 5 pL iodoacetamide for about 1 minute, about 2 minutes, about 3 minutes, about 4 minutes, about 5 minutes, about 6 minutes, about 7 minutes, about 8 minutes, about 9 minutes, about 10 minutes.

[0118] In another embodiment 45 pL pre-treated pancreatic protein mixture is allowed to react with 50 pL low pH phosphate buffer and 5 pL iodoacetamide preferably for 3 minutes at suitable temperature.

[0119] In yet another embodiment the temperature is selected from about 65°C, about 66°C, about 67°C, about 68°C, about 69°C, about 70°C, about 71°C, about 72°C, about 73°C, about 74°C, about 75°C. In yet another embodiment 45 pL pre-treated pancreatic protein mixture is allowed to react with 50 pL low pH phosphate buffer and 5 pL iodoacetamide for 3 minutes preferably at 70°C.

[0120] In one aspect of the invention, treated pancreatic protein mixture is optionally with a suitable technique for separating insoluble particles. In another aspect of the invention, treated pancreatic protein mixture is optionally centrifuged to separate insoluble particles such as cell debris.

[0121] In an embodiment, the invention provides a method for improving the separation of pancreatic proteins present in the pancreatic protein mixture comprising: a. pancreatic protein mixture comprising two or more pancreatic protein selected from protease, amylase, lipase, trypsin, chymotrypsin, elastase and kallikrein glandular and variant thereof, wherein the pancreatic protein mixture does not contain pancreatic protein having molecular weight more than 65 KDa; b. treating the pancreatic protein mixture with solvent and low pH phosphate buffer to form treated pancreatic protein mixture; c. injecting the treated protein mixture in CE-instrument, d. performing the CE-SDS; and, e. separating at least two enzymes present in pancreatin protein mixture; wherein, step (a) optionally further comprises use of centrifugation to separate undesirable amount, of insoluble particles from the treated pancreatic protein mixture.

[0122] In an embodiment, the invention provides a method for improving the quantification of pancreatic proteins present in the pancreatic protein mixture comprising: a. pancreatic protein mixture comprising two or more pancreatic protein selected from protease, amylase, lipase, trypsin, chymotrypsin, elastase and kallikrein glandular and variant thereof, wherein the pancreatic protein mixture does not contain pancreatic protein having molecular weight more than 65 KDa; b. treating the pancreatic protein mixture with solvent and low pH phosphate buffer to form treated pancreatic protein mixture; c. injecting the treated protein mixture in CE-instrument, d. performing the CE-SDS; and, e. detecting at least two enzymes present in pancreatin protein mixture; wherein, step (a) optionally further comprises use of centrifugation to separate undesirable amount, of insoluble particles from the treated pancreatic protein mixture. In an embodiment, the invention provides a method for improving the separation and quantification of pancreatic proteins present in the pancreatic protein mixture comprising: a. pancreatic protein mixture comprising two or more pancreatic protein selected from protease, amylase, lipase, trypsin, chymotrypsin, elastase and kallikrein glandular and variant thereof, wherein the pancreatic protein mixture does not contain pancreatic protein having molecular weight more than 65 KDa; b. treating the pancreatic protein mixture with solvent and low pH phosphate buffer to form treated pancreatic protein mixture; c. injecting the treated protein mixture in CE-instrument, d. performing the CE-SDS; and, e. separating and detecting at least two enzymes present in pancreatin protein mixture; wherein, step (a) optionally further comprises use of centrifugation to separate undesirable amount, of insoluble particles from the treated pancreatic protein mixture.

[0123] In an embodiment, the gel matrix having viscosity 75 cP and below is prepared by SDS-MW gel by adding suitable modulator in suitable ratio. In an embodiment the modulator for preparation of gel matrix is selected from but not limited to water, SDS-MW buffer, low pH phosphate buffer. In an embodiment the modulator for preparation of gel matrix is preferably low pH phosphate buffer.

[0124] In another embodiment, gel matrix having viscosity 75 cP and below is prepared by mixing SDS- MW gel with low pH phosphate buffer in a ratio of about 80:20, about 70:30, and about 40:60. In yet another embodiment separation gel is prepared by diluting SDS MW gel with low pH phosphate buffer in a preferable ratio of 80:20.

[0125] In an aspect, of the present invention, treated pancreatic protein mixture is injected in CE- instrument to separate two or more enzymes from pancreatic protein mixture, wherein CE- instrument comprising gel matrix has viscosity 75 cP and below suitable to separate pancreatic protein.

[0126] In an embodiment, the gel matrix has viscosity is about 75 cP, about 74 cP, about 73 cP, about 72 cP, about 71 cP, about 70 cP, about 69 cP, about 68 cP, about 67 cP, about 66 cP, about 65 cP, about 64 cP, about 63 cP, about 62 cP, about 61 cP, about 60 cP, about 59 cP, about 58 cP, about 57 cP, about 56 cP, about 55 cP. In certain embodiments, CE-instrument comprising gel matrix has preferable viscosity of about 70 cP, about 69 cP, about 68 cP, about 67 cP, about 65 cP, about 64 cP, about 63 cP, about 62 cP, about 61 cP, about 60 cP. In an embodiment, the gel matrix has viscosity is about 75 cP. In an embodiment, the present disclosure is related to an improved method for separation of pancreatic proteins present in the pancreatic protein mixture comprising two or more enzymes comprising: a. pancreatic proteins comprising two or more enzymes selected from protease, amylase, lipase trypsin, chymotrypsin, elastase and kallikrein glandular, wherein the protein mixture does not contain protein having molecular weight more than 65kDa; b. treating the pancreatic protein with suitable solvent and buffer to form treated pancreatic protein mixture; c. injecting the treated protein mixture in CE-instrument, comprises gel matrix having viscosity 75 cP and below; d. performing CE-SDS; and, e. separating the pancreatic enzymes; wherein, the separation of the pancreatic enzymes is improved by modulating gel matrix having viscosity 75 cP and below compared to pancreatic protein mixture separated through gel matrix having viscosity more than 75cP wherein, the separation of the pancreatic protein mixture improves resolution of the pancreatic proteins.

[0127] In an embodiment, the present disclosure is related to an improved method for quantification of pancreatic proteins present in the pancreatic protein mixture comprising two or more enzymes comprising: a. pancreatic proteins comprising two or more enzymes selected from protease, amylase, lipase trypsin, chymotrypsin, elastase and kallikrein glandular, wherein the protein mixture does not contain protein having molecular weight more than 65kDa; b. treating the pancreatic protein with suitable solvent and buffer to form treated pancreatic protein mixture; c. injecting the treated protein mixture in CE-instrument, comprises gel matrix having viscosity 75 cP and below; d. performing CE-SDS; and, e. detecting the pancreatic enzymes; wherein, the separation of the pancreatic enzymes is improved by modulating gel matrix having viscosity 75 cP and below compared to pancreatic protein mixture separated through gel matrix having viscosity more than 75cP wherein, the separation of the pancreatic protein mixture improves resolution of the pancreatic proteins. In an embodiment, the present disclosure is related to an improved method for separation and quantification of pancreatic proteins present in the pancreatic protein mixture comprising two or more enzymes comprising: a. pancreatic proteins comprising two or more enzymes selected from protease, amylase, lipase trypsin, chymotrypsin, elastase and kallikrein glandular, wherein the protein mixture does not contain protein having molecular weight more than 65kDa; b. treating the pancreatic protein with suitable solvent and buffer to form treated pancreatic protein mixture; c. injecting the treated protein mixture in CE-instrument, comprises gel matrix having viscosity 75 cP and below; d. performing CE-SDS; and, e. separating and detecting the pancreatic enzymes; wherein, the separation of the pancreatic enzymes is improved by modulating gel matrix having viscosity 75 cP and below compared to pancreatic protein mixture separated through gel matrix having viscosity more than 75cP wherein, the separation of the pancreatic protein mixture improves resolution of the pancreatic proteins.

[0128] In an embodiment, the present invention relates to a CE-SDS method for the separation of pancreatin protein presents in pancreatic protein mixture comprising; a. injecting the pancreatic protein mixture in CE-instrument, b. performing the CE-SDS; and, c. separating the at least more than two pancreatic proteins present in pancreatin protein mixture selected from protease, amylase, lipase, trypsin, chymotrypsin, elastase and kallikrein glandular; wherein, the CE-instrument comprises gel matrix having viscosity 75 cP and below; wherein the gel matrix having viscosity 75 cP and below improves the separation of the pancreatic protein mixture compared to pancreatic protein mixture separated through gel matrix having viscosity more than 75cP; wherein the separation of the pancreatic protein mixture improves resolution of the pancreatic proteins

[0129] In another embodiment the invention further improved resolution of pancreatic protein present in pancreatic mixture selected from co-lipase, phospholipase A2, elastase, Trypsin, Kallikrein glandular, chymotrypsin, carboxypeptidase B, Carboxypeptidase A, Amylase and Carboxy ester lipase (CEL) In an embodiment, the invention provides a method for improving the quantification of pancreatic proteins present in the pancreatic protein mixture comprising: a. pancreatic protein mixture comprising two or more pancreatic protein selected from protease, amylase, lipase, trypsin, chymotrypsin, elastase and kallikrein glandular and variant thereof, wherein the pancreatic protein mixture does not contain pancreatic protein having molecular weight more than 65 KDa; b. treating the pancreatic protein mixture with suitable solvent and buffer to form treated pancreatic protein mixture; c. injecting the treated protein mixture in CE-instrument, d. performing the CE-SDS; and, e. separating and detecting at least two enzymes present in pancreatin protein mixture; wherein, the CE-instrument comprising a gel matrix has viscosity 75 cP and below; wherein, the gel matrix comprises SDS and suitable modulator in suitable ratio comprising SDS in higher amount than suitable modulator; wherein, the suitable modulator comprises a buffer having pH below 7 ; wherein, the gel matrix is suitable to separate at least two enzymes present in pancreatin protein mixture selected from protease, amylase, lipase trypsin, chymotrypsin, elastase and kallikrein glandular.

[0130] In an embodiment, the invention provides a method for improving the separation of pancreatic proteins present in the pancreatic protein mixture comprising: a. pancreatic protein mixture comprising two or more pancreatic protein selected from protease, amylase, lipase, trypsin, chymotrypsin, elastase and kallikrein glandular colipase, phospholipase A2, elastase, Trypsin, Kallikrein glandular, chymotrypsin, carboxypeptidase B, Carboxypeptidase A, Amylase and Carboxy ester lipase (CEL) and variant thereof, wherein the pancreatic protein mixture does not contain pancreatic protein having molecular weight more than 65 KDa; b. treating the pancreatic protein mixture with suitable solvent and buffer to form treated pancreatic protein mixture; c. injecting the treated protein mixture in CE-instrument, d. performing the CE-SDS; and, e. separating at least two enzymes present in pancreatin protein mixture; wherein, the separation efficiency of the separated pancreatic enzymes is improved compared to pancreatic protein mixture separated through CE SDS without treating with suitable buffer and solvent; wherein, the buffer is low pH phosphate buffer comprising pH below 7.

[0131] In an embodiment, the invention provides a method for improving the quantification of pancreatic proteins present in the pancreatic protein mixture comprising: a. pancreatic protein mixture comprising two or more pancreatic protein selected from protease, amylase, lipase, trypsin, chymotrypsin, elastase and kallikrein glandular, colipase, phospholipase A2, elastase, Trypsin, Kallikrein glandular, chymotrypsin, carboxypeptidase B, Carboxypeptidase A, Amylase and Carboxy ester lipase (CEL)and variant thereof, wherein the pancreatic protein mixture does not contain pancreatic protein having molecular weight more than 65 KDa; b. treating the pancreatic protein mixture with solvent and low pH phosphate buffer to form treated pancreatic protein mixture; c. injecting the treated protein mixture in CE-instrument, d. performing the CE-SDS; and, e. separating and detecting at least two enzymes present in pancreatin protein mixture; wherein, the quantification of the separated pancreatic enzymes is improved compared to pancreatic protein mixture separated through CE SDS without treating with suitable buffer and solvent; wherein, the buffer is low pH phosphate buffer comprising pH below 7.

[0132] In an embodiment, the present disclosure is related to an improved method for separation of pancreatic proteins present in the pancreatic protein mixture comprising two or more enzymes comprising: a. pancreatic proteins comprising two or more enzymes selected from protease, amylase, lipase trypsin, chymotrypsin, elastase and kallikrein glandular, co-lipase, phospholipase A2, elastase, Trypsin, Kallikrein glandular, chymotrypsin, carboxypeptidase B, Carboxypeptidase A, Amylase and Carboxy ester lipase (CEL) wherein the protein mixture does not contain protein having molecular weight more than 65kDa; b. treating the pancreatic protein with suitable solvent and buffer to form treated pancreatic protein mixture; c. injecting the treated protein mixture in CE-instrument comprises gel matrix having viscosity 75 cP and below; d. performing CE-SDS; and, e. separating the pancreatic enzymes; wherein, the separation of the pancreatic enzymes is improved by modulating gel matrix having viscosity 75 cP and below compared to pancreatic protein mixture separated through gel matrix having viscosity more than 75cP; wherein the separation of the pancreatic protein mixture improves resolution of the pancreatic proteins.

[0133] In one aspect of this invention, treated pancreatic protein mixture is injected in prepared gel matrix in CE-instrument to separate and detect pancreatic enzymes at suitable capillary temperature and at suitable separation voltage.

[0134] In an embodiment, the CE-SDS is performed at capillary temperature below 25°C. In certain embodiment the capillary temperature is about 14°C, about 15°C, about 16°C, about 17°C, about 18°C, about 19°C, about 20°C, about 21°C, about 22°C, about 23°C, about 24°C and about 25°C. In an embodiment the capillary temperature is about 15 °C to about 22°C. In preferred embodiment the capillary temperature is about 17°C to about 22°C. In an embodiment the preferred capillary temperature is about 17°C.

[0135] In one aspect of this embodiment, the CE-SDS performed at a separation voltage below 10 kV. In certain embodiment the separation voltage is selected from about 1 kV, about 1.5 kV, about 2 kV, about 2.5 kV, about 3 kV, about 3.5 kV about 4 kV, about 4.5 kV, about 5 kV, about 5.5 kV, about 6 kV, about 6.5 kV, about 7 kV, about 7.5 kV, about 8 kV, about 8.5 kV, about 9 kV, about 9.5 kV, and about 10 kV. In an embodiment the separation voltage is about 3.5 kV to about 8kV. In another embodiment the separation voltage is about 5kV to 7.5 kV. In an embodiment the separation voltage is about 3.5 kV to about 7.5kV. In certain embodiments, the CE-SDS is performed at separation voltage about 7.5 kV.

[0136] In an embodiment, the present disclosure is related to a method to improve the separation and resolution of pancreatic proteins present in the pancreatic protein mixture comprising: a. pancreatic protein mixture comprising two or more pancreatic enzymes selected from protease, amylase, lipase, trypsin, chymotrypsin, elastase and kallikrein glandular wherein the pancreatic protein mixture does not contain pancreatic protein having molecular weight more than 65 KDa; b. treating the pancreatic protein mixture with suitable buffer and solvent to form treated pancreatic protein mixture; c. injecting the treated protein mixture in CE-instrument; d. performing the CE-SDS; and, e. separating the one or more enzymes present in pancreatin protein mixture; wherein the separation improves resolution of one or more pancreatic enzyme as follow;

[0137] (i) lipase peak is obtained in chromatogram at about 25 minutes to about 42 minutes during CE-SDS at separation voltage between 6 KV to about 10 KV;

[0138] (ii) protease peak is obtained in chromatogram at about 26 minutes to about 37 minutes during CE-SDS at separation voltage between 6 KV to about 10 KV;

[0139] (iii) amylase peak is obtained in chromatogram at about 32 minutes to about 42 minutes during CE-SDS at separation voltage between 6KV to about 10 KV;

[0140] (iv) trypsin peak is obtained in chromatogram at about 29 minutes to about 31 minutes during CE-SDS at separation voltage between 6KV to about 10 KV;

[0141] (v) elastase peak is obtained in chromatogram at about 29 minutes to about 31 minutes during CE-SDS at separation voltage between 6KV to about 10 KV;

[0142] (vi) chymotrypsin peak is obtained in chromatogram at about 29 minutes to about 31 minutes during CE-SDS at separation voltage between 6KV to about 10 KV;

[0143] (vii) kallikrein glandular peak is obtained in chromatogram at about 29 minutes to about 31 minutes during CE-SDS at separation voltage between 6KV to about 10 KV.

[0144] In an embodiment, the present disclosure is related to a method to improve the separation, detection of pancreatic proteins present, in the pancreatic protein mixture comprising: a. pancreatic protein mixture comprising two or more pancreatic enzymes selected from protease, amylase, lipase, trypsin, chymotrypsin, elastase and kallikrein glandular wherein the pancreatic protein mixture does not contain pancreatic protein having molecular weight more than 65 KDa; b. treating the pancreatic protein mixture with suitable buffer and solvent to form treated pancreatic protein mixture; c. injecting the treated protein mixture in CE-instrument; d. performing the CE-SDS; and, e. separating and detecting the one or more enzymes present in pancreatin protein mixture; wherein the separation improves resolution of one or more pancreatic enzyme as follow; i. lipase peak is obtained in chromatogram at about 25 minutes to about 42 minutes during CE- SDS at separation voltage between 6 KV to about 10 KV; ii. protease peak is obtained in chromatogram at about 26 minutes to about 37 minutes during CE- SDS at separation voltage between 6 KV to about 10 KV; iii. amylase peak is obtained in chromatogram at about 32 minutes to about 42 minutes during CE- SDS at separation voltage between 6KV to about 10 KV; iv. trypsin peak is obtained in chromatogram at about 29 minutes to about 31 minutes during CE- SDS at separation voltage between 6KV to about 10 KV; v. elastase peak is obtained in chromatogram at about 29 minutes to about 31 minutes during CE- SDS at separation voltage between 6KV to about 10 KV; vi. chymotrypsin peak is obtained in chromatogram at about 29 minutes to about 31 minutes during CE-SDS at separation voltage between 6KV to about 10 KV; vii. kallikrein glandular peak is obtained in chromatogram at about 29 minutes to about 31 minutes during CE-SDS at separation voltage between 6KV to about 10 KV.

[0145] In an embodiment, the present disclosure is related to a method to improve the separation, detection of pancreatic proteins present, in the pancreatic protein mixture comprising: a. pancreatic protein mixture comprising two or more pancreatic enzymes selected from protease, amylase, lipase, trypsin, chymotrypsin, elastase and kallikrein glandular wherein the pancreatic protein mixture does not contain pancreatic protein having molecular weight more than 65 KDa; b. treating the pancreatic protein mixture with suitable buffer and solvent to form treated pancreatic protein mixture; c. injecting the treated protein mixture in CE-instrument; d. performing the CE-SDS; and, e. separating and / or detecting the one or more enzymes present in pancreatin protein mixture according to figure 5(a). In an embodiment the invention discloses a CE SDS method provides major peaks for one or more pancreatic protein selected from protease, amylase, lipase, trypsin, chymotrypsin, elastase, kallikrein glandular, co-lipase, phospholipase A2 (PLA2), Carboxy ester lipase (CEL) and Carboxypeptidase B.

[0146] In an embodiment the invention discloses a CE SDS method provides at least major peaks for pancreatic protein selected from trypsin, chymotrypsin, elastase, kallikrein glandular.

[0147] In an embodiment, the invention provides a method for improving the resolution and separation efficiency of the enzymes present in the pancreatic protein, wherein the resolution of Triacylglycerol lipase, Co-lipase, Carboxy ester lipase (CEL), and phospholipase A2 (PLA 2) is obtained between about 25 minutes to about 42 minutes of performing CE-SDS at separation voltage between 6KV to about 10 KV as shown figure 5 (a).

[0148] In an embodiment, the resolution of Co-lipase is obtained at about 21 minutes, about 22 minutes, about 23 minutes, about 24 minutes, about 25 minutes, about 26 minutes, about 27 minutes, about 28 minutes, about 29 minutes of performing CE-SDS at separation voltage between 6KV to about 10 KV as shown figure 5 (a). In another embodiments the resolution of co-lipase is obtained at about 23 minutes, about 24 minutes, about 25 minutes, about 26 minutes, about 27 minutes of performing CE-SDS at separation voltage between 6KV to about 10 KV as shown figure 5 (a). In preferred embodiment, the resolution of Co-lipase is obtained between about 24 minutes to about 26 minutes of performing CE-SDS at separation voltage between 6KV to about 10 KV as shown figure 5 (a).

[0149] In an embodiment, the resolution of phospholipase A2 (PLA 2) is obtained at about 25 minutes, about 26 minutes, about 27 minutes, about 28 minutes, about 29 minutes, about 30 minutes, about 31 minutes of performing CE-SDS at separation voltage between 6KV to about 10 KV as shown figure 5 (a). In another embodiments the resolution of phospholipase A2 (PLA 2) is obtained at about 27 minutes, about 28 minutes, about 29 minutes, about 30 minutes of performing CE-SDS at separation voltage between 6KV to about 10 KV as shown figure 5 (a). In preferred embodiment, the resolution of phospholipase A2 (PLA 2) is obtained between about 28 minutes to about 29 minutes of performing CE-SDS at separation voltage between 6KV to about 10 KV as shown figure 5 (a).

[0150] In an embodiment, the resolution of Carboxy ester lipase (CEL) is obtained at about 40 minutes, about 41 minutes, and at about 42 minutes of performing CE-SDS at separation voltage between 6KV to about 10 KV as shown figure 5 (a). In another embodiments the resolution of Carboxy ester lipase (CEL) is obtained at about 40 minutes, to about 42 minutes of performing CE-SDS at separation voltage between 6KV to about 10 KV as shown figure 5 (a).

[0151] In preferred embodiment, the invention provides an improved method for improving the resolution and separation efficiency of the enzymes present in the pancreatic protein, wherein the resolution of trypsin, chymotrypsin, elastase, kallikrein glandular, Carboxypeptidase Al, Carboxypeptidase B, and chymotrypsin is obtained between about 26 minutes to about 37 minutes of performing CE- SDS at separation voltage between 6KV to about 10 KV as shown figure 5 (a).

[0152] In an embodiment, the resolution of Elastase is obtained at about 26 minutes, about 27 minutes, about 28 minutes, about 29 minutes, about 30 minutes, about 31 minutes, about 32 minutes, about 33 minutes of performing CE-SDS at separation voltage between 6KV to about 10 KV as shown figure 5 (a). In another embodiments the resolution of Elastase is obtained at about 28 minutes, about 29 minutes, about 30 minutes, about 31 minutes, about 32 minutes of performing CE-SDS at separation voltage between 6KV to about 10 KV as shown figure 5 (a). In preferred embodiment, the resolution of Elastase is obtained between about 29 minutes to about 31 minutes of performing CE-SDS at separation voltage between 6KV to about 10 KV as shown figure 5 (a).

[0153] In an embodiment, the resolution of chymotrypsin based on the molecular weight and as evident in figure 5 (a) is obtained at about 28 minutes, about 29 minutes, about 30 minutes, about 31 minutes, and about 32 minutes of performing CE-SDS at separation voltage between 6KV to about 10 KV. In an embodiment, the resolution of chymotrypsin based on the molecular weight and as evident in figure 5 (a) is obtained between about 29 minutes, to about 31minutes, of performing CE-SDS at separation voltage between 6KV to about 10 KV.

[0154] In an embodiment, the resolution of Kallikrein based on the molecular weight and as evident in figure 5 (a) is obtained at about 28 minutes, about 29 minutes, about 30 minutes, about 31 minutes, about 32 minutes of performing CE-SDS at separation voltage between 6KV to about 10 KV. In an embodiment, the resolution of Kallikrein based on the molecular weight and as evident in figure 5 (a) is obtained between about 29 minutes, to about 31minutes, of performing CE-SDS at separation voltage between 6KV to about 10 KV.

[0155] In an embodiment, the resolution of trypsin is obtained at about 26 minutes, about 27 minutes, about 28 minutes, about 29 minutes, about 30 minutes, about 31 minutes, about 32 minutes, about 33 minutes of performing CE-SDS at separation voltage between 6KV to about 10 KV. In another embodiments the resolution of trypsin is obtained at about 28 minutes, about 29 minutes, about 30 minutes, about 31 minutes, about 32 minutes of performing CE-SDS at separation voltage between 6KV to about 10 KV. In preferred embodiment, the resolution of trypsin is obtained between about 29 minutes to about 31 minutes of performing CE-SDS at separation voltage between 6KV to about 10 KV.

[0156] In an embodiment, the present invention provides a method for improving the separation efficiency and resolution of proteins selected from trypsin, chymotrypsin, elastase and Kallikrein. In an embodiment, the pancreatic proteins trypsin, chymotrypsin, elastase and Kallikrein have similar Molecular Weight which interrupts the separation and quantification of said proteins, the present invention relates to a method to improve the separation efficiency and resolution of proteins selected from trypsin, chymotrypsin, elastase and Kallikrein.

[0157] In an embodiment, the resolution of Carboxypeptidase B is obtained at about 30 minutes, about 31 minutes, about 32 minutes, about 33 minutes, about 34 minutes, about 35 minutes, about 36 minutes, about 37 minutes of performing CE-SDS at separation voltage between 6KV to about 10 KV. In another embodiments the resolution of Carboxypeptidase B is obtained at about 32 minutes, about 33 minutes, about 34 minutes, about 35 minutes of performing CE-SDS at separation voltage between 6KV to about 10 KV. In preferred embodiment, the resolution of Carboxypeptidase B is obtained between about 33 minutes to about 34 minutes of performing CE-SDS at separation voltage between 6KV to about 10 KV.

[0158] In preferred embodiment the invention provides an improved method for improving the resolution and separation efficiency of the enzymes present in the pancreatic protein, wherein the resolution of amylase is obtained between about 32 minutes to about 42 minutes of performing CE-SDS at separation voltage between 6KV to about 10 KV.

[0159] In an embodiment, the resolution of amylase is obtained at about 32 minutes, about 33 minutes, about 34 minutes, about 35 minutes, about 36 minutes, about 37 minutes, about 38 minutes, about 39 minutes, about 40 minutes, about 41 minutes, about 42 minutes of performing CE-SDS at separation voltage between 6KV to about 10 KV. In another embodiments the resolution of amylase is obtained at about 34 minutes, about 35 minutes, about 36 minutes, about 37 minutes, about 38 minutes, about 39 minutes, about 40 minutes of performing CE-SDS at separation voltage between 6KV to about 10 KV. In preferred embodiment, the resolution of amylase is obtained between about 36 minutes to about 38 minutes of performing CE-SDS at separation voltage between 6KV to about 10 KV.

[0160] In an embodiment, the present invention provides a method for improving the separation efficiency and resolution of pancreatic proteins through CE-SDS, wherein the pancreatic proteins are separated in the following order of co-lipase, phospholipase A2, elastase, Trypsin, Kallikrein glandular, chymotrypsin, carboxypeptidase B, Carboxypeptidase A, Amylase and Carboxy ester lipase (CEL).

[0161] In an embodiment, the present invention relates to a method for providing a stable pancreatic protein mixture comprising a. Isolating the pancreatin protein mixture comprising more than at least two enzymes selected from protease, amylase, lipase, trypsin, chymotrypsin, elastase and kallikrein glandular; b. treating the pancreatic protein mixture with low pH phosphate buffer; c. producing the stable pancreatic protein mixture; d. wherein the stable pancreatic protein mixture is suitable for the characterization of at least two enzymes selected from protease, amylase, lipase, trypsin, chymotrypsin, elastase and kallikrein glandular compared to pancreatic protein mixture not treated with low pH phosphate buffer; wherein, the characterization is performed by CE-SDS.

[0162] In an embodiment, the present invention relates to a stable pancreatic protein mixture comprising; a. treating the pancreatic protein mixture with suitable buffer to form stable pancreatic protein mixture; b. injecting the stable pancreatic protein mixture in CE-instrument, d. performing the CE-SDS; and, e. separating the at least more than two pancreatic proteins present in pancreatin protein mixture selected from protease, amylase, lipase, trypsin, chymotrypsin, elastase and kallikrein glandular; wherein, the suitable buffer is low pH phosphate buffer having pH below 7; wherein, the stable pancreatic protein mixture has reduced amount of digested pancreatic proteins compared to the pancreatic protein mixture not treated with low pH phosphate buffer.

[0163] In an embodiment, the present method inhibits the digestion of pancreatic proteins and thereby peptide formation, resulting in improved separation efficiency of the pancreatic proteins. In another embodiment, the present method prevents the increase in the peak percentage area of peptides by inhibiting the digestion of pancreatic proteins resulting in improved separation efficiency of the pancreatic proteins.

[0164] In an embodiment, the present invention provides a method for improving the separation of pancreatic proteins present in the pancreatic protein mixture comprising: a. pancreatic protein mixture comprising two or more pancreatic protein selected from protease, amylase, lipase, trypsin, chymotrypsin, and elastase wherein the pancreatic protein mixture does not contain pancreatic protein having molecular weight more than 65 KDa; b. treating the pancreatic protein mixture with suitable solvent and buffer to form treated pancreatic protein mixture; c. injecting the treated protein mixture in CE-instrument; d. performing the CE-SDS; and, separating the protease, amylase, lipase, trypsin, chymotrypsin, and elastase present in pancreatin protein mixture as shown in figure 5 (a). In an embodiment, the present invention provides a method for improving the quantification of pancreatic proteins present in the pancreatic protein mixture comprising: a. pancreatic protein mixture comprising two or more pancreatic protein selected from protease, amylase, lipase, trypsin, chymotrypsin, and elastase wherein the pancreatic protein mixture does not contain pancreatic protein having molecular weight more than 65 KDa; b. treating the pancreatic protein mixture with suitable solvent and buffer to form treated pancreatic protein mixture; c. injecting the treated protein mixture in CE-instrument; d. performing the CE-SDS; and, detecting the protease, amylase, lipase, trypsin, chymotrypsin, and elastase present in pancreatin protein mixture as shown in figure 5 (a).

[0165] In an embodiment, the present invention provides a method for improving the separation and quantification of pancreatic proteins present in the pancreatic protein mixture comprising: a. pancreatic protein mixture comprising two or more pancreatic protein selected from protease, amylase, lipase, trypsin, chymotrypsin, and elastase wherein the pancreatic protein mixture does not contain pancreatic protein having molecular weight more than 65 KDa; b. treating the pancreatic protein mixture with suitable solvent and buffer to form treated pancreatic protein mixture; c. injecting the treated protein mixture in CE-instrument; d. performing the CE-SDS; and, e. separating and detecting the protease, amylase, lipase, trypsin, chymotrypsin, and elastase present in pancreatin protein mixture as shown in figure 5 (a).

[0166] In an embodiment the invention discloses a CE SDS method provides major peaks for one or more pancreatic protein selected from protease, amylase, lipase, trypsin, chymotrypsin, elastase, kallikrein glandular, co-lipase, phospholipase A2 (PLA2), Carboxy ester lipase (CEL) and Carboxypeptidase B.

[0167] In an embodiment the invention discloses a CE SDS method provides at least major peaks for pancreatic protein selected from trypsin, chymotrypsin, elastase, kallikrein glandular. In an embodiment, the present invention relates to a method to improve the separation of trypsin, chymotrypsin, elastase and kallikrein glandular present in the pancreatic protein mixture comprising: a. pancreatic protein mixture comprising two or more pancreatic enzymes selected from protease, amylase and lipase, wherein the pancreatic protein mixture does not contain pancreatic protein having molecular weight more than 65 KDa; b. treating the pancreatic protein mixture to form a treated pancreatic protein mixture; c. injecting the treated protein mixture in CE-instrument, d. performing the CE-SDS; and e. separating trypsin, chymotrypsin, elastase and kallikrein glandular protein; wherein, the CE-instrument comprising a gel matrix has viscosity 75 cP and below; wherein the gel matrix comprises SDS and low pH phosphate buffer in a ratio of 80:20; wherein the low pH phosphate buffer in the gel matrix comprises pH below 7; wherein, the gel matrix having viscosity 75 cP and below improves the separation efficiency of trypsin, chymotrypsin, elastase and kallikrein glandular compared to separation efficiency of proteins when separated with gel matrix having viscosity above 75cP.

[0168] In an embodiment, the present invention relates to a method to improve the quantification of trypsin, chymotrypsin, elastase and kallikrein glandular present in the pancreatic protein mixture comprising: a. pancreatic protein mixture comprising two or more pancreatic enzymes selected from protease, amylase and lipase, wherein the pancreatic protein mixture does not contain pancreatic protein having molecular weight more than 65 KDa; b. treating the pancreatic protein mixture to form a treated pancreatic protein mixture; c. injecting the treated protein mixture in CE-instrument, d. performing the CE-SDS; and e. detecting trypsin, chymotrypsin, elastase and kallikrein glandular protein; wherein, the CE-instrument comprising a gel matrix has viscosity 75 cP and below; wherein the gel matrix comprises SDS and low pH phosphate buffer in a ratio of 80:20; wherein the low pH phosphate buffer comprises pH below 7; wherein, the gel matrix having viscosity 75 cP and below improves the separation efficiency of trypsin, chymotrypsin, elastase and kallikrein glandular compared to separation efficiency of proteins when separated with gel matrix having viscosity above 75cP.

[0169] In an embodiment, the present invention relates to a method to improve the separation and quantification of trypsin, chymotrypsin, elastase and kallikrein glandular present in the pancreatic protein mixture comprising: a. pancreatic protein mixture comprising two or more pancreatic enzymes selected from protease, amylase and lipase, wherein the pancreatic protein mixture does not contain pancreatic protein having molecular weight more than 65 KDa; b. treating the pancreatic protein mixture to form a treated pancreatic protein mixture; c. injecting the treated protein mixture in CE-instrument, d. performing the CE-SDS; and e. separating and detecting trypsin, chymotrypsin, elastase and kallikrein glandular protein; wherein, the CE-instrument comprising a gel matrix has viscosity 75 cP and below; wherein the gel matrix comprises SDS and low pH phosphate buffer in a ratio of 80:20; wherein the low pH phosphate buffer in the gel matrix comprises pH below 7; wherein, the gel matrix having viscosity 75 cP and below improves the separation efficiency of trypsin, chymotrypsin, elastase and kallikrein glandular compared to separation efficiency of proteins when separated with gel matrix having viscosity above 75cP.

[0170] In an embodiment, the present disclosure provides a method for improving separation profile of pancreatic protein mixture comprising at least two enzyme amylase, protease and lipase & variant thereof through capillary electrophoresis Sodium dodecyl sulfate (CE-SDS). In another aspect of the present invention the present disclosure provides separation profile of pancreatic protein mixture comprising trypsin, chymotrypsin, elastase, and kallikrein glandular through capillary electrophoresis Sodium dodecyl sulfate (CE-SDS).

[0171] The present invention provides below examples for illustrative purpose only and invention should not be considered limiting to below examples.

[0172] Example 1 Separation profile of pancreatic proteins using citrate phosphate buffer

[0173] The pancreatic protein mixture was mixed with citrate phosphate buffer at pH 6.2 and prepared for CE-SDS using SDS MW Sample Buffer (100 mM Tris-HCl, pH 9.0, 1% SDS), was injected on CE-SDS system. The separation was achieved by using Separation voltage of 15KV, detected by UV at 220 nm. The capillary cartridge temperature maintained at 25 °C and sample temperature kept at 25 °C. It is evident from Figure 1 that pancreatic protein mixture, when mixed with citrate phosphate buffer, pH 6.2, shows digestion of proteins or enzymes.

[0174] Example 2 - Separation profile of pancreatic proteins using different organic solvents

[0175] Pancreatic protein mixture was mixed with different organic solvents and prepared for CE-SDS by treating the pre-treated pancreatic protein mixture with SDS MW Sample Buffer (100 mM Tris- HC1, pH 9.0, 1% SDS), treated pancreatic protein mixture was injected on CE-SDS instrument. The separation was achieved by using Separation voltage of 15KV and detected by UV at 220 nm. The capillary cartridge was temperature maintained at 25 °C and sample temperature kept at 25 °C. It is evident from Figure 2 that pancreatic protein mixture mixed with 0.1% TFA in 30% Acetonitrile separated all the enzymes Additionally, this method can quantify, and separate proteolytic enzymatic products and autocatalytic products present in pancreatic protein mixture.

[0176] Example 3 - Separation profile of pancreatic proteins using buffer at different pH

[0177] Pancreatic protein mixture was mixed with 0.1% TFA in 25% Acetonitrile and prepared for CE- SDS using SDS MW Sample Buffer (100 mM Tris-HCl, pH 9.0, 1% SDS) , Pancreatic protein mixture was mixed with 0.2% TFA in 25% Acetonitrile and prepared for CE-SDS using SDS MW Sample Buffer (100 mM Tris-HCl, pH 9.0, 1% SDS) and Pancreatic protein mixture was mixed with 0.1% TFA in 25% Acetonitrile and prepared for CE-SDS using Low pH Phosphate SDS Sample Buffer, (40 mM Phosphate, pH 6.5, 1% SDS) , was injected on CE-SDS instrument. The separation was achieved by using Separation voltage of 15KV, detected by UV at 220 nm. The capillary cartridge temperature maintained at 25 °C and sample temperature kept at 25 °C.

[0178] It is evident from Figure 3 that pancreatic protein mixed with 0.1% TFA in 25% Acetonitrile and prepared with SDS MW Sample Buffer (100 mM Tris-HCl, pH 9.0, 1% SDS) may lead to proteolytic and autocatalytic activity while sample prepared with Low pH Phosphate SDS Sample Buffer, (40 mM Phosphate, pH 6.5, 1% SDS) not led to proteolytic and autocatalytic activity.

[0179] Example 4 - Comparative separation profile of pancreatic proteins separated through CE- SDS between in house pancreatic protein mixture and Reference product

[0180] In house pancreatic protein mixture was mixed with 0.1% TFA in 25% Acetonitrile and prepared for CE-SDS using Low pH Phosphate SDS Sample Buffer, (40 mM Phosphate, pH 6.5, 1% SDS), was injected on CE-SDS system where commercially available gel was replaced with differently modulated gel using Low pH Phosphate SDS Sample Buffer, (40 mM Phosphate, pH 6.5, 1% SDS). In the same manner, the Reference product mixed in 0.1% trifluoroacetic acid (TFA) in 25% acetonitrile (ACN) using Low pH Phosphate SDS Sample Buffer, (40 mM Phosphate, pH 6.5, 1% SDS), was injected on CE-SDS system is performed comprising a gel matrix is modulated. The separation profile of the pancreatic proteins in the both the samples was achieved by using Separation voltage of 7.5KV, detected by UV at 220 nm. The capillary cartridge temperature maintained at 17 °C and sample temperature kept at 25°C.

[0181] It is evident from figure 4, both the samples a) in house pancreatic protein mixture and b) Reference product mixture provided same peak pattern, which shows similarity between the inhouse pancreatic protein mixture and the reference product.

[0182] Example 5 - Comparative separation profile of pancreatic proteins separated through CE- SDS between a) in house pancreatic protein mixture with modulated gel matrix and b) in house pancreatic protein mixture with commercially available gel used in CE SDS

[0183] In house pancreatic protein mixture was mixed with 0.1% TFA in 25% Acetonitrile and prepared for CE-SDS using Low pH Phosphate SDS Sample Buffer, (40 mM Phosphate, pH 6.5, 1% SDS), was injected on CE-SDS system where commercially available gel was replaced with differently modulated gel using Low pH Phosphate SDS Sample Buffer, (40 mM Phosphate, pH 6.5, 1% SDS) having viscosity 75 cP and below. In the same manner, another in house pancreatic protein mixture was mixed with 0.1% TFA in 25% Acetonitrile and prepared for CE-SDS using Low pH Phosphate SDS Sample Buffer, (40 mM Phosphate, pH 6.5, 1% SDS), was injected on CE-SDS system is performed with commercially available gel having viscosity above 75cP. The separation profile of the pancreatic proteins in the both the samples was achieved by using Separation voltage of 7.5KV, detected by UV at 220 nm. The capillary cartridge temperature maintained at 17 °C and sample temperature kept at 25°C.

[0184] It is evident from Figure 5 (a) that sample extracted with 0.1% TFA in 25% Acetonitrile, prepared using Low pH Phosphate SDS Sample Buffer, (40 mM Phosphate, pH 6.5, 1% SDS) was injected on CE-SDS instrument with modulated gel wherein the gel is modulated using Low pH Phosphate SDS Sample Buffer, (40 mM Phosphate, pH 6.5, 1% SDS) comprising viscosity 75 cP and below, shows better resolution for the cluster of 20 to 35kDa enzymatic product and separate closely related molecular weight enzymes like trypsin, chymotrypsin, elastase, and kallikrein glandular.

[0185] On contrast to figure 5 (a); figure 5 (b) wherein the sample extracted with 0.1% TFA in 25% Acetonitrile, prepared using Low pH Phosphate SDS Sample Buffer, (40 mM Phosphate, pH 6.5, 1% SDS) was injected on CE-SDS instrument with commercially available gel comprising viscosity above 75cP fails to separate Elastase, Trypsin, Chymotrypsin and Kallikrein and provide a cluster of the Elastase, Trypsin, Chymotrypsin and Kallikrein glandular peaks having closely related molecular weight.

Claims

Claims1. A CE-SDS method for the separation of pancreatin protein presents in pancreatic protein mixture comprising; a. injecting the pancreatic protein mixture in CE-instrument, b. performing the CE-SDS; and, c. separating the at least more than two pancreatic proteins present in pancreatin protein mixture selected from protease, amylase, lipase, trypsin, chymotrypsin, elastase and kallikrein glandular; wherein, the CE-instrument comprises gel matrix having viscosity 75 cP and below; wherein the gel matrix having viscosity 75 cP and below improves the separation of the pancreatic protein mixture compared to pancreatic protein mixture separated through gel matrix having viscosity more than 75cP; wherein the separation of the pancreatic protein mixture improves resolution of the pancreatic proteins.

2. The CE-SDS method as claimed in claim 1, wherein the gel matrix is treated or modulated with suitable modulator; wherein the suitable modulator is selected from citrate phosphate, Ammonium phosphate, ammonium formate and low pH buffer having pH below 7.

3. The CE-SDS method as claimed in claim 2, wherein the buffer is low pH phosphate buffer having pH 6 to pH 7.

4. The CE-SDS method as claimed in claim 3, wherein the low pH buffer is low pH phosphate buffer having pH 6.

55. The CE-SDS method as claimed in claim 1, wherein the gel matrix comprises SDS and low pH phosphate buffer in suitable ratio comprising SDS in higher amount than low pH phosphate buffer.

6. The CE-SDS method as claimed in claim 5, wherein the SDS amount is at least two times or three times or four times higher amount than low pH phosphate buffer.

7. The CE-SDS method as claimed in claim 6, wherein the SDS-MW gel with low pH phosphate buffer in a ratio of about 80:20, about 70:30 and about 40:60.

8. The CE-SDS method as claimed in claim 1, wherein the gel matrix is having viscosity 75 cP and below, about 74 cP, about 73 cP, about 72 cP, about 71 cP, about 70 cP, about 69 cP, about 68 cP, about 67 cP, about 65 cP, about 64 cP, about 63 cP, about 62 cP, about 61 cP, about 60 cP, about 59 cP, about 58 cP , about 57 cP, about 56 cP, and about 55 cP.

9. The CE-SDS method as claimed in claim 1, wherein the CE SDS method is suitable for quantification of pancreatin protein presents in pancreatic protein mixture.

10. The CE-SDS method as claimed in claim 1, wherein the CE SDS method is suitable for analytical characterization of pancreatin protein presents in pancreatic protein mixture.

11. The CE-SDS method as claimed in claim 1, wherein the pancreatic protein mixture is obtained from semi purified, purified pancreatic mixture optionally further formulated in the form dosage selected from tablet, capsule, granules and powder.

12. The CE-SDS method as claimed in claim 1, wherein the pancreatic protein mixture is treated with a suitable solvent to form pre-treated pancreatic protein mixture.

13. The CE-SDS method as claimed in claim 12, wherein the solvent improves separation and / or resolution of pancreatic protein during CE-SDS.

14. The CE-SDS method as claimed in claim 12, wherein the solvent is carbon tetrachloride, trichloroethylene, Diethyl ether, Dichloroethane, chlorobenzene, cyclohexane, 1,2- dichloroethene, Dichloromethane, 1,2- Dichloromethane, N,N- Dimethylacetamide, 1,4- dioxane, 2- Ethoxyethanol, Ethylene glycol, Formamide, Hexane, Methanol, 2- Methoxyethanol, Methylbutylketone, pyridine, Sulfolane, Tetralin, Toluene, citrate phosphate buffer, and trifluoroacetic acid (TFA) in acetonitrile (ACN).

15. The CE-SDS method as claimed in claim 14, wherein the solvent is trifluoroacetic acid (TFA) in acetonitrile (ACN)16. The CE-SDS method as claimed in claim 15, wherein the trifluoroacetic acid (TFA) amount is 0.1%; Wherein the acetonitrile (ACN) amount is about 25%.

17. The CE-SDS method as claimed in claim 1, wherein capillary temperature is about 14°C, about 15°C, about 16°C, about 17°C, about 18°C, about 19°C, about 20°C, about 21°C, about 22°C, about 23°C, about 24°C and about 25°C.

18. The CE-SDS method as claimed in claim 1, wherein the CE-SDS is performed at separation voltage of about 1 kV, about 1.5 kV, about 2 kV, about 2.5 kV, about 3 kV, about 3.5 kV about 4 kV, about 4.5 kV, about 5 kV, about 5.5 kV, about 6 kV, about 6.5 kV, about 7 kV, about 7.5 kV, about 8 kV, about 8.5 kV, about 9 kV, about 9.5 kV, and about 10 kV.

19. The CE-SDS method as claimed in claim 1, wherein the CE-SDS is performed at capillary temperature about 25°C at a voltage below 10 kV.

20. The CE-SDS method as claimed in claim 1, wherein the method improves resolution of pancreatic proteins according to fig. 5(a).

21. A method for improving the separation of pancreatic proteins present in the pancreatic protein mixture comprising: a. pancreatic protein mixture comprising pancreatic protein selected from protease, amylase, lipase, trypsin, chymotrypsin, elastase and kallikrein glandular and variantthereof, wherein the pancreatic protein mixture does not contain pancreatic protein having molecular weight more than 65 KDa; b. treating the pancreatic protein mixture with suitable solvent and buffer to form treated pancreatic protein mixture; c. injecting the treated protein mixture in CE-instrument, d. performing the CE-SDS; and, e. separating at least two enzymes present in pancreatin protein mixture; wherein, the CE-instrument comprising a gel matrix has viscosity 75 cP and below; wherein, the gel matrix comprises SDS and low pH phosphate buffer in suitable ratio having SDS at least two time higher than low pH phosphate buffer. wherein, the low pH phosphate buffer having pH below 7 preferably about pH 6.5; wherein, the gel matrix is suitable to separate at least two enzymes present in pancreatin protein mixture selected from protease, amylase, lipase trypsin, chymotrypsin, elastase and kallikrein glandular.

22. The method as claimed in claim 21, wherein, the process provides an improved resolution of pancreatic proteins selected from lipase trypsin, chymotrypsin, elastase and kallikrein glandular.

23. The method as claimed in claim 1 and claim 21 wherein the method further improves the separation of one or more enzyme selected from co-lipase, phospholipase A2 (PLA2), Carboxy ester lipase (CEL) and Carboxypeptidase B.

24. A stable pancreatic protein mixture comprising; a. treating the pancreatic protein mixture with suitable buffer to form stable pancreatic protein mixture; b. injecting the stable pancreatic protein mixture in CE-instrument, d. performing the CE-SDS; and, e. separating the at least more than two pancreatic proteins present in pancreatin protein mixture selected from protease, amylase, lipase, trypsin, chymotrypsin, elastase and kallikrein glandular; wherein, the suitable buffer is low pH phosphate buffer having pH below 7; wherein, the stable pancreatic protein mixture has reduced amount of digested pancreatic proteins compared to the pancreatic protein mixture not treated with low pH phosphate buffer.

25. The stable pancreatic protein mixture as claimed in claim 2, wherein, the low pH phosphate buffer reduces proteolytic and autolytic activity in pancreatic protein mixture.

26. The stable pancreatic protein mixture as claimed in claim 25, wherein the low pH buffer is low pH phosphate buffer having pH 6 to pH 7; wherein the low pH buffer is low pH phosphate buffer having pH 6.5.

27. The stable pancreatic protein mixture as claimed in claim 24, wherein the CE instrument comprises a gel matrix comprising SDS and low pH phosphate buffer in suitable ratio comprising SDS in higher amount than low pH phosphate buffer; wherein the SDS-MW gel with low pH phosphate buffer in a ratio of about 80:20, about 70:30 and about 40:60.

28. The stable pancreatic protein mixture as claimed in claim 24, wherein the CE SDS method is suitable for quantification of pancreatin protein presents in pancreatic protein mixture; wherein the CE SDS method is suitable for analytical characterization of pancreatin protein presents in pancreatic protein mixture.

29. The stable pancreatic protein mixture as claimed in claim 24, improves the resolution of pancreatic protein or enzyme during CE-SDS; wherein the stable pancreatic protein mixture improves the resolution of trypsin, chymotrypsin, elastase and kallikrein glandular;30. The stable pancreatic protein mixture as claimed in claim 2, wherein the pancreatic protein mixture is further treated with solvent improves the resolution of pancreatic protein or enzyme during CE-SDS.

31. The stable pancreatic protein mixture as claimed in claim 30, wherein the pancreatic protein mixture is treated with suitable solvent; wherein the Wherein the solvent is carbon tetrachloride, trichloroethylene, Diethyl ether, Dichloroethane, chlorobenzene, cyclohexane, 1,2-dichloroethene, Dichloromethane, 1,2- Dichloromethane, N,N- Dimethylacetamide, 1,4- dioxane, 2- Ethoxyethanol, Ethylene glycol, Formamide, Hexane, Methanol, 2- Methoxyethanol, Methylbutylketone, pyridine, Sulfolane, Tetralin, Toluene, citrate phosphate buffer, and trifluoroacetic acid (TFA) in acetonitrile (CAN.)32. The stable pancreatic protein mixture as claimed in claim 24, wherein the CE instrument comprises a gel matrix having viscosity about 75 cP, about 74 cP, about 73 cP, about 72 cP, about 71 cP, about 70 cP, about 69 cP, about 68 cP, about 67 cP, about 65 cP, about 64 cP, about 63 cP, about 62 cP, about 61 cP, about 60 cP, about 59 cP, about 58 cP , about 57 cP, about 56 cP, and about 55 cP.

33. A method to improve the separation, and detection of pancreatic proteins present, in the pancreatic protein mixture comprising: a. pancreatic protein mixture comprising two or more pancreatic enzymes selected from protease, amylase, lipase, trypsin, chymotrypsin, elastase, kallikrein glandular, co-lipase,phospholipase A2 (PLA2), Carboxy ester lipase (CEL) and Carboxypeptidase B wherein the pancreatic protein mixture does not contain pancreatic protein having molecular weight more than 65 KDa; b. treating the pancreatic protein mixture with suitable buffer and solvent to form treated pancreatic protein mixture; c. injecting the treated protein mixture in CE-instrument; d. performing the CE-SDS; and, e. separating and detecting the one or more enzymes present in pancreatin protein mixture; wherein the separation improves resolution of one or more pancreatic enzyme as follow; viii. lipase peak is obtained in chromatogram at about 25 minutes to about 42 minutes during CE-SDS at separation voltage between 6 KV to about 10 KV; ix. protease peak is obtained in chromatogram at about 26 minutes to about 37 minutes during CE-SDS at separation voltage between 6 KV to about 10 KV; x. amylase peak is obtained in chromatogram at about 32 minutes to about 42 minutes during CE-SDS at separation voltage between 6KV to about 10 KV; xi. trypsin peak is obtained in chromatogram at about 29 minutes to about 31 minutes during CE-SDS at separation voltage between 6KV to about 10 KV; xii. elastase peak is obtained in chromatogram at about 29 minutes to about 31 minutes during CE-SDS at separation voltage between 6KV to about 10 KV; xiii. chymotrypsin peak is obtained in chromatogram at about 29 minutes to about 31 minutes during CE-SDS at separation voltage between 6KV to about 10 KV; xiv. kallikrein glandular peak is obtained in chromatogram at about 29 minutes to about 31 minutes during CE-SDS at separation voltage between 6KV to about 10 KV.

34. The method as claimed in claim 33, wherein co-lipase peak is obtained in chromatogram at about 24 minutes to about 26 minutes during CE-SDS at separation voltage between 6KV to about 10 KV.

35. The method as claimed in claim 33, wherein phospholipase A2 (PLA 2) peak is obtained in chromatogram at about 28 minutes to about 29 minutes during CE-SDS at separation voltage between 6KV to about 10 KV.

36. The method as claimed in claim 33, wherein Carboxy ester lipase (CEL) is obtained in chromatogram at about 40 minutes, to about 42 minutes during CE-SDS at separation voltage between 6KV to about 10 KV.

37. The method as claimed in claim 33, wherein Carboxypeptidase B peak is obtained in chromatogram at about 33 minutes to about 34 minutes during CE-SDS at separation voltage between 6KV to about 10 KV.

38. The method as claimed in claim 1, claim 21 and claim 33, wherein the pancreatic proteins are separated in the following order of co-lipase, phospholipase A2, elastase, Trypsin, Kallikrein glandular, chymotrypsin, carboxypeptidase B, Carboxypeptidase A, Amylase and Carboxy ester lipase (CEL).

39. The method as claimed in any preceding claim wherein the CE SDS provides major peaks for one or more pancreatic protein selected from protease, amylase, lipase, trypsin, chymotrypsin, elastase, kallikrein glandular, co-lipase, phospholipase A2 (PLA2), Carboxy ester lipase (CEL) and Carboxypeptidase B.

40. The method as claimed in any preceding claim wherein the CE SDS provides at least major peaks for pancreatic protein selected from trypsin, chymotrypsin, elastase, kallikrein glandular.

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