Quantification of misfolded tnfr2:fc

Patent metadata

Field	Value
Publication number	US-2016017403-A1
Application number	US-201414455183-A
Country	US
Kind code	A1
Filing date	Aug 8, 2014
Priority date	Jul 18, 2014
Publication date	Jan 21, 2016
Grant date	—

Abstract

The present invention is directed to methods for determining the relative amount of wrongly disulphide bridged TNFR2:Fc in a sample of TNFR2:Fc, a fusion protein which is used in a variety of therapeutic applications. In addition, the invention pertains to a method for purifying TNFR2:Fc using said method for determining the percentage of wrongly disulphide bridged TNFR2:Fc, and to TNFR2:Fc compositions obtained thereby.

First claim

1 . A method for determining Cys 78 -Cys 88 disulfide bridged TNFR2:Fc in a sample comprising Cys 74 -Cys 88 /Cys 78 -Cys 96 disulfide bridged TNFR2:Fc and Cys 78 -Cys 88 disulfide bridged TNFR2:Fc, wherein the method comprises the steps of:
(f) providing a sample comprising a mixture of Cys 78 -Cys 88 disulfide bridged TNFR2:Fc and Cys 74 -Cys 88 /Cys 78 -Cys 96 disulfide bridged TNFR2: Fc; (g) denaturing and alkylating the sample of step (a); (h) subjecting the sample resulting from step (b) to tryptic digestion; (i) subjecting the sample resulting from step (c) to HPLC, thereby separating fragments indicative of Cys 78 -Cys 88 disulfide bridged TNFR2:Fc; and (j) conducting a peak integration for the peak indicative of Cys 78 -Cys 88 disulfide bridged TNFR2:Fc and for a peak not affected by disulfide bridging of Cys 74 , Cys 78 , Cys 88 and Cys 96 , as obtained from step (d); wherein the amino acid sequence of the TNFR2 part of TNFR2:Fc has at least 97% identity to the amino acids 1-235 of the amino acid sequence of SEQ ID NO: 1.

2 . The method of claim 1 , wherein the amino acid sequence of the TNFR2:Fc applied to step (a) has at least 97% identity to the amino acid sequence of SEQ ID NO: 3 (etanercept).

3 . The method of claim 1 , wherein the peak not affected by disulfide bridging of Cys 74 , Cys 78 , Cys 88 and Cys 96 is not affected by disulfide bridging at all and indicative of the total TNFR:Fc in the sample.

4 . The method of claim 3 , wherein the fragments indicative of Cys 78 -Cys 88 disulfide bridged TNFR2:Fc comprise the amino acid sequence shown in SEQ ID NO: 4 (“T7”); and
wherein the fragments indicative of total TNFR2:Fc comprise the amino acid sequence shown in SEQ ID NO: 5 (“T27”).

5 . The method of claim 4 , wherein the fragments indicative of Cys 78 -Cys 88 disulfide bridged TNFR2:Fc consist of the amino acid sequence shown in SEQ ID NO: 4 (“T7”); and
wherein the fragments indicative of total TNFR2:Fc consist of the amino acid sequence shown in SEQ ID NO: 5 (“T27”).

6 . The method of claim 4 , wherein the relative amount of Cys 78 -Cys 88 disulfide bridged TNFR2:Fc is determined by
(iii) integrating the peak areas in the HPLC chromatogram indicative of Cys 78 -Cys 88 disulfide bridged TNFR2:Fc (“T7 area”) and indicative of total TNFR2:Fc (“T27 area”); and (iv) calculating the relative amount according to formula (1).

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100

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7 . The method of claim 1 , wherein step (b) is carried out in a buffer having a pH in the range of 7 to 9, comprising at least one of
10-100 mM TRIS, 0.5-1.5M iodoacetamide, and 0.02%-0.5% of a cleavable surfactant.

8 . The method of claim 7 , wherein step (b) is carried out in a buffer having a pH in the range of 7.5 to 8.5, comprising at least one of
20-80 mM TRIS, 0.9-1.2M iodoacetamide, and 0.1%-0.2% of a cleavable surfactant.

9 . The method of claim 8 , wherein the buffer comprises a cleavable surfactant which is selected from
sodium 3-[(2-methyl-2-undecyl-1,3-dioxolan-4-yl)methoxy]-1-propanesulfonate, sodium 3-((1-(furan-2-yl)undecyloxy)carbonylamino)propane-1-sulfonate, and sodium 3-(4-(1,1-bis(hexyloxy)ethyl)pyridinium-1-yl)propane-1-sulfonate.

10 . The method of claim 9 , wherein the cleavable surfactant is sodium 3-[(2-methyl-2-undecyl-1,3-dioxolan-4-yl)methoxy]-1-propanesulfonate.

11 . The method of claim 1 , wherein step (b) is carried out at 40 to 70° C. for 30 to 60 min.

12 . The method of claim 1 , wherein step (b) is carried out at 50 to 60° C. for 30 to 45 min.

13 . The method of claim 1 , wherein step (c) is carried out in a digestion buffer having a pH in the range of 5 to 7; and wherein the digestion buffer comprises
MES as the buffering agent; or 0.02%-0.5% of a cleavable surfactant; or MES as the buffering agent and 0.02%-0.5% of a cleavable surfactant.

14 . The method of claim 13 , wherein step (c) is carried out in a digestion buffer having a pH in the range of 5.5 to 6.5; and wherein the digestion buffer comprises
10-100 mM MES as the buffering agent; or 0.1%-0.2% of a cleavable surfactant; or 10-100 mM MES as the buffering agent and 0.1%-0.2% of a cleavable surfactant.

15 . The method of claim 13 , wherein the buffer comprises a cleavable surfactant which is selected from
sodium 3-[(2-methyl-2-undecyl-1,3-dioxolan-4-yl)methoxy]-1-propanesulfonate, sodium 3-((1-(furan-2-yl)undecyloxy)carbonylamino)propane-1-sulfonate, and sodium 3-(4-(1,1-bis(hexyloxy)ethyl)pyridinium-1-yl)propane-1-sulfonate.

16 . The method of claim 15 , wherein the cleavable surfactant is sodium 3-[(2-m

Assignees

• Sandoz Ag

Inventors

• Rupprechter Alfred
• Fuchs Michael
• Holzmann Johann
• Lamanna William
• Posch Christopher
• Toll Hansjorg
• Mayer Robert

Classifications

• C12Q1/37 Primary

  involving peptidase or proteinase · CPC title

• G01N21/33

  using ultraviolet light (G01N21/39 takes precedence) · CPC title

• C07K14/7151 Primary

  for tumor necrosis factor [TNF], for lymphotoxin [LT] · CPC title

• G01N33/6863 Primary

  Cytokines, i.e. immune system proteins modifying a biological response such as cell growth proliferation or differentiation, e.g. TNF, CNF, GM-CSF, lymphotoxin, MIF or their receptors · CPC title

• G01N2440/20

  formation of disulphide bridges · CPC title