Building IP: BMY Patent Appl "METHOD OF PURITY DETERMINATION BY CAPILLARY ELECTRO... | BMY Message Board Posts


Bristol-Myers Squibb Co.

  BMY website

BMY   /  Message Board  /  Read Message

 

 






Keyword
Subject
Between
and
Rec'd By
Authored By
Minimum Recs
  
Previous Message  Next Message    Post Message    Post a Reply return to message boardtop of board
Msg  12091 of 12418  at  12/7/2023 9:29:01 AM  by

JBWIN


Building IP: BMY Patent Appl "METHOD OF PURITY DETERMINATION BY CAPILLARY ELECTRO...

 

METHOD OF PURITY DETERMINATION BY CAPILLARY ELECTROPHORESIS

DOCUMENT ID

US 20230393094 A1

DATE PUBLISHED

2023-12-07

INVENTOR INFORMATION

NAME

CITY

STATE

ZIP CODE

COUNTRY

BECKMAN; Jeff W
Northborough
MA
N/A
US
GUAN; Qian
Newton
MA
N/A
US

APPLICANT INFORMATION

NAME
BRISTOL-MYERS SQUIBB COMPANY
CITY
Princeton
STATE
NJ
ZIP CODE
N/A
COUNTRY
US
AUTHORITY
N/A
TYPE
assignee

ASSIGNEE INFORMATION

NAME
BRISTOL-MYERS SQUIBB COMPANY
CITY
Princeton
STATE
NJ
ZIP CODE
N/A
COUNTRY
US
TYPE CODE
02

APPLICATION NO

18/324079

DATE FILED

2023-05-25

DOMESTIC PRIORITY (CONTINUITY DATA)

parent US continuation 16645059 20200306 parent-grant-document US 11698358 WO continuation PCT/US2018/049992 20180907 child US 18324079

us-provisional-application US 62555335 20170907

US CLASS CURRENT:

1/1

CPC CURRENT

TYPE

CPC

DATE

CPCI
2013-01-01

Abstract

This disclosure provides protein separation techniques using detergents that have alkyl chains longer than 12 carbon atoms, which improves protein separation during electrophoresis.

Background/Summary

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation of U.S. application Ser. No. 16/645,059, filed Sep. 17, 2018, which is a U.S. 371 filing of PCT/US2018/049992 filed Sep. 7, 2018, which claims the benefit of U.S. Provisional application No. 62/555,335, filed Sep. 7, 2017, the disclosure of which is incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

[0002] A method capable of improving protein peak separation efficiency (PSE) by capillary electrophoresis. The method includes increasing detergent hydrophobicity to improve protein separation efficiency (PSE) by increasing the affinity of the detergent to the protein.

BACKGROUND OF THE INVENTION

[0003] Throughout this application, various publications are referenced in parentheses by author name and date, or by Patent No. or Patent Publication No. The disclosures of these publications are hereby incorporated in their entireties by reference into this application in order to more fully describe the state of the art as known to those skilled therein as of the date of the disclosure described and claimed herein. However, the citation of a reference herein should not be construed as an acknowledgement that such reference is prior art to the present disclosure.

[0004] Commercialization of therapeutic proteins require analytical techniques that can measure product heterogeneity from the complexity of biosynthesis (Chirino, A. J., et.al. (2004) Characterizing Biological Products and Assessing Comparability Following Manufacturing Changes. Nat Biotechnol. 22, 1383-1391, 2; Zhao, S., et.al. (2014) Applications of Capillary Electrophoresis in Characteriziong Recombinant Protein Therapeutics. Electrophoresis 35, 96-108). Towards this end, Capillary Gel Electrophoresis (CGE) contributes to the understanding of protein size heterogeneity by separating and quantitatively detecting proteins by molecular weight and hydrodynamic radius (Rustandi, R. et.al. (2008) Applications of CE SDS Gel in Development of Biopharmaceutical Antibody-Based Products. Electrophoresis 29, 3612-3620-4; Chen, T., et.al. (2016) Antibody-Drug Conjugate Characterization by Chromatographic and Electrophoretic Techniques. J. Chromatogr. B 1032, 39-50). In this procedure, the protein is denatured with charged detergent to produce protein-detergent complexes with a uniform mass/charge ratio that is separated by molecular weight while sieving through a capillary filled with a hydrophilic gel buffer solution. This is followed by UV detection ideally at a point along the capillary when quantification of peaks can occur, which requires adequate protein peak separation efficiency (PSE), as defined by high plate counts and resolution. CGE works well for most therapeutic proteins in this regard, specifically for IgGs, and has been accepted as standard for the evaluation of product purity in the biotechnology industry (Nunally, B., et.al. (2006) A Series of Collaborations Between Various Pharmaceutical Companies and Regulatory Authorities Concerning the Analysis of Biomolecules Using Capillary Electrophoresis. Chromatographia 64, 359-368).

[0005] Sodium dodecyl sulfate (SDS) has been used as the default detergent for CGE separations largely because of its establishment in conventional polyacrylamide gel electrophoresis (SDS-PAGE) (Laemmli, U. K. (1970) Cleavage of Structural Proteins During the Assembly of the Head of Bacteriophage T4. Nature 227, 680-685; Otzen, D. E. (2015) Proteins in a Brave New Surfactant World. Curr. Opin. Colloid Interface Sci. 20, 161-169). In addition, SDS is able to uniformly bind to a typical protein at a ratio of 1.4 g SDS to 1 g protein, ensuring uniform mass/charge ratios of SDS:Protein complexes in most cases (Reynolds, J. A., et.al. (1970) Binding of Dodecyl Sulfate to Proteins at High Binding Ratios. Possible Implications for the State of Proteins in Biological Membrane. Proc. Natl. Acad. Sci. USA 66, 1002-1007). Hence CGE is commonly referred to as SDS-CGE or CE-SDS.

SUMMARY OF THE INVENTION

[0006] An embodiment of the invention is a method for analyzing a protein sample, comprising separating denatured protein of interest in the sample by capillary electrophoresis, comprising a hydrophobic detergent gel buffer.

[0007] An embodiment of the invention is a method for improving capillary electrophoresis protein peak separation efficiency (PSE), comprising separating denatured protein of interest in the sample in a hydrophobic detergent gel buffer.

[0008] An embodiment of the invention is a method for improving protein purity determination by capillary electrophoresis comprising separating denatured protein of interest in the sample in a hydrophobic detergent gel buffer.

[0009] Another embodiment of the invention is a method for analyzing a protein sample, comprising separating denatured protein of interest in the sample by capillary electrophoresis, comprising hydrophobic detergent gel buffer, wherein the hydrophobic detergent has the same charged sulfate head group and sodium counter-ion with alkyl chain lengths selected from 11, 14, and 16.

[0010] Another embodiment of the invention is a method for improving capillary electrophoresis protein peak separation efficiency (PSE), comprising separating denatured protein of interest in the sample in a hydrophobic detergent gel buffer, wherein the hydrophobic detergent has the same charged sulfate head group and sodium counter-ion with alkyl chain lengths selected from 11, 14, and 16.

[0011] Another embodiment of the invention is a method for improving protein purity determination by capillary electrophoresis comprising separating denatured protein of interest in the sample in a hydrophobic detergent gel buffer, wherein the hydrophobic detergent has the same charged sulfate head group and sodium counter-ion with alkyl chain lengths selected from 11, 14, and 16.

[0012] Another embodiment of the invention is a method for analyzing a protein sample, comprising separating denatured protein of interest in the sample by capillary electrophoresis, comprising a hydrophobic detergent gel buffer selected from the group consisting of sodium undecyl sulfate (SUS), sodium tetradecyl sulfate (STS), and sodium hexadecyl sulfate (SHS).

[0013] Another embodiment of the invention is a method for improving capillary electrophoresis protein peak separation efficiency (PSE), comprising separating denatured protein of interest in the sample in a hydrophobic detergent gel buffer selected from the group consisting of sodium undecyl sulfate (SUS), sodium tetradecyl sulfate (STS), and sodium hexadecyl sulfate (SHS).

[0014] Another embodiment of the invention is a method for improving protein purity determination by capillary electrophoresis comprising separating denatured protein of interest in the sample in a hydrophobic detergent gel buffer selected from the group consisting of sodium undecyl sulfate (SUS), sodium tetradecyl sulfate (STS), and sodium hexadecyl sulfate (SHS).

[0015] Another embodiment of the invention is a method for analyzing a protein sample, comprising separating denatured protein of interest in the sample by capillary electrophoresis, comprising a sodium hexadecyl sulfate (SHS) gel buffer.

[0016] Another embodiment of the invention is a method for improving capillary electrophoresis protein peak separation efficiency (PSE), comprising separating denatured protein of interest in the sample in a sodium hexadecyl sulfate (SHS) gel buffer.

[0017] Another embodiment of the invention is a method for improving protein purity determination by capillary electrophoresis comprising separating denatured protein of interest in the sample in a sodium hexadecyl sulfate (SHS) gel buffer.

[0018] Another embodiment of the invention is a capillary electrophoresis gel buffer for protein separation analysis that comprises a hydrophobic detergent.

[0019] In some embodiments, an electrophoresis buffer composition useful for the present disclosure comprises a hydrophobic detergent comprising a sulfate head group and a hydrophobic tail comprising an alkyl chain having greater than 12 carbon atoms.

[0020] In some embodiments, the hydrophobic detergent has an alkyl chain with carbon atoms less than 19 or 20. In some embodiments, the alkyl chain for the hydrophobic detergent has carbon atoms of 13, 14, 15, 16, 17, or 18. In some embodiments, the buffer composition comprises a hydrophobic detergent that is more hydrophobic than sodium dodecyl sulfate.

[0021] The present methods can be effective at improving protein peak separation efficiency. In some embodiments, the hydrophobic detergent is capable of inducing improved protein peak separation efficiency by capillary sieving electrophoresis compared to sodium dodecyl sulfate. In other embodiments, the hydrophobic detergent is capable of removing HMW species artifacts.

[0022] In some embodiments, the buffer composition comprises a detergent selected from a group consisting of sodium tridecyl sulfate, sodium tetradecyl sulfate (STS), sodium pentadecyl sulfate, sodium hexadecyl sulfate (SHS), sodium heptadecyl sulfate, and sodium octadecyl sulfate (SOS). In some embodiments, the hydrophobic detergent is sodium hexadecyl sulfate (SHS). In some embodiments, the hydrophobic detergent is at a concentration of from about 0.02% to about 4% w/v.

[0023] In some embodiments, the buffer composition can further comprise additional components In some embodiments, the buffer composition further comprises one or more additional components selected from a group consisting of a buffering component, an organic additive, a hydrophilic polymer, a metal chelator, and any combination thereof. In some embodiments, the buffering component comprises a tris(hydroxymethyl)aminomethane buffer, a phosphate buffer, a citrate buffer, or any combination thereof. In some embodiments, the organic additive is mannitol, glycerol, ethylene glycol, ethanol, methanol, or any combination thereof. In some embodiments, the hydrophilic polymer is dextran, polyacrylamide, polyethylene glycol, or any combination thereof. In some embodiments, the metal chelator is ethylenediaminetetraacetic acid, diethylenetriaminepentaacetic acid, or any combination thereof.

[0024] The present methods are useful for separating a protein of interest via electrophoresis wherein the electrophoresis buffer is added to the sample buffer, the running buffer, and/or the gel itself. In some embodiments, the method for separating a protein of interest via electrophoresis comprises denaturing the protein of interest in a sample buffer. In some embodiments, the method further comprises running an electrophoresis gel in a running buffer. In some embodiments, the running buffer and/or the electrophoresis gel comprise the electrophoresis buffer composition.

[0025] The present methods are useful for improving the protein peak separation efficiency (PSE) as compared to the protein peak separation efficiency (PSE) of a separation conducted using sodium dodecyl sulfate (SDS). In some embodiments, the method for improving peak separation efficiency of a protein of interest via electrophoresis comprises denaturing the protein of interest in a sample buffer and/or running a sample in a running buffer electrophoretically, wherein the protein peak separation efficiency (PSE) is improved as compared to the protein peak separation efficiency (PSE) of a separation conducted using sodium dodecyl sulfate (SDS). In some embodiments, the electrophoresis is capillary gel electrophoresis. In some embodiments, the electrophoresis gel results show less artificial high molecular weight species compared to an electrophoresis gel conducted with sodium dodecyl sulfate.

[0026] In some embodiments, the denaturing is performed at a temperature of at least about 60° C., at least about 65° C., at least about 70° C., at least about 75° C., or at least about 80° C. In some embodiments, the denaturing is performed at a temperature between about ° C. and about 70° C., between about 65° C. and about 70° C., or about 60° C. and about 65° C. In some embodiments, the denaturing is performed for at least about 3 minutes, at least about 4 minutes, at least about 5 minutes, at least about 6 minutes, at least about 7 minutes, at least about 8 minutes, at least about 9 minutes, at least about 10 minutes.

[0027] In some embodiments, the protein of interest is an antibody. In some embodiments, the antibody is an isotype selected from IgM, IgA, IgE, IgD, and IgG. In some embodiments, the IgG antibody is selected from IgG1, IgG2, IgG3, and IgG4. In some embodiments, the protein of interest comprises an enzyme, a hormone, a cytokine, a cell surface receptor, a protease, a cytokine receptor, or any combination thereof. In some embodiments, the protein of interest is a fusion protein. In some embodiments, the fusion protein is fused to a heterologous moiety. In some embodiments, the heterologous moiety is a half-life extending moiety. In some embodiments, the half-life extending moiety comprises an Fc.



     e-mail to a friend      printer-friendly     add to library      
|  
Recs: 1  
   Views: 0 []
Previous Message  Next Message    Post Message    Post a Reply return to message boardtop of board




Financial Market Data provided by
.
Loading...