Quantification of misfolded TNFR2:Fc

Patent metadata

Field	Value
Publication number	US-9598718-B2
Application number	US-201414455183-A
Country	US
Kind code	B2
Filing date	Aug 8, 2014
Priority date	Jul 18, 2014
Publication date	Mar 21, 2017
Grant date	Mar 21, 2017

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

The invention claimed is:

1. A method for determining a relative amount of 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:
(a) 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;
(b) denaturing and alkylating the sample of step (a);
(c) subjecting the sample resulting from step (b) to tryptic digestion under non-reducing conditions;
(d) determining by HPLC the amount of a fragment indicative of Cys 78 -Cys 88 disulfide bridged TNFR2:Fc in the sample resulting from step (c); and
(e) determining the relative amount of the Cys 78 -Cys 88 disulfide bridged TNFR2:Fc in the sample based on the amount of the fragment indicative of Cys 78 -Cys 88 disulfide bridged TNFR2:Fc relative to the amount of a fragment not affected by disulfide bridging of Cys 74 , Cys 78 , Cys 88 and Cys 96 ;
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, and wherein the fragment indicative of Cys 78 -Cys 88 disulfide bridged TNFR2:Fc consists of SEQ ID NO:4 (“T7”).

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 is indicative of the total TNFR2:Fc in the sample.

4. The method of claim 3 , wherein the fragment indicative of total TNFR2:Fc comprises the amino acid sequence shown in SEQ ID NO: 5 (“T27”).

5. The method of claim 4 , wherein the fragment indicative of total TNFR2:Fc consists 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
(i) determining 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
(ii) calculating the relative amount according to formula (1)

x
⁡

(

i
⁢

⁢
n
⁢

⁢
%

)

=

[

T
⁢

⁢
7
⁢

⁢
area

]

[

T
⁢

⁢
7
⁢

⁢
area

]

+

[

T
⁢

⁢
27
⁢

⁢
area

]

×
100.

(
1
)

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, and wherein the buffer comprises at least one of:
10-100 mM TRIS,
0.5-1.5 M 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, and wherein the buffer comprises at least one of:
20-80 mM TRIS,
0.9-1.2 M 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 the group consisting of:
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 the group consisting of:
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

Assignees

• Sandoz Ag

Inventors

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

Classifications

• G01N21/33

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

• C07K14/7151 Primary

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

• C12Q1/37 Primary

  involving peptidase or proteinase · 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

• C07K2319/30

  Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto · CPC title