title: Applications of an Automated and Quantitative CE-Based Size and Charge Western Blot for Therapeutic Proteins and Vaccines.
abstract: Capillary Electrophoresis (CE) is a versatile and indispensable analytical tool that can be applied to characterize proteins. In recent years, labor-intensive SDS-PAGE and IEF slab gels have been replaced with CE-SDS (CGE) and CE-IEF methods, respectively, in the biopharmaceutical industry. These two CE-based methods are now an industry standard and are an expectation of the regulatory agencies for biologics characterization. Another important and traditional slab gel technique is the western blot, which detects proteins using immuno-specific reagents after SDS-PAGE separation. This technique is widely used across industrial and academic laboratories, but it is very laborious, manual, time-consuming, and only semi-quantitative. Here, we describe the applications of a relatively new CE-based western blot technology which is automated, fast, and quantitative. We have used this technology for both charge- and size-based CE westerns to analyze biotherapeutic and vaccine products. The size-based capillary western can be used for fast antibody screening, clone selection, product titer, identity, and degradation while the charge-based capillary western can be used to study product charge heterogeneity. Examples using this technology for monoclonal antibody (mAb), Enbrel, CRM197, and Clostridium difficile (C. difficile) vaccine proteins are presented here to demonstrate the utility of the capillary western techniques. Details of sample preparation and experimental conditions for each capillary western mode are described in this chapter.
authors.affiliation: Merck Research Laboratories, Vaccine Analytical Development, 770 Sumneytown Pike, WP42A-30, P.O. Box 4, West Point, PA, 19486, USA. richard_rustandi@merck.com.
authors.forename: Richard R
authors.initials: RR
authors.lastname: Rustandi
authors.affiliation: Merck Research Laboratories, Vaccine Analytical Development, 770 Sumneytown Pike, WP42A-30, P.O. Box 4, West Point, PA, 19486, USA.
authors.forename: Melissa
authors.initials: M
authors.lastname: Hamm
authors.affiliation: Merck Research Laboratories, Vaccine Analytical Development, 770 Sumneytown Pike, WP42A-30, P.O. Box 4, West Point, PA, 19486, USA.
authors.forename: Catherine
authors.initials: C
authors.lastname: Lancaster
authors.affiliation: Merck Research Laboratories, Vaccine Analytical Development, 770 Sumneytown Pike, WP42A-30, P.O. Box 4, West Point, PA, 19486, USA.
authors.forename: John W
authors.initials: JW
authors.lastname: Loughney
mesh_terms: D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000302:isolation & purification*; D057205:Automation, Laboratory; D001426:Bacterial Proteins / Q000737:chemistry / Q000302:isolation & purification; D015153:Blotting, Western / Q000379:methods*; D019075:Electrophoresis, Capillary / Q000379:methods*; D000068800:Etanercept / Q000737:chemistry / Q000302:isolation & purification; D014612:Vaccines / Q000737:chemistry / Q000302:isolation & purification*
chemical_list: D000911:Antibodies, Monoclonal; D001426:Bacterial Proteins; D014612:Vaccines; C028581:CRM197 (non-toxic variant of diphtheria toxin); D000068800:Etanercept
keywords: CRM197; Capillary western; Clostridium difficile vaccine; Mab; Western blot