Name: capillary electrophoresis separation of monoclonal antibody isoforms using a neutral capillary
Uploaded: 2017-01-16T14:00:00
Description: Watch this Scientific Journal Video about Capillary Electrophoresis Separation of Monoclonal Antibody Isoforms Using a Neutral Capillary at JoVE.com

The authors thank Wiley for the granted permission to use the concepts of the following publication for this tutorial. Carlos E. Espinosa-de la Garza, Francisco C. Perdomo-Ab&#250;ndez, Jes&#250;s Padilla-Calder&#243;n, Jaime M. Uribe-Wiechers, N&#233;stor O. P&#233;rez, Luis F. Flores-Ortiz, Emilio Medina-Rivero: Analysis of recombinant monoclonal antibodies by capillary zone electrophoresis. Electrophoresis. 2013. 34. 1133-1140. Copyright Wiley-VCH Verlag GmbH &amp; Co. KGaA. This work was supported by CONACyT, Mexico, grant 230551.

1. Preparation of Solutions
<ol>
	<li>Prepare the BGE solution.
	<ol>
		<li>Prepare 100 ml of a solution composed of 0.05% (m/v) hydroxy propyl methyl cellulose (HPMC), 200 mM &#949;-amino n-caproic acid (EACA) and 30 mM lithium acetate. 
		NOTE: As HPMC is a viscoelastic polymer, pour the powder into a glass beaker, add 80 mL of water and finally add the stirring bar. Continue adding the remaining reagents as normally. Wear safety glasses when handling lithium acetate as it can cause eye irritati...

<ol>
	<li>Bruno, V., Battaglia, G., Nicoletti, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+advent+of+monoclonal+antibodies+in+the+treatment+of+chronic+autoimmune+diseases.">The advent of monoclonal antibodies in the treatment of chronic autoimmune diseases.</a> Neurol. Sci. 31, 283-288 (2011).</li><li>Liu, H., Gaza-Bulseco, G., Faldu, D., Chumsae, C., Sun, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterogeneity+of+monoclonal+antibodies.">Heterogeneity of monoclonal antibodies.</a> J. Pharm. Sci. 97 (7), 2426-2447 (2008).</li><li>Creamer, J. S., Oborny, N. J., Lunte, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+advances+in+the+analysis+of+therapeutic+proteins+by+capillary+and+microchip+electrophoresis.">Recent advances in the analysis of therapeutic proteins by capillary and microchip electrophoresis.</a> Anal. Methods. 6 (15), 5427-5449 (2014).</li><li>Fekete, S., Guillarme, D., Sandra, P., Sandra, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromatographic,+Electrophoretic,+and+Mass+Spectrometric+Methods+for+the+Analytical+Characterization+of+Protein+Biopharmaceuticals.">Chromatographic, Electrophoretic, and Mass Spectrometric Methods for the Analytical Characterization of Protein Biopharmaceuticals.</a> Anal. Chem. 88 (1), 480-507 (2016).</li><li>He, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+identity,+charge+variants,+and+disulfide+isomers+of+monoclonal+antibodies+with+capillary+zone+electrophoresis+in+an+uncoated+capillary+column.">Analysis of identity, charge variants, and disulfide isomers of monoclonal antibodies with capillary zone electrophoresis in an uncoated capillary column.</a> Anal. Chem. 82 (8), 3222-3230 (2010).</li><li>He, Y., Isele, C., Hu, W., Ruesch, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+analysis+of+charge+variants+of+monoclonal+antibodies+with+capillary+zone+electrophoresis+in+dynamically+coated+fused-silica+capillary.">Rapid analysis of charge variants of monoclonal antibodies with capillary zone electrophoresis in dynamically coated fused-silica capillary.</a> J. Sep. Sci. 34 (5), 548-555 (2011).</li><li>Zhao, S. S., Chen, D. D. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Applications+of+capillary+electrophoresis+in+characterizing+recombinant+protein+therapeutics.">Applications of capillary electrophoresis in characterizing recombinant protein therapeutics.</a> Electrophoresis. 35 (1), 96-108 (2014).</li><li>&#352;t&#283;p&#225;nov&#225;, S., Ka&#353;i&#269;ka, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+impurities+and+counterions+of+pharmaceuticals+by+capillary+electromigration+methods.">Determination of impurities and counterions of pharmaceuticals by capillary electromigration methods.</a> J. Sep. Sci. 37 (15), 2039-2055 (2014).</li><li>&#352;t&#283;p&#225;nov&#225;, S., Ka&#353;i&#269;ka, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+applications+of+capillary+electromigration+methods+to+separation+and+analysis+of+proteins.">Recent applications of capillary electromigration methods to separation and analysis of proteins.</a> Anal. Chim. Acta. 933, 23-42 (2016).</li><li>Espinosa-de la Garza, C. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+recombinant+monoclonal+antibodies+by+capillary+zone+electrophoresis.">Analysis of recombinant monoclonal antibodies by capillary zone electrophoresis.</a> Electrophoresis. 34 (8), 1133-1140 (2013).</li><li>Staub, A., Guillarme, D., Schappler, J., Veuthey, J. L., Rudaz, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intact+protein+analysis+in+the+biopharmaceutical+field.">Intact protein analysis in the biopharmaceutical field.</a> J. Pharm. Biomed. Anal. 55 (4), 810-822 (2011).</li><li>Altria, K. D., Altria, K. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Troubleshooting.">Troubleshooting.</a> Methods in Molecular Biology, Vol 52. Capillary Electrophoresis Guidebook: Principles, Operation and Applications. , (1996).</li><li>Ma, S., Nashabeh, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+protein+therapeutics+by+capillary+electrophoresis.">Analysis of protein therapeutics by capillary electrophoresis.</a> Chromatographia. 53 (5), 75-89 (2001).</li><li>Jaccoulet, E., Smadja, C., Prognon, P., Taverna, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Capillary+electrophoresis+for+rapid+identification+of+monoclonal+antibodies+for+routine+application+in+hospital.">Capillary electrophoresis for rapid identification of monoclonal antibodies for routine application in hospital.</a> Electrophoresis. 36 (17), 2050-2056 (2015).</li></ol>

In this tutorial, we highlight the importance of proper practices when conducting CZE analyses of mAbs in order to increase the probability to succeed. However, when CZE is used on a routine basis, issues arise inevitably12.
For best results, it is important to follow the notes that were included throughout the protocol, as they will help the analyst to overcome and troubleshoot difficult steps. A major consideration to obtain optimum resolution at a given set of conditions is the c...

Monoclonal antibodies (mAbs) are biotherapeutic proteins with increasing interest due to their capacity to act against several chronic and degenerative diseases1. Like other biomolecules, mAbs are prone to undergo several physicochemical modifications at all stages of their life cycle (i.e., from biosynthesis to the final product). Such modifications include, but are not limited to: deamidation, glycosylation, oxidation, cyclization, isomerization, aggregation and proteolytic cleavage2. Hence, analytical techniques capable to resolve intrinsic isoforms are needed to monitor mAbs heterogeneity and stability in order to establish quality specifications.
Capillary electrophoresis (CE) is a high-performance separation technology carried outinside of a narrow fused-silica tube (&#181;m range) filled with a background electrolyte (BGE). Upon application of an electrical field (up to 30,000 V), charged molecules migrate towards the electrode with opposite charge (i.e., electro-driven separation). The use of high voltages in CE permits fast analyses and increased efficiency, which are superior to classical gel electrophoresis. Capillary zone electrophoresis (CZE) is a CE-based technique routinely used in the biopharmaceutical industry for product quality assessment3-9. Unlike other modes of CE (e.g., capillary gel electrophoresis, capillary isoelectric focusing) or chromatography-based methods, CZE can be conducted without using denaturants or solid-phase interfaces, allowing the analysis of the inherent heterogeneity of mAbs close to their native state10. CZE separation of mAb isoforms occurs inside of a fused-silica capillary covered with a hydrophilic polymer (neutral capillary) and is based on their different electrophoretic mobility, which is ruled by charge, mass, size and shape (or hydrodynamic volume)11. mAb moieties are detected when they are mobilized and pass through the detection window, which is sensed by an ultraviolet (UV) absorbance detector at 214 nm4.
The successful implementation of this analytical technique will depend on proper attention to details before and during the experiment. Acting otherwise will increase the cost and time to conduct the analysis, ultimately leading to constant failure and frustration.
Here, we present a step-by-step guide to conduct a successful analysis of mAb heterogeneity by CZE through the detailed explanation of the preparation of solutions and samples, the preparation of CE system, the instrument methods set up, the data acquisition, and the processing. For the purpose of this tutorial, a recombinant fully human anti-tumor necrosis factor alpha (anti-TNF&#945;) mAb is used as protein model; however, this protocol can be easily customized for the analysis of other proteins considering brief modifications. Additionally, several recommendations to mitigate potential issues are proposed. The reader is encouraged to strictly follow the proposed protocol, as the probability to succeed will increase.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Glacial acetic acid</td>
			<td>Tecsiquim</td>
			<td>AT0035-7</td>
		</tr>
		<tr>
			<td>ACS grade hydrochloric acid</td>
			<td>J.T. Baker</td>
			<td>9535-05</td>
		</tr>
		<tr>
			<td>Histamine dihydrochloride</td>
			<td>Fluka</td>
			<td>53300</td>
		</tr>
		<tr>
			<td>(Hydroxypropyl) methyl cellulose&#160;</td>
			<td>Fluka</td>
			<td>09963</td>
		</tr>
		<tr>
			<td>Lithium acetate</td>
			<td>Sigma-Aldrich</td>
			<td>517992</td>
		</tr>
		<tr>
			<td>6-Aminocaproic acid</td>
			<td>Sigma-Aldrich</td>
			<td>A2504</td>
		</tr>
		<tr>
			<td>eCAP Tris Buffer, 50.0 mM,&#160; pH 8</td>
			<td>Beckman Coulter</td>
			<td>477427</td>
		</tr>
		<tr>
			<td>PA 800 Plus Pharmaceutical Analysis System</td>
			<td>Beckman Coulter</td>
			<td>A66528</td>
		</tr>
		<tr>
			<td>eCAP Neutral capillary&#160;</td>
			<td>Beckman Coulter</td>
			<td>477441</td>
		</tr>
		<tr>
			<td>Vial, Micro, 200 &#181;l</td>
			<td>Beckman Coulter</td>
			<td>144709</td>
		</tr>
		<tr>
			<td>Universal Vial Caps</td>
			<td>Beckman Coulter</td>
			<td>A62250</td>
		</tr>
		<tr>
			<td>Universal Vials</td>
			<td>Beckman Coulter</td>
			<td>A62251</td>
		</tr>
		<tr>
			<td>Cable, Optics, UV/Vis</td>
			<td>Beckman Coulter</td>
			<td>144093</td>
		</tr>
		<tr>
			<td>UV/Vis Detector Module</td>
			<td>Beckman Coulter</td>
			<td>144733</td>
		</tr>
		<tr>
			<td>Cartridge Assembly Kit, Blank</td>
			<td>Beckman Coulter</td>
			<td>144738</td>
		</tr>
	</tbody>
</table>

capillary electrophoresis separation of monoclonal antibody isoforms using a neutral capillary

Biotherapeutic proteins, such as monoclonal antibodies (mAbs), are feasible alternatives for the treatment of chronic-degenerative diseases. The biological activity of these proteins depends on their physicochemical properties. The use of high-performance techniques like chromatography and capillary electrophoresis has been described for the analysis of physicochemical heterogeneity of mAbs. Nowadays, capillary zone electrophoresis (CZE) technique constitutes one of the most resolutive and sensitive assays for the analysis of biomolecules. Besides, the electro-driven separation in CZE is governed by extensive properties of matter and offers the advantage of analyzing proteins close to their native state. However, the successful implementation of this technique for routine analysis depends on the skills of the analyst at the critical steps during sample and system preparation. The purpose of this tutorial is to detail the steps to succeed in the CZE analysis of mAbs. Further, this protocol can be used for the development and improvement of skills of the personnel involved in protein analytical chemistry laboratories.

Figure 2 shows the typical electrical current profile of a 200 mM EACA, 30 mM lithium acetate, pH 4.8 BGE with anti-TNF&#945; mAb sample diluted with Tris buffer (50 mM, pH 8.0). As can be observed, the current is stable throughout the analysis and can oscillate between values of 30 to 35 &#181;A. Figure 3 shows the CZE electropherogram of a blank sample where the detected peak corresponds to the histamine internal standard. It is expected for histamine t...

Watch this Scientific Journal Video about Capillary Electrophoresis Separation of Monoclonal Antibody Isoforms Using a Neutral Capillary at JoVE.com

Capillary Electrophoresis Separation of Monoclonal Antibody Isoforms Using a Neutral Capillary

Here, we present a comprehensive capillary zone electrophoresis protocol for the assessment of intrinsic physicochemical heterogeneity of monoclonal antibodies as a quality attribute.

Biotherapeutic proteins, such as monoclonal antibodies (mAbs), are feasible alternatives for the treatment of chronic-degenerative diseases. The ...

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