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 irritation.</li>
		<li>Adjust to pH value 4.8 &#177; 0.1 with 50% (v/v) acetic acid. Allow the solution to stabilize for 5 min and adjust as necessary.</li>
		<li>Add water to make up to a final volume of 100 ml in volumetric flask.</li>
		<li>Filter through a hydrophilic membrane with a 0.2 &#181;m pore size.</li>
		<li>Store at 2-8 &#176;C for up to 7 days.</li>
	</ol>
	</li>
	<li>Prepare the internal standard.
	<ol>
		<li>Prepare 1 mL of a solution composed of 1% (m/v) histamine.</li>
		<li>Filter through a hydrophilic membrane with a 0.2 &#181;m pore size.</li>
		<li>Store at 2-8 &#176;C for up to 1 day.</li>
	</ol>
	</li>
</ol>
2. Sample Preparation
<ol>
	<li>Prepare 180 &#181;l of mAb sample diluted with Tris buffer (50 mM tris(hydroxymethyl)aminomethane, pH 8.0) to a final concentration of 1 mg/ml.</li>
	<li>Add 20 &#181;l of 1% (m/v) histamine.</li>
	<li>Mix and centrifuge at 1,000 x g for 5 sec.</li>
	<li>Place a micro vial inside a universal vial.</li>
	<li>Transfer the sample into the micro vial and cap the universal vial. Dispense the sample upwards in order to avoid introducing any bubbles.</li>
	<li>Store at 2-8 &#176;C for up to 1 day.</li>
	<li>Place the universal vial (with micro vial containing the sample solution) in the sample inlet tray.</li>
</ol>
3. Preparation of CE System.
<ol>
	<li>CE system cleaning. 
	NOTE: Conduct this procedure at least once a week in order to avoid electric current leakage derived from accumulation of dust and debris. However, depending on the application (e.g., use of a BGE with increased viscosity or with high salt concentration, samples with increased viscosity) electrodes and opening levers may require to be cleaned more frequently to prevent cross contamination and sample carryover.
	<ol>
		<li>Turn off the power switch of the CE instrument and open the front door.</li>
		<li>Clean the surface of the sample cover, sample holding system (sample and buffer trays), cartridge cover, clamp bar, interface block and electrodes with a water-dampened nonwoven wipe. Repeat the procedure with an ethanol-dampened wipe and dry before use.</li>
		<li>Rinse the opening levers thoroughly with water. Clean their surface with a nonwoven wipe. Repeat the procedure with an ethanol-dampened wipe and dry before installation.</li>
		<li>Clean the two ends of the fiber optic cable carefully with a water-dampened microfiber cloth. Repeat the procedure with an ethanol-dampened cloth.</li>
	</ol>
	</li>
	<li>Cartridge assembly.
	<ol>
		<li>Remove a new neutral capillary (50 &#181;m internal diameter) from its package.</li>
		<li>Tape down one end of the capillary protective tubing to the workbench. Uncoil, straighten and pull the capillary out the tubing.</li>
		<li>Stick a piece of tape or paper to the workbench and add measurement marks as follows: length to the detector (30 cm), capillary window (0.2 cm) and length to the outlet end (10 cm) (i.e., 40.2 cm total length, 30.0 cm effective length).</li>
		<li>Align the capillary window to the 0.2 cm measurement mark in the reference paper. Fix the capillary with tape and mark the capillary ends 2 mm outside the measurement marks.</li>
		<li>Cut the protective cap at the capillary inlet end (the end farthest from the window) in a single straight movement using the flush edge of the cleaving stone.
		<ol>
			<li>Immerse the capillary end into a capped universal vial filled with water in order to avoid permanent damage to the capillary inner coating. Repeat this procedure each time the capillary end is exposed to the ambient for more than 1 min.</li>
		</ol>
		</li>
		<li>Insert the capillary inlet end into the outlet side of the cartridge.</li>
		<li>Push and pull the capillary through the cartridge as necessary until the capillary window is centered to the cartridge window.</li>
		<li>Insert the aperture plug (100 &#181;m x 200 &#181;m) into the cartridge window. 
		NOTE: Confirm that white light passes through the window when exposed to a white source of light. If the light that passes through has a brownish appearance, adjust as necessary.</li>
		<li>Insert the capillary into the pre-formed coolant tubing for a total capillary length of 40.2 cm (with pre-installed tubing nut, ferrule and O-ring at both ends in that order).</li>
		<li>Push the capillary through the coolant tubing as necessary until the capillary appears at the other side of the tubing.</li>
		<li>Insert the end of the coolant tubing into the outlet side of the cartridge and tighten the tubing nut.</li>
		<li>Insert the capillary inlet end into the inlet side of the cartridge.</li>
		<li>Push and pull the capillary through the cartridge as necessary until the capillary appears at the inlet side of the cartridge.</li>
		<li>Insert the end of the coolant tubing into the inlet side of the cartridge and tighten the tubing nut.</li>
		<li>Cut the protective cap at the capillary outlet end (the end nearest from the window) in a single straight movement using the flush edge of the cleaving stone.</li>
		<li>Insert the seal-retainer clips over the capillary ends and press to snap into position. Visually inspect the capillary ends. If they are not even, repeat the procedure.</li>
		<li>Place the capillary-length template on the edge of the workbench.</li>
		<li>Place the cartridge facing down against the capillary-length template and align the capillary ends with the reference lines on the template. If the capillary measurement marks (see 3.2.4) do not align with the reference lines, adjust as necessary.</li>
		<li>Hold the capillary against the capillary-length template and cut both ends of the capillary using the flush edge of the cleaving stone 2 mm below the reference lines on the template.</li>
		<li>Visually inspect the capillary ends with a magnifying glass. If capillary ends are not smooth, burnish them using the soft face of the cleaving stone.</li>
		<li>Insert the aperture O-ring into the aperture plug hole using the O-ring insertion tool.</li>
		<li>Install capped universal vials filled with water into the cartridge case and place cartridge into case immediately with capillary ends inserted into vials. For short and long term storage keep at 2-8 &#176;C.</li>
	</ol>
	</li>
	<li>Preparation of buffer trays.
	<ol>
		<li>Fill and place capped universal vials in the buffer inlet and outlet trays (Figure 1). Repeat vial positions in lanes 2, 3, 4 and 5 according with the number of samples to be analyzed, placing one lane for every six sample injections. 
		NOTE: Avoid wetting the vial caps in order to prevent current leakage.</li>
	</ol>
	</li>
</ol>
<img alt="Figure 1" src="/files/ftp_upload/55082/55082fig1.jpg" /> 
Figure 1: Position and fill level of universal vials in the buffer inlet and outlet trays. Fill levels: 1/1 = 1400 &#181;L, 3/4 = 1300 &#181;L and 1/2 = 1000 &#181;L. Abbreviations: W = water, HCl = 0.1 M hydrochloric acid. <a href="https://cloudfront-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55082/55082fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol start="4">
	<li>CE system components assembly.
	<ol>
		<li>Install the UV detector.</li>
		<li>Install the opening levers by pressing up into the interface block.</li>
		<li>Install the fiber optic cable. Insert the corresponding end to the clamp bar and, while holding both ends, rotate clockwise. Connect the other end to the UV detector. 
		NOTE: Handle the fiber optic cable with care during this procedure since bending could cause its fracture.</li>
		<li>Place sample and buffer trays into the sample holding system and snap into position.</li>
		<li>Place the capillary cartridge into the interface block and, while pressing the clamp bar at both ends, tighten the knobs.</li>
	</ol>
	</li>
</ol>
4. Instrument Methods Set-up
<ol>
	<li>Create the conditioning, running and shutdown instrument methods according with the following parameters and Table 1: Voltage, max: 30.0 kV; Current, max: 300.0 &#181;A; Cartridge temperature: 20.0 &#176;C; Sample storage temperature: 10.0 &#176;C; UV detector initial conditions: Wavelength: 214 nm; Data rate: 4 Hz; Filter: Normal; Peak width (points): 16-25; Absorbance signal: Direct. 
	NOTE: Data acquisition rate can be increased up to 25 Hz in order to improve the coverage of narrow separation zones.</li>
</ol>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td colspan="8">Conditioning method time program</td>
		</tr>
		<tr>
			<td>Time (min)</td>
			<td>Event</td>
			<td>Value</td>
			<td>Duration</td>
			<td>Inlet vial</td>
			<td>Outlet vial</td>
			<td>Summary</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>-</td>
			<td>Rinse - Pressure</td>
			<td>2068 mbar</td>
			<td>1.0 min</td>
			<td>BI:C1</td>
			<td>BO:C1</td>
			<td>Forward</td>
			<td>HCl 0.1 M</td>
		</tr>
		<tr>
			<td>-</td>
			<td>Rinse - Pressure</td>
			<td>2068 mbar</td>
			<td>2.0 min</td>
			<td>BI:B1</td>
			<td>BO:C1</td>
			<td>Forward</td>
			<td>Water</td>
		</tr>
		<tr>
			<td>-</td>
			<td>Rinse - Pressure</td>
			<td>2068 mbar</td>
			<td>10.0 min</td>
			<td>BI:E1</td>
			<td>BO:C1</td>
			<td>Forward</td>
			<td>BGE</td>
		</tr>
		<tr>
			<td>0.00</td>
			<td>Separate - Voltage</td>
			<td>15.0 KV</td>
			<td>10.0 min</td>
			<td>BI:D1</td>
			<td>BO:D1</td>
			<td>0.17 Min ramp, normal polarity</td>
			<td>Separate</td>
		</tr>
		<tr>
			<td>10.00</td>
			<td>Rinse - Pressure</td>
			<td>2758 mbar</td>
			<td>10.0 min</td>
			<td>BI:E1</td>
			<td>BO:C1</td>
			<td>Forward</td>
			<td>BGE</td>
		</tr>
		<tr>
			<td>20.01</td>
			<td>Wait</td>
			<td>-</td>
			<td>0.0 min</td>
			<td>BI:A1</td>
			<td>BO:A1</td>
			<td>-</td>
			<td>Rinse tips</td>
		</tr>
		<tr>
			<td colspan="8">Running method time program</td>
		</tr>
		<tr>
			<td>Time (min)</td>
			<td>Event</td>
			<td>Value</td>
			<td>Duration</td>
			<td>Inlet vial</td>
			<td>Outlet vial</td>
			<td>Summary</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>-</td>
			<td>Rinse - Pressure</td>
			<td>2068 mbar</td>
			<td>1.0 min</td>
			<td>BI:C1</td>
			<td>BO:C1</td>
			<td>Forward, In / Out vial inc 6</td>
			<td>HCl 0.1 M</td>
		</tr>
		<tr>
			<td>-</td>
			<td>Rinse - Pressure</td>
			<td>2068 mbar</td>
			<td>2.0 min</td>
			<td>BI:B1</td>
			<td>BO:C1</td>
			<td>Forward, In / Out vial inc 6</td>
			<td>Water</td>
		</tr>
		<tr>
			<td>-</td>
			<td>Rinse - Pressure</td>
			<td>2068 mbar</td>
			<td>4.0 min</td>
			<td>BI:E1</td>
			<td>BO:C1</td>
			<td>Forward, In / Out vial inc 6</td>
			<td>BGE</td>
		</tr>
		<tr>
			<td>-</td>
			<td>Inject - Pressure</td>
			<td>34 mbar</td>
			<td>20.0 sec</td>
			<td>SI:A1</td>
			<td>BO:B1</td>
			<td>Override, Forward</td>
			<td>Sample Injection</td>
		</tr>
		<tr>
			<td>-</td>
			<td>Wait</td>
			<td>-</td>
			<td>0.4 min</td>
			<td>BI:A1</td>
			<td>BO:A1</td>
			<td>In / Out vial inc 6</td>
			<td>Wash tips</td>
		</tr>
		<tr>
			<td>0</td>
			<td>Separate - Voltage</td>
			<td>15.0 KV</td>
			<td>30.0 min</td>
			<td>BI:D1</td>
			<td>BO:D1</td>
			<td>0.17 Min ramp, normal polarity, In / Out vial inc 6</td>
			<td>Sample Separation</td>
		</tr>
		<tr>
			<td>0.5</td>
			<td>Autozero</td>
			<td>-</td>
			<td>-</td>
			<td>-</td>
			<td>-</td>
			<td>-</td>
			<td>-</td>
		</tr>
		<tr>
			<td>30.01</td>
			<td>Stop data</td>
			<td>-</td>
			<td>-</td>
			<td>-</td>
			<td>-</td>
			<td>-</td>
			<td>-</td>
		</tr>
		<tr>
			<td>30.02</td>
			<td>Rinse - Pressure</td>
			<td>2068 mbar</td>
			<td>2.0 min</td>
			<td>BI:B1</td>
			<td>BO:C1</td>
			<td>Forward, In / Out vial inc 6</td>
			<td>Water</td>
		</tr>
		<tr>
			<td>32.03</td>
			<td>Wait</td>
			<td>-</td>
			<td>0.0 min</td>
			<td>BI:A1</td>
			<td>BO:A1</td>
			<td>In / Out vial inc 6</td>
			<td>Rinse tips</td>
		</tr>
		<tr>
			<td>32.04</td>
			<td>End</td>
			<td>-</td>
			<td>-</td>
			<td>-</td>
			<td>-</td>
			<td>-</td>
			<td>End</td>
		</tr>
		<tr>
			<td colspan="8">Shutdown method time program</td>
		</tr>
		<tr>
			<td>Time (min)</td>
			<td>Event</td>
			<td>Value</td>
			<td>Duration</td>
			<td>Inlet vial</td>
			<td>Outlet vial</td>
			<td>Summary</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>-</td>
			<td>Rinse - Pressure</td>
			<td>2068 mbar</td>
			<td>1.0 min</td>
			<td>BI:E6</td>
			<td>BO:E6</td>
			<td>Forward</td>
			<td>HCl 0.1 M</td>
		</tr>
		<tr>
			<td>-</td>
			<td>Rinse - Pressure</td>
			<td>2068 mbar</td>
			<td>6.0 min</td>
			<td>BI:F6</td>
			<td>BO:F6</td>
			<td>Forward</td>
			<td>Water</td>
		</tr>
		<tr>
			<td>-</td>
			<td>Lamp - Off</td>
			<td>-</td>
			<td>-</td>
			<td>-</td>
			<td>-</td>
			<td>-</td>
			<td>-</td>
		</tr>
		<tr>
			<td>-</td>
			<td>Wait</td>
			<td>-</td>
			<td>0.0 min</td>
			<td>BI:A1</td>
			<td>BO:A1</td>
			<td>-</td>
			<td>Rinse tips</td>
		</tr>
	</tbody>
</table>
Table 1: Conditioning, running and shutdown time programs.
5. Data Acquisition and Processing
<ol>
	<li>Program the sample set.
	<ol>
		<li>Program the capillary for conditioning using the conditioning instrument method. When the capillary is used for the first time, program four repetitions; otherwise program only two repetitions of the conditioning method.</li>
		<li>Program the samples to be analyzed using the running instrument method.</li>
		<li>Program the capillary for storage using the shutdown instrument method and repeat the procedure indicated in section 3.2.22.</li>
	</ol>
	</li>
	<li>Run the experiment.</li>
	<li>Export the electropherograms.</li>
	<li>Calculate the migration time and the percentage content of basic, main and acidic isoforms using vertical drop line integration of the electropherogram profile.</li>
</ol>

<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. 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The authors Carlos E. Espinosa-de la Garza, Rodolfo D. Salazar-Flores, N&#233;stor O. P&#233;rez, Luis F. Flores-Ortiz and Emilio Medina-Rivero are employees of Probiomed S.A. de C.V., which is developing, manufacturing and marketing biosimilar products. All authors are involved in the development of biosimilar products for Probiomed.

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 correct preparation of BGE solution. This process requires having strict control over ionic strength of buffering agents, concentration of non-buffering additives and pH. Overall, the CE system requires care and attention to detail. It can be easily damaged if any step concerning the system cleaning or assembly is ignored.
The present method considers the use of a neutral capillary. Unlike bare fused-silica capillaries, neutral capillaries reduce protein adsorption onto the capillary inner wall, thus improving separation efficiency, accuracy and repeatability9. However, its use is limited by the amount of injections that can be performed. In our experience, 120 to 150 injections can be carried out without effect on resolution due to the progressive degradation of the inner coating. Once the capillary has reached this limit, a new capillary will be required.
Other protocols have been reported for the analysis of the physicochemical heterogeneity of mAbs by CZE, which include the use of permanently- and dynamically-coated capillaries13,14. However, these studies propose the application of a fixed set of conditions to analyze the wide diversity of mAbs. In contrast to existing methods, we believe that optimum performance can only be achieved by tailoring experimental conditions. For instance, the protocol presented here has been used and adjusted in the past to analyze the isoform heterogeneity of four other different mAbs and a mAb fragment10. In order to adapt this protocol to evaluate other mAbs, we recommend the modulation of BGE ionic strength and pH according to the variants diversity and isoelectric point of the molecule to be analyzed.
Further steps after mastering this technique should include the evaluation of the method performance. Parameters such as selectivity and repeatability (in terms of the relative standard deviation of migration time and area percentage) must be tested in order to confirm that the method is suitable for the intended use.

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><tbody><tr><td>Glacial acetic acid</td><td>Tecsiquim</td><td>AT0035-7</td><td></td></tr><tr><td>ACS grade hydrochloric acid</td><td>J.T. Baker</td><td>9535-05</td><td></td></tr><tr><td>Histamine dihydrochloride</td><td>Fluka</td><td>53300</td><td></td></tr><tr><td>(Hydroxypropyl) methyl cellulose&amp;nbsp;</td><td>Fluka</td><td>09963</td><td></td></tr><tr><td>Lithium acetate</td><td>Sigma-Aldrich</td><td>517992</td><td></td></tr><tr><td>6-Aminocaproic acid</td><td>Sigma-Aldrich</td><td>A2504</td><td></td></tr><tr><td>eCAP Tris Buffer, 50.0 mM,&amp;nbsp; pH 8</td><td>Beckman Coulter</td><td>477427</td><td></td></tr><tr><td>PA 800 Plus Pharmaceutical Analysis System</td><td>Beckman Coulter</td><td>A66528</td><td></td></tr><tr><td>eCAP Neutral capillary&amp;nbsp;</td><td>Beckman Coulter</td><td>477441</td><td></td></tr><tr><td>Vial, Micro, 200 &amp;micro;l</td><td>Beckman Coulter</td><td>144709</td><td></td></tr><tr><td>Universal Vial Caps</td><td>Beckman Coulter</td><td>A62250</td><td></td></tr><tr><td>Universal Vials</td><td>Beckman Coulter</td><td>A62251</td><td></td></tr><tr><td>Cable, Optics, UV/Vis</td><td>Beckman Coulter</td><td>144093</td><td></td></tr><tr><td>UV/Vis Detector Module</td><td>Beckman Coulter</td><td>144733</td><td></td></tr><tr><td>Cartridge Assembly Kit, Blank</td><td>Beckman Coulter</td><td>144738</td><td></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 to have a migration time of 3.7 to 4.1 min. Figure 4 shows the CZE electropherogram of anti-TNF&#945; mAb. As can be noted, the main peak is preceded and followed by peaks of smaller intensity. Since separation is conducted under normal polarity (i.e., from positive to negative), the analysis reveals basic isoforms (mAb variants with positive charge) migrating faster and acidic isoforms (mAb variants with negative charge) migrating slower than the main isoform.
<img alt="Figure 2" src="/files/ftp_upload/55082/55082fig2.jpg" /> 
Figure 2: Electrical current profile of a typical CZE run. The BGE current is stable around 30 &#181;A when the mAb sample is diluted with Tris buffer (50 mM, pH 8.0). <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55082/55082fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/55082/55082fig3.jpg" /> 
Figure 3: CZE electropherogram of blank sample. Note that no peaks are detected apart from that of the histamine internal standard. AU = absorbance units. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55082/55082fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/55082/55082fig4.jpg" /> 
Figure 4: Physicochemical heterogeneity of anti-TNF&#945; mAb determined by CZE. This sample is composed of basic, main and acidic isoforms. Inset shows an expanded view of the variants resolved by CZE. AU = absorbance units. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55082/55082fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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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Biochemistry

12.6K Views.  Unidad de Investigación y Desarrollo, Probiomed S.A. de C.V.. Here, we present a comprehensive capillary zone electrophoresis protocol for the assessment of intrinsic physicochemical heterogeneity of monoclonal antibodies as a quality attribute.

Video: Capillary Electrophoresis Separation of Monoclonal Antibody Isoforms Using a Neutral Capillary

Highly Sensitive and Quantitative Detection of Proteins and Their Isoforms by Capillary Isoelectric Focusing Method

Immunoblotting has become a routine technique in many laboratories for protein characterization from biological samples. The following protocol provides an alternative strategy, capillary isoelectric focusing (cIEF), with many advantages compared to conventional immunoblotting. This is an antibody-based, automated, rapid, and quantitative method in which a complete western blotting procedure takes place inside an ultrathin capillary. This technique does not require a gel to transfer to a membrane, stripping of blots, or x-ray films, which are typically required for conventional immunoblotting. Here, proteins are separated according to their charge (isoelectric point; pI), using less than a microliter (400 nL) of total protein lysate. After electrophoresis, proteins are immobilized onto the capillary walls by ultraviolet light treatment, followed by primary and secondary (horseradish peroxidase (HRP) conjugated) antibody incubation, whose binding is detected through enhanced chemiluminescence (ECL), generating a light signal that can be captured and recorded by a charge-coupled device (CCD) camera. The digital image can be analyzed and quantified (peak area) using software. This high throughput procedure can handle 96 samples at a time; is highly sensitive, with protein detection in the picogram range; and produces highly reproducible results because of automation. All of these aspects are extremely valuable when the quantity of samples (e.g., tissue samples and biopsies) is a limiting factor. The technique has wider applications as well, including screening of drugs or antibodies, biomarker discovery, and diagnostic purposes.

Capillary isoelectric focusing is an antibody-based, ultrasensitive, high throughput technique, enabling detailed characterization of proteins and their isoforms from extremely small biological samples. The following describes a protocol for detection and quantification of specific proteins and their isoforms in an automated and robotized manner.

Immunoblotting has become a routine technique in many laboratories for protein characterization from biological samples. The following protocol provides ...

Isolation and Characterization Of Chimeric Human Fc-expressing Proteins Using Protein A Membrane Adsorbers And A Streamlined Workflow

Laboratory scale to industrial scale purification of biomolecules from cell culture supernatants and lysed cell solutions can be accomplished using affinity chromatography.&#160;While affinity chromatography using porous protein A agarose beads packed in columns is arguably the most common method of laboratory scale isolation of antibodies and recombinant proteins expressing Fc fragments of IgG, it can be a time consuming and expensive process.&#160;Time and financial constraints are especially daunting in small basic science labs that must recover hundreds of micrograms to milligram quantities of protein from dilute solutions, yet lack access to high pressure liquid delivery systems and/or personnel with expertise in bioseparations. Moreover, product quantification and characterization may also excessively lengthen processing time over several workdays and inflate expenses (consumables, wages, etc.).&#160;Therefore, a fast, inexpensive, yet effective protocol is needed for laboratory scale isolation and characterization of antibodies and other proteins possessing an Fc fragment.&#160;To this end, we have devised a protocol that can be completed by limited-experience technical staff in less than 9 hr (roughly one workday) and as quickly as 4 hr, as opposed to traditional methods that demand 20+ work hours. Most required equipment is readily available in standard biomedical science, biochemistry, and (bio)chemical engineering labs, and all reagents are commercially available.&#160;To demonstrate this protocol, representative results are presented in which chimeric&#160;murine galectin-1 fused to human Fc (Gal-1hFc) from cell culture supernatant was isolated using a protein A membrane adsorber.&#160;Purified Gal-1hFc was quantified using an expedited Western blotting analysis procedure and characterized using flow cytometry. The streamlined workflow can be modified for other Fc-expressing proteins, such as antibodies, and/or altered to incorporate alternative quantification and characterization methods.

Compared with traditional affinity chromatography using protein A agarose bead-packed columns, protein A membrane adsorbers can significantly speed laboratory-scale isolation of antibodies and other Fc fragment-expressing proteins.&#160;Appropriate analysis and quantification methods can further accelerate protein processing, allowing isolation/characterization to be completed in one workday, instead of 20+ work hours.

Laboratory scale to industrial scale purification of biomolecules from cell culture supernatants and lysed cell solutions can be accomplished using ...

Bioengineering

Peptide Scanning-assisted Identification of a Monoclonal Antibody-recognized Linear B-cell Epitope

The identification of an antigenic epitope by the immune system allows for the understanding of the protective mechanism of neutralizing antibodies that may facilitate the development of vaccines and peptide drugs. Peptide scanning is a simple and efficient method that straightforwardly maps the linear epitope recognized by a monoclonal antibody (mAb). Here, the authors present an epitope determination methodology involving serially truncated recombinant proteins, synthetic peptide design, and dot-blot hybridization for the antigenic recognition of nervous necrosis virus coat protein using a neutralizing mAb. This technique relies on the dot-blot hybridization of synthetic peptides and mAbs on a polyvinylidene fluoride (PVDF) membrane. The minimum antigenic region of a viral coat protein recognized by the RG-M56 mAb can be narrowed down by step-by-step trimmed peptide mapping onto a 6-mer peptide epitope. In addition, alanine scanning mutagenesis and residue substitution can be performed to characterize the binding significance of each amino acid residue making up the epitope. The residues flanking the epitope site were found to play critical roles in peptide conformation regulation. The identified epitope peptide may be used to form crystals of epitope peptide-antibody complexes for an x-ray diffraction study and functional competition, or for therapeutics.

Here, the authors present a simple and efficient protocol to define a linear antigenic epitope using a purified monoclonal antibody and peptide scanning through dot-blot hybridization. The identified epitope can then be used in therapeutic and diagnostic applications.

The identification of an antigenic epitope by the immune system allows for the understanding of the protective mechanism of neutralizing antibodies that ...

Immunology and Infection

Purification and Analytics of a Monoclonal Antibody from Chinese Hamster Ovary Cells Using an Automated Microbioreactor System

Monoclonal antibodies (mAbs) are one of the most popular and well-characterized biological products manufactured today. Most commonly produced using Chinese hamster ovary (CHO) cells, culture and process conditions must be optimized to maximize antibody titers and achieve target quality profiles. Typically, this optimization uses automated microscale bioreactors (15 mL) to screen multiple process conditions in parallel. Optimization criteria include culture performance and the critical quality attributes (CQAs) of the monoclonal antibody (mAb) product, which may impact its efficacy and safety. Culture performance metrics include cell growth and nutrient consumption, while the CQAs include the mAb's N-glycosylation and aggregation profiles, charge variants, and molecular weight. This detailed protocol describes how to purify and subsequently analyze HCCF samples produced by an automated microbioreactor system to gain valuable performance metrics and outputs. First, an automated protein A fast protein liquid chromatography (FPLC) method is used to purify the mAb from harvested cell culture samples. Once concentrated, the glycan profiles are analyzed by mass spectrometry using a specific platform (refer to the Table of Materials). Antibody molecular weights and aggregation profiles are determined using size exclusion chromatography-multiple angle light scattering (SEC-MALS), while charge variants are analyzed using microchip capillary zone electrophoresis (mCZE). In addition to the culture performance metrics captured during the bioreactor process (i.e., culture viability, cell counts, and common metabolites including glutamine, glucose, lactate, and ammonia), spent media is analyzed to identify limiting nutrients to improve the feeding strategies and overall process design. Therefore, a detailed protocol for the absolute quantification of amino acids by liquid chromatography-mass spectrometry (LC-MS) of spent media is also described. The methods used in this protocol take advantage of high-throughput platforms that are compatible for large numbers of small-volume samples.

A detailed protocol for the purification and subsequent analysis of a monoclonal antibody from harvested cell culture fluid (HCCF) of automated microbioreactors has been described. Use of analytics to determine critical quality attributes (CQAs) and maximizing limited sample volume to extract vital information is also presented.

Monoclonal antibodies (mAbs) are one of the most popular and well-characterized biological products manufactured today. Most commonly produced using ...

Procedure and Key Optimization Strategies for an Automated Capillary Electrophoretic-based Immunoassay Method

New technologies that utilize capillary-based immunoassays promise faster and more quantitative protein assessment compared to traditional immunoassays. However, similar to other antibody-based protein assays, optimization of capillary-based immunoassay parameters, such as protein concentration, antibody dilution, and exposure time is an important prerequisite to the generation of meaningful and reliable data. Measurements must fall within the linear range of the assay where changes in signal are directly proportional to changes in lysate concentration. The process of choosing appropriate lysate concentrations, antibody dilutions, and exposure times in the human bronchial epithelial cell line, BEAS-2B, is demonstrated here. Assay linearity is shown over a range of whole cell extract protein concentrations with p53 and &#945;-tubulin antibodies. An example of signal burnout is seen at the highest concentrations with long exposure times, and an &#945;-tubulin antibody dilution curve is shown demonstrating saturation. In addition, example experimental results are reported for doxorubicin-treated cells using optimized parameters.

A capillary-based immunoassay using a commercial platform to measure target proteins from total protein preparations is demonstrated. In addition, assay parameters of exposure time, protein concentration, and antibody dilution are optimized for a cell culture model system.

New technologies that utilize capillary-based immunoassays promise faster and more quantitative protein assessment compared to traditional immunoassays. ...

Biology

Capillary Electrophoresis (CE)

Source: Laboratory of Dr. B. Jill Venton - University of Virginia
Capillary electrophoresis (CE) is a separation technique that separates molecules in an electric field according to size and charge. CE is performed in a small glass tube called a capillary that is filled with an electrolyte solution. Analytes are separated due to differences in electrophoretic mobility, which varies with charge, solvent viscosity, and size. Traditional electrophoresis in gels is limited in the amount of voltage that can be applied because Joule heating effects will ruin the gel and the separation. Capillaries have a large surface area-to-volume ratio and thus dissipate heat better. Therefore, the voltages applied for a capillary electrophoresis experiment are quite large, often 10,000&#8211;20,000 V.
Capillary electrophoresis is useful for high-performance separations. Compared to liquid chromatography, CE separations are often faster and more efficient. However, capillary electrophoresis works best to separate charged molecules, which is not a limitation of liquid chromatography. CE has a greater peak capacity than high-performance liquid chromatography (HPLC), meaning the separations are more efficient and more peaks can be detected. The instrumentation can be very simple. However, HPLC is more versatile and many stationary and mobile phases have been developed for different types of molecules.

Capillary Electrophoresis: Principle, Procedure and Applications

Source: Laboratory of Dr. B. Jill Venton - University of Virginia
Capillary electrophoresis (CE) is a separation technique that separates molecules in an ...

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Analytical Chemistry

An Affinity Chromatography Technique for the Purification of Monoclonal Antibodies

Source: Elgundi, Z., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/55153">Laboratory Scale Production and Purification of a Therapeutic Antibody.</a> J. Vis. Exp. (2017).
This video demonstrates a technique for purifying monoclonal antibodies from the culture supernatant obtained from an antibody-producing cell culture. The process involves using a matrix of crosslinked agarose beads with conjugated Protein A, which immobilizes the antibodies in the supernatant on the beads, allowing for effective separation from contaminants. By applying an elution buffer with low pH, the protein A-antibody interaction is loosened, facilitating the collection of antibody-containing fractions.

1. Antibody Purification by Affinity Chromatography Using an Automated Fast Protein Liquid Chromatography (FPLC) System
NOTE: The following procedure can ...

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Immunology

Antibody-Based Technologies

Production and Purification

DFE-Based Affinity Chromatography: A Technique for Purification of 2F5 Monoclonal Antibodies from a Crude Sample Using High Ionic Strength-Based Elution Conditions

Source: Kn&#246;dler, M. et al., <a href="https://www-jove-com.remotexs.ntu.edu.sg/v/59933">Activated Cross-linked Agarose for the Rapid Development of Affinity Chromatography Resins - Antibody Capture as a Case Study</a>. J. Vis. Exp. (2019).
This video describes an affinity chromatography technique for purifying 2F5 monoclonal antibodies using the DsRed-2F5-Epitope or DFE ligands. The purified 2F5 monoclonal antibodies are essential drugs in immunotherapy.

Source: Kn&#246;dler, M. et al., Activated Cross-linked Agarose for the Rapid Development of Affinity Chromatography Resins - Antibody Capture as a Case ...

Biological Techniques

Chromatography Techniques

A Capillary Electrophoresis Immunoassay for Protein Biomarker Detection

Source: Sage, J. M., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/60638">Use of Capillary Electrophoresis Immunoassay to Search for Potential Biomarkers of Amyotrophic Lateral Sclerosis in Human Platelets.</a>&#160;J. Vis. Exp.&#160;(2020)
This video demonstrates a capillary electrophoresis immunoassay protocol for detecting a protein biomarker associated with amyotrophic lateral sclerosis (ALS) from human blood platelets. Confirmation of the presence of the ALS biomarker protein is achieved through size-based separation of the platelet proteins followed by chemiluminescent detection.

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Preparation of buffers and ...

Characterization and Functional Analysis

Electrophoresis: Overview

Electrophoresis is a powerful analytical separation technique that relies on the differential migration of charged species when subjected to an electric field. The core strength of electrophoresis lies in its ability to separate high-molecular-weight species in complex mixtures. It has found widespread use in biochemistry, molecular biology, and analytical chemistry, allowing the separation of compounds like amino acids, nucleotides, carbohydrates, and proteins with excellent resolution.
There are two main formats for electrophoretic separations: slab electrophoresis and capillary electrophoresis. Slab Electrophoresis uses a gel matrix, such as agarose or polyacrylamide, which acts as a sieve, allowing molecules to move through the pores. Capillary electrophoresis employs a narrow capillary tube filled with a buffer solution, providing high-resolution separation with rapid analysis time and minimal sample requirements. Although slab electrophoresis is widely used in biochemistry and biology to separate high-molecular-mass species, capillary electrophoresis is a more recent development with several advantages.
Capillary electrophoresis is a separation technique used to analyze complex mixtures of analytes based on their differential migration rates in an applied electric field. In this method, a sample is injected into a narrow capillary tube filled with a buffered solution, and an electric field is applied to facilitate separation. The migration rate of the analyte depends on its charge and size. The smaller, more highly charged ions migrate faster than the larger, less charged ones. In capillary electrophoresis, the migration of sample components is influenced by two types of mobility: electrophoretic mobility and electroosmotic mobility. Electrophoretic mobility is the solute&#39;s response to the applied electric field, with cations moving toward the negatively charged cathode, anions moving toward the positively charged anode, and neutral species remaining stationary. Electroosmotic mobility occurs when the buffer solution moves through the capillary in response to the electric field, typically toward the cathode.
Under normal conditions, capillary electrophoresis separates cations first, followed by neutral species, and finally, the anions. This separation is based on their respective electrophoretic mobilities and electroosmotic flow velocities. Notably, all separated species are eluted from the same end of the capillary, allowing their detection using quantitative detectors. The detector signals produce an electropherogram resembling a chromatogram but feature narrower peaks.
Capillary electrophoresis is a powerful tool for qualitative and quantitative analysis of complex mixtures. It provides information similar to that obtained from other separation techniques, such as gas chromatography (GC) or high-performance liquid chromatography (HPLC).

Electrophoresis is a powerful analytical separation technique that relies on the differential migration of charged species when subjected to an electric ...

Capillary Electrophoresis Separation of Monoclonal Antibody Isoforms Using a Neutral Capillary

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