Optimization, Validation, and Use of Capillary Gel Electrophoresis for Quality Control Testing of Synagis®, a Monoclonal Antibody

Abstract

Capillary electrophoresis methods offer many advantages over their conventional electrophoresis counterparts including: greater precision, higher throughput, on-line detection and less waste. Capillary gel electrophoresis (CGE), using replaceable sieving polymers, is Widely used as an alternative to SDS-PAGE and densitometry CGE offers greater automation capabilities than SDS-PAGE and can be operated using conventional chromatography system software. This software for handling and analyzing data makes the validation of CGE methods look quite similar to the validation of an HPLC method. The same parameters can be examined, including: precision, accuracy, linearity, selectivity, range, robustness, and system suitability. There are, however, certain key elements that are critical to a successful CGE method validation. The CGE quantification method must be shown to be equivalent to SDS-PAGE with densitometry and should be presented such that the densitometry plots look like the electropherograms. In addition, the sensitivity of the methods must be compared and the CGE method must have comparable or better sensitivity to SDS-PAGE with Coomassie staining. The validation must also be performed using materials that might be tested during the course of a typical process (in-process samples) in their particular matrices. The other vital element is assuring that acceptance criteria for the validation have been set to reasonable ranges. These ranges must be determined in pilot studies that are executed prior to the validation studies. Under most CGE conditions, the acceptance criteria will be quite similar to those seen in HPLC validations. Overall, CGE methods can be validated to the same level of assurance as HPLC methods. The CGE method described here has been approved by the FDA and is used routinely for testing of Synagis®.

Capillary electrophoresis of proteins 1999-2001

“…Capillary SDS-electrophoresis was used to analyze rubisco in plant leaves [117,208] and to study its dissociation [209], to analyze murine monoclonal antibodies and antibody fragments [15], seed 2S albumins [201], and a monoclonal antibody Synagis [210]. CIEF was employed to analyze erythropoietin glycoforms [120], BSA and its monoclonal antibody [211,212], heme proteins [85], Enbrel isoforms [125], proteins in single cell embryo of nematode Caenorhabditis elegans [213], cellobiohydrolase I in extracts from Trichoderma reesei [128], major erythrocyte membrane proteins [214], noncovalent protein-protein complexes [132], and isoenzymes of human alcohol dehydrogenase [133].…”

Applications of CE SDS gel in development of biopharmaceutical antibody‐based products

“…Owing to certain intrinsic limitations of the SDS PAGE assay, many biopharmaceutical laboratories have shifted efforts into developing CE SDS gel [1,2] assays. CE SDS gel assays have been successfully applied to many therapeutic proteins including monoclonal antibody (mAb) [3][4][5] and protein-based vaccines [6,7], and it is now routinely used in the quality-control environment to assess purity and integrity including the assay validation requirements that are easily achieved [1,8].…”

Capillary electrophoresis of proteins 2001–2003

“…If the freezedried samples were stored at room temperature for prolonged time, a minor decrease of enzymatic activity was observed accompanied by formation of oligomers in electropherogram. In another case, a CE method was developed for testing of Synagis  and approved by the FDA [172].…”