A PROCESS FOR SEPARATION AND QUANTITATION OF PROTEINS USING CAPILLARY ELECTROPHORESIS
The present disclosure provides a method for analyzing, detecting and separating at least one low molecular weight impurity from a protein mixture using capillary electrophoresis, e.g., capillary electrophoresis-sodium dodecyl sulfate (CE-SDS). The present disclosure further provides methods to improve protein peak separation efficiency and quantification of a protein. Furthermore, the present disclosure provides an improved reduced CE-SDS method for analyzing a protein mixture comprising protein of interest which is pegylated and separates LMW or HMW fragments present in the protein mixture.
1 . A process for the quantification and/or detection of impurity in the protein mixture comprises;
a. Protein mixture comprising at least the impurity and the protein of interest, wherein the protein mixture does not contain an antibody;
b. Mixing the protein mixture with a suitable buffer and detergent;
c. Optionally incorporating a 10 kDa marker into the treated protein mixture;
d. Injecting the treated protein mixture in CE-instrument;
e. Performing CE SDS by injecting the treated protein mixture in a CE-instrument at suitable temperature; and
f. Detecting and/or quantifying the impurity;
wherein the impurity is selected from LMW impurity and HMW impurity.
2 . The process according to claim 1 further comprises mixing the protein mixture in step (b) with reducing agent capable of denaturing the protein and reducing noncovalent bonds in the protein mixture.
3 . The process according to claim 2 wherein the reducing agent is selected from DTT, B-mercaptoethanol and TCEP.
4 . The process according to claim 1 wherein the detergent is sodium dodecyl sulphate or SDS.
5 . The process according to claim 1 wherein the SDS concentration is 1%.
6 . The process according to claim 1 wherein the protein mixture further comprises water or buffer.
7 . The process according to claim 1 wherein the protein mixture is at least 2 mg/ml.
8 . The process according to claim 7 wherein the protein mixture is at least 2 mg/ml to 5 mg/ml.
9 . The process according to claim 1 wherein the protein mixture does not comprise an antibody and/or fragment thereof.
10 . The process according to claim 1 wherein the suitable temperature of CE-SDS capillary is below 25° C.
11 . The process according to claim 10 wherein the capillary temperature is selected from 15° C., 16° C., 17° C., 18° C., 19° C., 20° C., 21° C., 22° C.
12 . The process according to claim 11 wherein the capillary temperature is 15° C.
13 . The process according to claim 1 wherein the quantification or detection of low molecular weight or high molecular weight impurity is improved compared to CE-SDS performed at 25° C.
14 . The process according to claim 1 wherein the CE-SDS is performed at separation voltage at about 15 kV or −15 kV for at least about 30 minutes.
15 . The process according to claim 1 wherein the CE-SDS is performed at an injection voltage of about 5 kV or −5 kV for at least about 10 seconds.
16 . The process according to claim 1 wherein the CE-SDS is performed with Tris buffer at pH 9.
17 . The process according to claim 1 wherein the suitable buffer is Tris.
18 . The process according to claim 16 wherein the Tris concentration is selected from 60 mM to 90 mM.
19 . The process according to claim 1 wherein the low molecular weight impurity is less than about 38 KDa.
20 . The process according to claim 1 wherein the low molecular weight impurity is GCSF or any other fragments derived from PEG-GCSF.
21 . The process according to claim 1 wherein the high molecular weight impurity is higher than about 38 KDa.
22 . A process for an improving protein peak separation efficiency in CE-SDS comprising:
a. Providing a protein mixture comprising a LMW impurity having a molecular weight of at least about 18 kDa, and a protein of interest having a molecular weight of about 38 kDa;
b. Mixing the protein mixture with Tris, SDS and 2-mercaptoethanol to form a treated protein mixture;
c. Injecting the treated protein mixture in CE-SDS;
d. Performing the CE-SDS with capillary temperature below 25° C. to separate the LMW impurity from the protein having a molecular weight of about 38 kDa; and
e. Separating the LMW impurity from the protein having a molecular weight of about 38 kDa through CE-SDS
wherein the separation is improved compared to CE-SDS performed at 25° C.
23 . A process for the quantification and/or detection of GCSF in the protein mixture comprises;
a. Protein mixture comprising the GCSF, pegylated GCSF and suitable buffer or water;
b. Mixing the protein mixture with Tris buffer, SDS and 2-mercaptoethanol to form a treated protein mixture;
c. Optionally incorporating a 10 kDa marker into the treated protein mixture;
d. Injecting the treated protein mixture in CE-instrument;
e. Performing the CE-SDS to separate the GCSF from pegylated GCSF; and
f. Detecting and/or quantifying the GCSF;
wherein the CE-SDS provide improved quantification and/or detection of GCSF or any other fragment compared to CE-SDS performed at 25° C.
24 . A process for the analysis of PEG-GCSF comprising;
a. Preparing a protein mixture comprising the pegylated GCSF in suitable buffer or water;
b. Mixing the protein mixture with Tris buffer, SDS and 2-mercaptoethanol to form a treated protein mixture;
c. Optionally incorporating a 10 kDa marker into the treated protein mixture;
d. Injecting the treated protein mixture in CE-instrument;
e. Performing the CE-SDS with keeping capillary temperature below 25° C.; and
f. Analyzing the protein mixture to evaluate presence of unpegylated GCSF;
wherein the CE-SDS provide improved analysis of unpegylated GCSF compared to CE-SDS performed at 25° C.