Quantification of misfolded TNFR2:Fc

摘要

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.

主权项

1. A composition comprising Cys74-Cys88/Cys78-Cys96 disulfide bridged TNFR2:Fc and Cys78-Cys88 disulfide bridged TNFR2:Fc;
wherein the relative amount of Cys78-Cys88 disulfide bridged TNFR2:Fc is less than 2.2% of the total TNFR2:Fc in the composition when determined according to formula (1);x⁡(in⁢⁢%)=[T⁢⁢7⁢⁢area][T⁢⁢7⁢⁢area]+[T⁢⁢27⁢⁢area]⨯100(1) wherein [T7 area] and [T27 area] are obtained by a method comprising the steps of:
(a) providing a sample comprising a mixture of Cys78-Cys88 disulfide bridged TNFR2:Fc and Cys74-Cys88/Cys78-Cys96 disulfide bridged TNFR2:Fc;(b) denaturing and alkylating the sample of step (a);(c) subjecting the sample resulting from step (b) to tryptic digestion;(d) subjecting the sample resulting from step (c) to HPLC, thereby separating fragments indicative of Cys78-Cys88 disulfide bridged TNFR2:Fc; and(e) conducting a peak integration for the peak indicative of Cys78-Cys88 disulfide bridged TNFR2:Fc and for a peak indicative of the total TNFR:Fc in the sample, as obtained from step (d), wherein the fragments indicative of Cys78-Cys88 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”); wherein the amino acid sequence of the TNFR2:Fc has at least 97% identity to the amino acid sequence of SEQ ID NO: 3.