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="http://cloudfront.jove.com/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>
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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 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 ...

capillary-electrophoresis-separation-monoclonal-antibody-isoforms

Research

JoVE Journal

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

Sheathless Capillary Electrophoresis&#8211;Mass Spectrometry for Metabolic Profiling of Biological Samples

In metabolomics, a wide range of analytical techniques is used for the global profiling of (endogenous) metabolites in complex samples. In this paper, a protocol is presented for the analysis of anionic and cationic metabolites in biological samples by capillary electrophoresis&#8211;mass spectrometry (CE-MS). CE is well-suited for the analysis of highly polar and charged metabolites as compounds are separated on the basis of their charge-to-size ratio. A recently developed sheathless interfacing design, i.e., a porous tip interface, is used for coupling CE to electrospray ionization (ESI) MS. This interfacing approach allows the effective use of the intrinsically low-flow property of CE in combination with MS, resulting in nanomolar detection limits for a broad range of polar metabolite classes. The protocol presented here is based on employing a bare fused-silica capillary with a porous tip emitter at low-pH separation conditions for the analysis of a broad array of metabolite classes in biological samples. It is demonstrated that the same sheathless CE-MS method can be used for the profiling of cationic metabolites, including amino acids, nucleosides and small peptides, or anionic metabolites, including sugar phosphates, nucleotides and organic acids, by only switching the MS detection and separation voltage polarity. Highly information-rich metabolic profiles in various biological samples, such as urine, cerebrospinal fluid and extracts of the glioblastoma cell line, can be obtained by this protocol in less than 1 hr of CE-MS analysis.

A protocol for metabolic profiling of biological samples by capillary electrophoresis&#8211;mass spectrometry using a sheathless porous tip interface design is presented.

In metabolomics, a wide range of analytical techniques is used for the global profiling of (endogenous) metabolites in complex samples. In this paper, a ...

Characterization of Multi-subunit Protein Complexes of Human MxA Using Non-denaturing Polyacrylamide Gel-electrophoresis

The formation of oligomeric complexes is a crucial prerequisite for the proper structure and function of many proteins. The interferon-induced antiviral effector protein MxA exerts a broad antiviral activity against many viruses. MxA is a dynamin-like GTPase and has the capacity to form oligomeric structures of higher order. However, whether oligomerization of MxA is required for its antiviral activity is an issue of debate. We describe here a simple protocol to assess the oligomeric state of endogenously or ectopically expressed MxA in the cytoplasmic fraction of human cell lines by non-denaturing polyacrylamide gel electrophoresis (PAGE) in combination with Western blot analysis. A critical step of the protocol is the choice of detergents to prevent aggregation and/or precipitation of proteins particularly associated with cellular membranes such as MxA, without interfering with its enzymatic activity. Another crucial aspect of the protocol is the irreversible protection of the free thiol groups of cysteine residues by iodoacetamide to prevent artificial interactions of the protein. This protocol is suitable for a simple assessment of the oligomeric state of MxA and furthermore allows a direct correlation of the antiviral activity of MxA interface mutants with their respective oligomeric states.

This article describes a simple and rapid protocol to evaluate the oligomeric state of the dynamin-like GTPase MxA protein from lysates of human cells using a combination of non-denaturing PAGE with western blot analysis.

The formation of oligomeric complexes is a crucial prerequisite for the proper structure and function of many proteins. The interferon-induced antiviral ...

Laboratory Scale Production and Purification of a Therapeutic Antibody

Ensuring the successful production of a therapeutic antibody begins early on in the development process. The first stage is vector expression of the antibody genes followed by stable transfection into a suitable cell line. The stable clones are subjected to screening in order to select those clones with desired production and growth characteristics. This is a critical albeit time-consuming step in the process. This protocol considers vector selection and sourcing of antibody sequences for the expression of a therapeutic antibody. The methods describe preparation of vector DNA for stable transfection of a suspension variant of human embryonic kidney 293 (HEK-293) cell line, using polyethylenimine (PEI). The cells are transfected as adherent cells in serum-containing media to maximize transfection efficiency, and afterwards adapted to serum-free conditions. Large scale production, setup as batch overgrow cultures is used to yield antibody protein that is purified by affinity chromatography using an automated fast protein liquid chromatography (FPLC) instrument. The antibody yields produced by this method can provide sufficient protein to begin initial characterization of the antibody. This may include in vitro assay development or physicochemical characterization to aid in the time-consuming task of clonal screening for lead candidates. This method can be transferable to the development of an expression system for the production of biosimilar antibodies.

This protocol describes the production of a therapeutic antibody in a mammalian expression system. The methods described include preparation of vector DNA, stable transfection and serum-free adaptation of a human embryonic kidney 293 cell line, set up of large scale cultures and purification using affinity chromatography.

Ensuring the successful production of a therapeutic antibody begins early on in the development process. The first stage is vector expression of the ...

Determination of High-affinity Antibody-antigen Binding Kinetics Using Four Biosensor Platforms

Label-free optical biosensors are powerful tools in drug discovery for the characterization of biomolecular interactions. In this study, we describe the use of four routinely used biosensor platforms in our laboratory to evaluate the binding affinity and kinetics of ten high-affinity monoclonal antibodies (mAbs) against human proprotein convertase subtilisin kexin type 9 (PCSK9). While both Biacore T100 and ProteOn XPR36 are derived from the well-established Surface Plasmon Resonance (SPR) technology, the former has four flow cells connected by serial flow configuration, whereas the latter presents 36 reaction spots in parallel through an improvised 6 x 6 crisscross microfluidic channel configuration. The IBIS MX96 also operates based on the SPR sensor technology, with an additional imaging feature that provides detection in spatial orientation. This detection technique coupled with the Continuous Flow Microspotter (CFM) expands the throughput significantly by enabling multiplex array printing and detection of 96 reaction sports simultaneously. In contrast, the Octet RED384 is based on the BioLayer Interferometry (BLI) optical principle, with fiber-optic probes acting as the biosensor to detect interference pattern changes upon binding interactions at the tip surface. Unlike the SPR-based platforms, the BLI system does not rely on continuous flow fluidics; instead, the sensor tips collect readings while they are immersed in analyte solutions of a 384-well microplate during orbital agitation.
Each of these biosensor platforms has its own advantages and disadvantages. To provide a direct comparison of these instruments&#39; ability to provide quality kinetic data, the described protocols illustrate experiments that use the same assay format and the same high-quality reagents to characterize antibody-antigen kinetics that fit the simple 1:1 molecular interaction model.

We describe here protocols for the measurement of antibody-antigen binding affinity and kinetics using four commonly used biosensor platforms.

Label-free optical biosensors are powerful tools in drug discovery for the characterization of biomolecular interactions. In this study, we describe the ...

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 ...

Analysis of AtHIRD11 Intrinsic Disorder and Binding Towards Metal Ions by Capillary Gel Electrophoresis and Affinity Capillary Electrophoresis

Plants are strongly dependent on their environment. In order to adjust to stressful changes (e.g., drought and high salinity), higher plants evolve classes of intrinsically disordered proteins (IDPs) to reduce oxidative and osmotic stress. This article uses a combination of capillary gel electrophoresis (CGE) and mobility shift affinity electrophoresis (ACE) in order to describe the binding behavior of different conformers of the IDP AtHIRD11 from Arabidopsis thaliana. CGE is used to confirm the purity of AtHIRD11 and to exclude fragments, posttranslational modifications, and other impurities as reasons for complex peak patterns. In this part of the experiment, the different sample components are separated by a viscous gel inside a capillary by their different masses and detected with a diode array detector. Afterward, the binding behavior of the sample towards various metal ions is investigated by ACE. In this case, the ligand is added to the buffer solution and the shift in migration time is measured in order to determine whether a binding event has occurred or not. One of the advantages of using the combination of CGE and ACE to determine the binding behavior of an IDP is the possibility to automate the gel electrophoresis and the binding assay. Furthermore, CGE shows a lower limit of detection than the classical gel electrophoresis and ACE is able to determine the manner of binding a ligand in a fast manner. In addition, ACE can also be applied to other charged species than metal ions. However, the use of this method for binding experiments is limited in its ability to determine the number of binding sites. Nevertheless, the combination of CGE and ACE can be adapted for characterizing the binding behavior of any protein sample towards numerous charged ligands.

This protocol combines the characterization of a protein sample by capillary gel electrophoresis and a fast-binding screening for charged ligands by affinity capillary electrophoresis. It is recommended for proteins with a flexible structure, such as intrinsically disordered proteins, to determine any differences in binding for different conformers.

Plants are strongly dependent on their environment. In order to adjust to stressful changes (e.g., drought and high salinity), higher plants evolve ...

Detection of Heterodimerization of Protein Isoforms Using an in Situ Proximity Ligation Assay

Regulated protein-protein interactions are a guiding principle for many signaling events, and the detection of such events is an important element in understanding how such pathways are organized and how they function. There are many methods to detect protein-protein interactions in cells, but relatively few can be used to detect interactions between endogenous proteins. One such method, the proximity ligation assay (PLA), has several advantages to recommend its use. Compared to other common methods of protein-protein interaction analysis, PLA has relatively high sensitivity and specificity, can be performed with minimal cell manipulation, and, in the protocol described herein, requires only two target-specific antibodies derived from different species (e.g., from mouse and rabbit) and one specialized reagent: a set of secondary antibodies that are covalently linked to specific oligonucleotides that, when brought in close proximity of one another, create an amplifiable platform for in situ PCR or rolling circle amplification. In this presentation, we show how to apply the PLA technique to visualize changes in MST1 and MST2 proximity in fixed cells. The technique described in this manuscript is particularly applicable for the analysis of cell signaling studies.

Detection of Heterodimerization of Protein Isoforms Using an in Situ Proximity Ligation Assay

Here, we show how to use a Proximity Ligation Assay (PLA) to visualize MST1/MST2 heterodimerization in fixed cells with high sensitivity.

Regulated protein-protein interactions are a guiding principle for many signaling events, and the detection of such events is an important element in ...

Hybrid Clear/Blue Native Electrophoresis for the Separation and Analysis of Mitochondrial Respiratory Chain Supercomplexes

Complexes of the oxidative phosphorylation machinery form supramolecular protein arrangements named supercomplexes (SCs), which are believed to confer structural and functional advantages to mitochondria. SCs have been identified in many species, from yeast to mammal, and an increasing number of studies report disruption of their organization in genetic and acquired human diseases. As a result, an increasing number of laboratories are interested in analyzing SCs, which can be methodologically challenging. This article presents an optimized protocol that combines the advantages of Blue- and Clear-Native PAGE methods to resolve and analyze SCs in a time-effective manner. With this hybrid CN/BN-PAGE method, mitochondrial SCs extracted with optimal amounts of the mild detergent digitonin are exposed briefly to the anionic dye Coomassie Blue (CB) at the beginning of the electrophoresis, without exposure to other detergents. This short exposure to CB allows to separate and resolve SCs as effectively as with traditional BN-PAGE methods, while avoiding the negative impact of high CB levels on in-gel activity assays, and labile protein-protein interactions within SCs. With this protocol it is thus possible to combine precise and rapid in gel activity measurements with analytical techniques involving 2D electrophoresis, immuno-detection, and/or proteomics for advanced analysis of SCs.

Here we present a protocol to extract, resolve and identify mitochondrial supercomplexes which minimizes exposure to detergents and Coomassie Blue. This protocol offers an optimal balance between resolution, and preservation of enzyme activities, while minimizing the risk of losing labile protein-protein interactions.

Complexes of the oxidative phosphorylation machinery form supramolecular protein arrangements named supercomplexes (SCs), which are believed to confer ...

DNA Electrophoresis Using Thiazole Orange Instead of Ethidium Bromide or Alternative Dyes

DNA gel electrophoresis using agarose is a common tool in molecular biology laboratories, allowing separation of DNA fragments by size. After separation, DNA is visualized by staining. This article demonstrates how to use thiazole orange to stain DNA. Thiazole orange compares favorably to common staining methods, in that it is sensitive, inexpensive, excitable with UV or blue light (to prevent sample damage), and safer than ethidium bromide. Labs already equipped to run DNA electrophoresis experiments using ethidium bromide can generally switch dyes with no additional changes to existing protocols, using UV light for detection. Blue-light detection to avoid sample damage can additionally be achieved with a blue-light source and emission filter. Labs already equipped for blue-light detection can simply switch dyes with no additional changes to existing protocols.

Here, we present a protocol to use thiazole orange for the detection of DNA in gel electrophoresis experiments. The use of thiazole orange allows elimination of ethidium bromide, and fluorescence detection can be achieved with either UV or blue light.

DNA gel electrophoresis using agarose is a common tool in molecular biology laboratories, allowing separation of DNA fragments by size. After separation, ...

Identifying the Binding Proteins of Small Ligands with the Differential Radial Capillary Action of Ligand Assay (DRaCALA)

The past decade has seen tremendous progress in the understanding of small signaling molecules in bacterial physiology. In particular, the target proteins of several nucleotide-derived secondary messengers (NSMs) have been systematically identified and studied in model organisms. These achievements are mainly due to the development of several new techniques including the capture compound technique and the differential radial capillary action of ligand assay (DRaCALA), which were used to systematically identify target proteins of these small molecules. This paper describes the use of the NSMs, guanosine penta- and tetraphosphates (p)ppGpp, as an example and video demonstration of the DRaCALA technique. Using DRaCALA, 9 out of 20 known and 12 new target proteins of (p)ppGpp were identified in the model organism, Escherichia coli K-12, demonstrating the power of this assay. In principle, DRaCALA could be used for studying small ligands that can be labeled by radioactive isotopes or fluorescent dyes. The critical steps, pros, and cons of DRaCALA are discussed here for further application of this technique.

Identifying the Binding Proteins of Small Ligands with the Differential Radial Capillary Action of Ligand Assay DRaCALA

The Differential Radial Capillary Action of Ligand Assay (DRaCALA) can be used to identify small ligand binding proteins of an organism by using an ORFeome library.

The past decade has seen tremendous progress in the understanding of small signaling molecules in bacterial physiology. In particular, the target proteins ...

Capillary Electrophoresis Separation of Monoclonal Antibody Isoforms Using a Neutral Capillary

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Rozdziały w tym wideo

Sheathless Capillary Electrophoresis–Mass Spectrometry for Metabolic Profiling of Biological Samples

Characterization of Multi-subunit Protein Complexes of Human MxA Using Non-denaturing Polyacrylamide Gel-electrophoresis

Laboratory Scale Production and Purification of a Therapeutic Antibody

Determination of High-affinity Antibody-antigen Binding Kinetics Using Four Biosensor Platforms

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

Analysis of AtHIRD11 Intrinsic Disorder and Binding Towards Metal Ions by Capillary Gel Electrophoresis and Affinity Capillary Electrophoresis

Detection of Heterodimerization of Protein Isoforms Using an in Situ Proximity Ligation Assay

Hybrid Clear/Blue Native Electrophoresis for the Separation and Analysis of Mitochondrial Respiratory Chain Supercomplexes

DNA Electrophoresis Using Thiazole Orange Instead of Ethidium Bromide or Alternative Dyes

Identifying the Binding Proteins of Small Ligands with the Differential Radial Capillary Action of Ligand Assay (DRaCALA)