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EoE Sub Articles

1. Purification of antibody
NOTE: The equilibration buffer for the in-house antibody is 25 mM Tris, 100 mM NaCl, pH 7.5. The elution buffer used is 0.1 M acetic acid. The buffers and resin (Protein A) are dependent on the specific antibody purified. Column volume is equivalent to the bed height of the resin. The amount of mobile phase used is determined in terms of column volume.
<ol>
	<li>Initializing the purification system
	<ol>
		<li>Open the software attached to the purification system. Using manual instructions, equilibrate the column with the equilibration buffer at a flow rate of 2 mL/min for 40 min. Stop the manual run after equilibration.</li>
		<li>In the fraction collector, place 15 mL conical tubes to collect purified antibody eluate and 50 mL conical tubes to collect flow-through during high salt wash. Ensure that the fraction collector is reset to the start position by opening and closing the fraction collector before the beginning the run. The fraction collector is maintained at 7 &#176;C. 
		NOTE: The fraction collector can be reset manually under the Fraction Collector tab in Settings, for both 15 mL and 50 mL tubes.</li>
	</ol>
	</li>
	<li>Sample injection 
	NOTE: The harvested cell culture fluid used in the following procedures has been obtained from chinese hamster ovary cells cultured in automated micro-bioreactors.
	<ol>
		<li>Add the 0.22 &#181;m filtered harvest cell culture fluid to an empty 12 mL syringe whose nozzle end is capped.</li>
		<li>Holding the syringe with the nozzle facing down, insert the syringe plunger till a small portion of the plunger is in. Making sure that the fluid is not leaking, turn the syringe with the nozzle facing up and remove the cap.</li>
		<li>Still holding the syringe with the nozzle facing up, push the cylinder to dispel any air until the cell culture fluid is at the tip of the nozzle. Insert the syringe nozzle into the manual injection port on the purification system and twist to tighten.</li>
		<li>Push down on the plunger until all the sample is injected and is visible in the attached 10 mL large volume sample loop.</li>
		<li>Open the saved method file. Save the result file in the required location and specify file name when prompted. Hit run after the sample has been injected into the large volume sample loop.</li>
	</ol>
	</li>
	<li>Running the purification method
	<ol>
		<li>Select the saved method and click run when prompted by instrument software (step 1.2.5). 
		NOTE: The system is set up to run the following steps. The user needs not do anything while the instrument is running.</li>
		<li>Equilibrate the column with three column volumes (CVs) of equilibration buffer at a flow rate of 2 mL/min. Once the column is equilibrated, the system, using the large volume sample loop, will inject the sample onto the column at a flow rate of 1 mL/min.</li>
		<li>A drop in UV signal at 280 nm indicates that the sample is finished loading. Wash the column with equilibration buffer at a flow rate of 2 mL/min until the UV signal drops below 25 mAU.</li>
		<li>Use four CVs of 25 mM Tris with 1 M NaCl at pH 7.5 to perform a secondary high salt wash at a flow rate of 2 mL/min. The system fraction collector will collect any protein/DNA that comes off the column during the salt wash in 50 mL tubes.</li>
		<li>Apply five CVs of elution buffer at a flow rate of 1 mL/min to elute the antibody off the column. Collect the eluate in 15 mL tubes based on UV signal; when the UV 280 signal is above 35 mAU, collection starts; collection ends when the signal drops below 50 mAU; this is called peak cutting. 
		NOTE: Peak cutting ensures normalization of elution profiles and to avoid elution peak tailing which may contain protein aggregates.</li>
		<li>Wash the column using three CVs of equilibration buffer. The run ends after the wash step.</li>
		<li>After elution immediately neutralize the purified protein using 1 M Tris base to a pH of ~5.5. Measure the protein concentration using a microvolume UV-Vis spectrophotometer at 280 nm and 260 nm and store at 4 &#176;C.</li>
	</ol>
	</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>CHO DG44 Cell Line</td>
			<td>Invitrogen</td>
			<td>A1100001</td>
			<td></td>
		</tr>
		<tr>
			<td>Akta Avant 25</td>
			<td>General Electric Life Sciences</td>
			<td>28930842</td>
			<td></td>
		</tr>
		<tr>
			<td>Pro Sep vA Ultra Chromatography Resin</td>
			<td>Millipore Sigma</td>
			<td>115115830</td>
			<td>Purification Stationary Phase</td>
		</tr>
		<tr>
			<td>Omnifit 10cm Column</td>
			<td>Diba Fluid Intelligence</td>
			<td>006EZ-06-10-AA</td>
			<td>Housing for Stationary Phase</td>
		</tr>
		<tr>
			<td>Tris Base</td>
			<td>Fisher Scientific</td>
			<td>BP154-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Superloop 10 mL</td>
			<td>GE Healthcare</td>
			<td>18-1113-81</td>
			<td></td>
		</tr>
		<tr>
			<td>&#181;Dawn Multi Angle Light Scattering Detector</td>
			<td>Wyatt</td>
			<td>WUDAWN-01</td>
			<td></td>
		</tr>
		<tr>
			<td>0.22 &#181;m Millex GV Filter Unit PVDF Membrane</td>
			<td>Merck Millipore</td>
			<td>SLGV033RB</td>
			<td></td>
		</tr>
		<tr>
			<td>10X Phosphate Buffered Saline</td>
			<td>Corning</td>
			<td>46-013-CM</td>
			<td></td>
		</tr>
		<tr>
			<td>12 mL Syringe</td>
			<td>Covidien</td>
			<td>8881512878</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL Falcon tube</td>
			<td>Corning Inc.</td>
			<td>352070</td>
			<td></td>
		</tr>
		<tr>
			<td>96-Well Plate</td>
			<td>Bio-Rad</td>
			<td>127737</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetic Acid</td>
			<td>Sigma-Aldrich</td>
			<td>695072</td>
			<td></td>
		</tr>
		<tr>
			<td>Amicon Ultra-4 100 kDa centrifugal filters</td>
			<td>Merck Millipore</td>
			<td>UFC810096</td>
			<td></td>
		</tr>
		<tr>
			<td>Amino Acid Standard, 1 nmol/&#181;L</td>
			<td>Agilent Technologies</td>
			<td>5061-3330</td>
			<td></td>
		</tr>
		<tr>
			<td>Amino Acid Supplement</td>
			<td>Agilent Technologies</td>
			<td>5062-2478</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium Formate Solution - Glycan Analysis</td>
			<td>Waters Corporation</td>
			<td>186007081</td>
			<td></td>
		</tr>
		<tr>
			<td>Blue Screw Caps with Septa</td>
			<td>Agilent Technologies</td>
			<td>5182-0717</td>
			<td></td>
		</tr>
		<tr>
			<td>Chromatography Water (MS Grade)</td>
			<td>Fisher Chemical</td>
			<td>W6-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrochloric Acid</td>
			<td>Fisher Scientific</td>
			<td>A144-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Intact mAb Mass Check Standard</td>
			<td>Waters Corporation</td>
			<td>186006552</td>
			<td></td>
		</tr>
		<tr>
			<td>Intrada Amino Acid Column 150 x 2 mm</td>
			<td>Imtakt</td>
			<td>WAA25</td>
			<td></td>
		</tr>
		<tr>
			<td>NanoDrop One Microvolume UV-Vis Spectrophotometer</td>
			<td>Thermo Fisher Scientific</td>
			<td>840274100</td>
			<td></td>
		</tr>
	</tbody>
</table>

a technique to purify monoclonal antibodies produced by chinese hamster ovary cells

Source: Velugula-Yellela, S. R., et al. <a href="https://app.jove.com/v/58947">Purification and Analytics of a Monoclonal Antibody from Chinese Hamster Ovary Cells Using an Automated Microbioreactor System.</a> J. Vis. Exp. (2019).
This video demonstrates an assay for purifying monoclonal antibodies from cell culture fluid harvested from Chinese Hamster Ovarian cells. It employs a porous glass resin with immobilized protein A, which binds to the Fc region of the antibodies in the sample, effectively separating them from contaminants. The use of a low-pH elution buffer weakens the interaction, facilitating the retrieval of purified antibodies.

A Technique to Purify Monoclonal Antibodies Produced by Chinese Hamster Ovary Cells

1. Purification of antibody
NOTE: The equilibration buffer for the in-house antibody is 25 mM Tris, 100 mM NaCl, pH 7.5. The elution buffer used is 0.1 M ...

Take an FPLC column containing a porous glass resin with immobilized Protein A &#8212; an antibody-binding bacterial protein. Equilibrate the column with a neutral pH buffer.
Add cell culture fluid harvested from Chinese hamster ovarian cells, containing monoclonal antibodies and contaminating proteins and DNA.
Protein A binds to the antibody&#39;s Fc region, immobilizing them.
Some contaminants non-specifically attach to the resin, while the rest flow through.
In the chromatogram, an increased UV absorbance during sample loading indicates a high protein concentration in the flow through.
Wash with the equilibration buffer to remove unbound contaminants, indicated by a decrease in the absorbance.
Flush with a high-salt buffer, disrupting the non-specific interactions and removing the contaminants, indicated by a small impurity peak.
Add a low-pH elution buffer to disrupt the Protein A-antibody interaction and elute the antibodies, which are collected by tracking the corresponding peak.
Neutralize the pH of the collected antibodies to prevent degradation.

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Research

JoVE Encyclopedia of Experiments

Immunology

Antibody-Based Technologies

Production and Purification

114 Views. Source: Velugula-Yellela, S. R., et al. Purification and Analytics of a Monoclonal Antibody from Chinese Hamster Ovary Cells Using an Automated Microbioreactor System. J. Vis. Exp. (2019). This video demonstrates an assay for purifying monoclonal antibodies from cell culture fluid harvested from Chinese Hamster Ovarian cells. It employs a porous glass resin with immobilized protein A, which binds to the Fc region of the antibodies in the sample, effectively separating them from contaminants. Th...

Video: A Technique to Purify Monoclonal Antibodies Produced by Chinese Hamster Ovary Cells - Concept

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 ...

JoVE Journal

Biochemistry

A GPC3-targeting Bispecific Antibody, GPC3-S-Fab, with Potent Cytotoxicity

This protocol describes the construction and functional studies of a bispecific antibody (bsAb), GPC3-S-Fab. bsAbs can recognize two different epitopes through their two different arms. bsAbs have been actively studied for their ability to directly recruit immune cells to kill tumor cells. Currently, the majority of bsAbs are produced in the form of recombinant proteins, either as Fc-containing bsAbs or as smaller bsAb derivatives without the Fc region. In this study, GPC3-S-Fab, an antibody fragment (Fab) based bispecific antibody, was designed by linking the Fab of anti-GPC3 antibody GC33 with an anti-CD16 single domain antibody. The GPC3-S-Fab can be expressed in Escherichia coli and purified by two affinity chromatographies. The purified GPC3-S-Fab can specifically bind to and kill GPC3 positive liver cancer cells by recruiting natural killer cells, suggesting a potential application of GPC3-S-Fab in liver cancer therapy.

Here, we present a protocol to produce a bispecific antibody GPC3-S-Fab in Escherichia coli. The purified GPC3-S-Fab has potent cytotoxicity against GPC3 positive liver cancer cells.

This protocol describes the construction and functional studies of a bispecific antibody (bsAb), GPC3-S-Fab. bsAbs can recognize two different epitopes ...

Cancer Research

Activated Cross-linked Agarose for the Rapid Development of Affinity Chromatography Resins - Antibody Capture as a Case Study

The purification of monoclonal antibodies (mAbs) is commonly achieved by Protein A affinity chromatography, which can account for up to 25% of the overall process costs. Alternative, cost-effective capture steps are therefore valuable for industrial-scale manufacturing, where large quantities of a single mAb are produced. Here we present a method for the immobilization of a DsRed-based epitope ligand to a cross-linked agarose resin allowing the selective capture of the HIV-neutralizing antibody 2F5 from crude plant extracts without using Protein A. The linear epitope ELDKWA was first genetically fused to the fluorescent protein DsRed and the fusion protein was expressed in transgenic tobacco (Nicotiana tabacum) plants before purification by immobilized metal-ion affinity chromatography. Furthermore, a method based on activated cross-linked agarose was optimized for high ligand density, efficient coupling and low costs. The pH and buffer composition and the soluble ligand concentration were the most important parameters during the coupling procedure, which was improved using a design-of-experiments approach. The resulting affinity resin was tested for its ability to selectively bind the target mAb in a crude plant extract and the elution buffer was optimized for high mAb recovery, product activity and affinity resin stability. The method can easily be adapted to other antibodies with linear epitopes. The new resins allow gentler elution conditions than Protein A and could also reduce the costs of an initial capture step for mAb production.

In this procedure, a DsRed-based epitope ligand is immobilized to produce a highly selective affinity resin for the capture of monoclonal antibodies from crude plant extracts or cell culture supernatants, as an alternative to Protein A.

The purification of monoclonal antibodies (mAbs) is commonly achieved by Protein A affinity chromatography, which can account for up to 25% of the overall ...

A Technique to Purify Monoclonal Antibodies Produced by Chinese Hamster Ovary Cells

Transcript

A Technique to Purify Monoclonal Antibodies Produced by Chinese Hamster Ovary Cells

Laboratory Scale Production and Purification of a Therapeutic Antibody

A GPC3-targeting Bispecific Antibody, GPC3-S-Fab, with Potent Cytotoxicity

Activated Cross-linked Agarose for the Rapid Development of Affinity Chromatography Resins - Antibody Capture as a Case Study