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In no event shall the Royal Society of Chemistry be held responsible for any errors or omissions in this Accepted Manuscript or any consequences arising from the use of any information it contains. www.rsc.org/methods Page 1 of 54 Analytical Methods 1 2 3 4 5 6 7 8 9 10 11 12 13 14 Manuscript 15 16 17 18 19 20 Accepted 21 22 CEC 23 CIEF 24 CGE 25 CZE 26 Methods 27 28 29 30 31 32 33 Analytical 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Analytical Methods Page 2 of 54 1 2 3 Review 4 5 6 Recent advances in the analysis of therapeutic proteins by capillary and microchip electrophoresis 7 1,3 2,3 1,2,3 8 Jessica S. Creamer, Nathan J. Oborny, Susan M. Lunte 9 10 1Department of Pharmaceutical Chemistry, University of Kansas, Lawrence, KS, USA 11 12 2Department of Bioengineering, University of Kansas, Lawrence, KS, USA 13 14 3Ralph N. Adams Institute for Bioanalytical Chemistry, University of Kansas, Lawrence, KS, USA 15 16 17 18 19 Susan M. Lunte, Ph.D. 20 21 Ralph N. Adams Professor of Chemistry and Pharmaceutical Chemistry Manuscript 22 23 Ralph N. Adams Institute for Bioanalytical Chemistry 24 25 2030 Becker Dr. Lawrence, KS, 66044 26 27 [email protected] 28 29 30 Fax: 785-864-1916 31 Accepted 32 List of abbreviations in alphabetical order: 33 + 34 1-vinyl-3-octylimidazolium (ViOcIm ), 8-aminopyrene-1,3,6-trisulfonic acid (APTS), background 35 − electrolyte (BGE), bis-trifluoromethanesulfonylimide (NTf 2 ), bovine serum albumin (BSA), capillary 36 37 electrochromatography (CEC), capillary electrophoresis (CE), capillary gel electrophoresis (CGE), capillary 38 isoelectric focusing (CIEF), capillary isotachophoresis (CITP), capillary zone electrophoresis (CZE), 39 diazoresin (DR), electroosmotic flow (EOF), electrospray ionization (ESI), erythropoietin (EPO), galactose- 40 Methods α-1,3-galactose (α1,3-Gal), gold nanoparticles (AuNP), graphene (G), graphene oxide (GO), hydrophilic 41 42 interaction chromatography (HILIC), hydroxyproplymethylcellulose (HPMC), imaging capillary isoelectric 43 focusing (iCIEF), ionic liquid (IL), isoelectric point (pI), laser-induced fluorescence (LIF), liquid 44 chromatography (LC), mass spectrometry (MS), matrix-assisted laser desorption ionization (MALDI), 45 46 microchip electrophoresis (ME), microchip gel electrophoresis (MGE), microchip isoelectric focusing 47 (MIEF), molecular weight (MW), monoclonal antibodies (mAbs), nanoparticles (NP), N- 48 glycolylneuraminic acid (Neu5Gc), N-methyl-2-pyrrolidonium methyl sulfonate ([NMP] +CH SO – ), open- 49 3 3 50 tubular capillary electrochromatography (OTCEC), pentaerythritol (PETA), phospholipid bilayers (PLB), Analytical 51 polyamidoamine-grafted silica nanoparticles (PAMAM-SNP), polybrene (PB), polyethylene glycol (PEG), 52 polyvinyl alcohol (PVA), post translational modifications (PTMs), pseudostationary phase (PSP), 53 54 quaternized celluoses (QC), sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), silica 55 nanoparticles (SNP), sodium dodecyl sulfate (SDS), stabilized phospholipid bilayer (SPB), sulfobutyl ether 56 β-cyclodextrins (SBE β-CD) 57 58 59 60 1 Page 3 of 54 Analytical Methods 1 2 3 Abstract 4 5 6 The development of therapeutic proteins and peptides is an expensive and time-intensive process. 7 8 Biologics, which have become a multi-billion dollar industry, are chemically complex products that 9 10 require constant observation during each stage of development and production. Post-translational 11 12 13 modifications along with chemical and physical degradation from oxidation, deamidation, and 14 15 aggregation, lead to high levels of heterogeneity that affect drug quality and efficacy. The various 16 17 separation modes of capillary electrophoresis (CE) are commonly utilized to perform quality control and 18 19 assess protein heterogeneity. This review attempts to highlight the most recent developments and 20 21 Manuscript 22 applications of CE separation techniques for the characterization of protein and peptide therapeutics by 23 24 focusing on papers accepted for publication in the in the two-year period between January 2012 and 25 26 December 2013. The separation principles and technological advances of CE, capillary gel 27 28 29 electrophoresis, capillary isoelectric focusing, capillary electrochromatography and CE-mass 30 31 spectrometry are discussed, along with exciting new applications of these techniques to relevant Accepted 32 33 pharmaceutical issues. Also included is a small selection of papers on microchip electrophoresis to show 34 35 36 the direction this field is moving with regards to the development of inexpensive and portable analysis 37 38 systems for on-site, high-throughput analysis. 39 40 Methods 41 42 43 44 45 46 47 48 49 50 51 Analytical 52 53 54 55 56 57 58 59 60 2 Analytical Methods Page 4 of 54 1 2 3 1. Introduction 4 5 6 The characterization of protein therapeutics presents a unique analytical challenge due to the inherent 7 8 heterogeneity of recombinant protein expression. Even small changes in the manufacturing process can 9 10 lead to vastly different active pharmaceutical ingredients. Additionally, numerous physical and chemical 11 12 13 degradation pathways can occur during manufacturing and storage that compromise protein integrity, 14 15 leading to a potentially harmful, unstable product [1]. Thorough characterization of protein therapeutics 16 17 is necessary at every step of the research and development process, from drug discovery to lot release. 18 19 Due to the potential complexity of product degradation during preformulation and formulation 20 21 Manuscript 22 studies, additional separation techniques are needed to complement the more widely used column 23 24 liquid chromatography (LC) methods. To address this issue, capillary electrophoresis (CE) has become a 25 26 popular choice for the separation and analysis of therapeutic proteins and peptides. 27 28 29 CE provides several distinct advantages over LC. First, due to the faster separation times and the use 30 31 of multi-capillary arrays, hundreds of samples can be processed by CE per day. Second, CE is capable of Accepted 32 33 achieving very high efficiency separations due to the low diffusion coefficients of biomolecules. Lastly, 34 35 36 the small dimensions of the capillary and the low sample volume requirements keep reagent and 37 38 analyte use to a minimum, reducing the cost-per-test. The benefits of CE for the analysis of therapeutic 39 40 peptides and proteins have been addressed in several excellent reviews to date [2-5]. Methods 41 42 This review is aimed at highlighting the advances made in the field of CE therapeutic protein analysis 43 44 45 during 2012 and 2013 by expanding on a paper that was recently published by Zhao et al. [5]. Following 46 47 brief descriptions of the working principles of the different CE separation and detection methods, the 48 49 recent technological improvements and novel applications are discussed. Two additional sections have 50 51 Analytical 52 been included to further explore the use of CE for the determination of protein glycosylation and the 53 54 comparison of biosimilars. Finally, a brief introduction into microfluidic approaches to protein analysis is 55 56 given. Microchip electrophoresis (ME) has the additional advantages of increased speed, high- 57 58 59 60 3 Page 5 of 54 Analytical Methods 1 2 3 throughput capabilities, and portability for on-site analyses. Tables are presented in each section to 4 5 6 highlight the relevant CE and ME application-based citations. 7 8 2. Techniques 9 10 Historically, capillary zone electrophoresis (CZE) has been the most commonly employed form of CE. Yet, 11 12 13 the principles of electrophoretic separations and the benefits of capillary-based techniques are 14 15 applicable to other CE separation modes as well. Protein analysis based on size can be accomplished by 16 17 capillary gel electrophoresis (CGE), capillary isoelectric focusing (CIEF) can be used to determine 18 19 isoelectric points and charge heterogeneity, and capillary electrochromatography (CEC), which combines 20 21 Manuscript 22 the high efficiency electrophoretic separation with chromatographic retention, can be used for more 23 24 selective separations and analysis of neutral species. Depending on the properties of the analyte and 25 26 requirements of the assay, each of these separation modes can be coupled to a number of detection 27 28 29 methods such as UV-Vis absorbance, laser-induced fluorescence (LIF), and mass spectrometry (MS). 30 31 2.1 Capillary zone electrophoresis Accepted 32 33 Of the electrophoresis-based separation techniques, CZE is most frequently used for the analysis of