The research was supported by the University of Sydney. pVITRO1-Trastuzumab-IgG1/&#954; was a gift from Andrew Beavil (Addgene plasmid # 61883). We thank Tihomir S. Dodev for useful discussions regarding pVITRO1-Trastuzumab-IgG1/&#954;.

NOTE: A suitable mammalian expression vector must be used for this protocol. Here, a single construct containing two expression cassettes is used (i.e. heavy and light chain expression is driven by separate promoters). Trastuzumab heavy and light chains were previously cloned into the vector. This vector was a gift from Andrew Beavil, obtained through a not-for-profit plasmid repository 13.
1. Recovery and Scale-up of Vector DNA
NOTE: Vector DNA was received as a soft agar stab culture in Escherichia coli XL-1 Blue strain; vector carries hygromycin resistance.
<ol>
	<li>Insert a sterile inoculation stab or loop into the soft agar of the stab culture then streak a Luria Bertani (LB) agar plate prepared with 75 &#181;g/ml hygromycin for isolated colonies. Incubate the plate at 37 &#176;C for 18-24 hr.</li>
	<li>Inoculate a single colony into 5 ml Terrific broth (TB) containing 75 &#181;g/ml hygromycin. Incubate the culture at 37 &#176;C for 18-24 hr with 225 rpm shaking.</li>
	<li>Use the overnight culture to prepare a glycerol stock of vector DNA by gently mixing 800 &#181;l of culture with 200 &#181;l 80% glycerol in a cryovial and freeze at -80 &#176;C.
	<ol>
		<li>Add 100 &#181;l of the overnight culture to 100 ml TB containing 75 &#181;g/ml hygromycin in a baffled shaker flask (i.e. 1/1,000 dilution). Incubate the culture at 37 &#176;C for 18-24 hr with 225 rpm shaking.</li>
	</ol>
	</li>
	<li>After overnight culture, extract and purify the DNA according to manufacturer&#39;s instructions of midi/maxi preparation kit with the following exception; during the final step, elute or resuspend DNA with water (pH 7.0-8.5).</li>
	<li>Check concentration and purity of DNA by absorbance readings at 260 and 280 nm then store DNA at -20 &#176;C. 
	NOTE: Optional (highly recommended): Sequence DNA using vector-specific primers to confirm identity.</li>
</ol>
2. Stable Transfection of HEK-293 Cells
<ol>
	<li>Grow and maintain HEK-293 cells (suspension cells) according to standard protocols in serum-free media supplemented with 0.1% non-ionic surfactant in Erlenmeyer flasks. Maintain at 2 x 105 cells/ml and subculture every fourth day. Culture cells at 37 &#176;C with 5% CO2 and 120 rpm rotation. 
	NOTE: Cells should have been in culture for no less than 4 days and no more than 4 weeks prior to transfection. HEK-293 cells grown as monolayers in serum-containing media can also be used for this procedure.</li>
	<li>On the day prior to transfection, seed HEK-293 cells at 3 x 105 cells/ml in wells of a 12-well plate in 2 ml of Dulbecco&#39;s Modified Eagle Media (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS). On the day of transfection, check that cells have reached 80-90% confluency</li>
	<li>Dilute DNA and polyethylenimine (PEI) separately in transfection media then mix together.
	<ol>
		<li>Dilute 1.25 &#181;g vector DNA per well (15 &#181;g for 12 wells) in 300 &#181;l transfection media. Incubate at room temperature for 5 min.</li>
		<li>Dilute 2.5 &#181;l of 1 mg/ml PEI solution (30 &#181;l for 12 wells; i.e. 1:2 ratio of DNA to PEI) in 300 &#181;l transfection media. Incubate at room temperature for 5 min.</li>
		<li>Add diluted DNA to diluted PEI, gently mix and incubate at room temperature for 15 min.</li>
	</ol>
	</li>
	<li>Add 50 &#181;l of DNA/PEI mixture dropwise to each well of the plate. Rock the plate gently to distribute the transfection mixture then place the plate at 37 &#176;C, 5% CO2 for 24 hr.</li>
	<li>Add 50 &#181;g/ml hygromycin B per well and return plate to 37 &#176;C, 5% CO2 for 10 days. 
	NOTE: By Day 10, un-transfected cells will have died and detached from the surface of the plate, whereas stably-transfected cells will be viable and attached to the plate.</li>
	<li>Replace the media in wells with 1/4 volume serum-free media and 3/4 volume DMEM supplemented with 10% FBS (i.e. 0.5 ml serum-free media + 1.5 ml DMEM = 7.5% final serum concentration) and incubate for 4 days.</li>
	<li>Replace the media in wells with 1/2 volume serum-free media and 1/2 volume DMEM supplemented with 10% FBS (i.e. 1 ml serum-free media + 1 ml DMEM = 5% final serum concentration) and incubate for 4 days.</li>
	<li>Replace the media in wells with 3/4 volume serum-free media and 1/4 volume DMEM supplemented with 10% FBS (i.e. 1.5 ml serum-free media + 0.5 ml DMEM = 2.5% final serum concentration) and incubate for 4 days.</li>
	<li>Replace the media in wells with 2 ml of serum-free media supplemented with 0.1% non-ionic surfactant (i.e. 0% final serum concentration) and incubate for 4 days. 
	NOTE: Maintain 50 &#181;g/ml hygromycin B selective pressure on cells during serum-free adaptation. Cells adapted to serum-free media detach from the surface of wells and may cluster in suspension. Refer to step 2.1 for culture conditions with the additional supplement of 50 &#181;g/ml hygromycin B.</li>
	<li>Using a pipette, gently mix the suspension cultures in the 12-well plate and pool cells into a separate tube.
	<ol>
		<li>Using a hemocytometer, enumerate pooled cells and seed in 30 ml serum-free media in a Erlenmeyer flask at 2 x 105 cells/ml. Culture cells at 37 &#176;C with 5% CO2 and 120 rpm rotation. Subculture and expand every fourth day.</li>
	</ol>
	</li>
	<li>Continue to expand culture size to required cell density to obtain 10 vials at 1 x 107 cells/vial for cryopreservation in liquid nitrogen. Freeze the cells in serum-free media containing 10% dimethylsulfoxide (DMSO). 
	NOTE: Conditioned media containing antibody can be harvested at each subculture to confirm protein production or used to optimize purification conditions. To harvest conditioned media and maintain cells for subculture, centrifuge the culture at 300 x g for 5 min then filter-sterilize supernatant through a 0.22 &#181;m filter. Filtered supernatant may be kept at 4 &#176;C for 1-2 weeks or -20 &#176;C for longer term storage.</li>
</ol>
3. Large Scale Antibody Production from Batch Overgrow Culture
NOTE: Antibody production can be followed on from step 2.11 where existing cells are expanded to the required cell density based on the batch overgrow culture volume to be setup. Otherwise, cells that have been thawed from cryopreservation begin at this stage once expanded to an appropriate cell density and volume. Various culture supplements may be used to optimize antibody production; the use of tryptone to increase antibody yield is demonstrated in this protocol.
<ol>
	<li>Seed cells at 2 x 105 cells/ml in 100 ml serum-free media in two Erlenmeyer flasks (to compare antibody yields from un-supplemented and nutrient-supplemented cultures). Culture at 37 &#176;C, 5% CO2 and 120 rpm.</li>
	<li>After 24 hr, add tryptone to a final concentration of 0.5% to nutrient-supplemented culture. Equalize volume of un-supplemented culture with serum-free media.</li>
	<li>From day 8, perform cell counts of the cultures daily using a hemocytometer to monitor cell viability. Harvest the culture supernatants once cell viability is less than 80%.
	<ol>
		<li>Harvest supernatant by centrifugation of the cultures at 3,000 x g for 15 min then filter-sterilize the supernatant (containing antibody) through a 0.22 &#181;m filter. Store the supernatants at 4 &#176;C (short-term) or freeze at -20 &#176;C (long-term).</li>
	</ol>
	</li>
</ol>
4. Antibody Purification by Affinity Chromatography Using an Automated Fast Protein Liquid Chromatography (FPLC) System
NOTE: The following procedure can generally be applied to most automated systems. Purification can be performed at room temperature or at 4 &#176;C (if FPLC system is kept in a cool room). A series of scouting tests can be performed to identify the optimal purification conditions including appropriate column matrix, binding buffer, elution buffer and pH to ensure maximum recovery of purified antibody from conditioned media (refer to Results section). The optimal conditions are dependent on the antibody or protein being purified. Purifications were performed on an automated FPLC system. Purifications were performed at room temperature using a 5 ml Protein A column.
<ol>
	<li>Prepare the following buffers using ultrapure water and adjust to the recommended pH then filter through a 0.22 &#181;m filter.
	<ol>
		<li>Prepare 1 L of phosphate buffered saline (PBS) pH 7.4 (binding buffer) by mixing the following: 0.14 M NaCl, 0.0027 M KCl, 0.01 M Na2HPO4 and 0.001 M KH2PO4. Adjust pH if necessary before filtering the buffer.</li>
		<li>Prepare 500 ml of 0.1 M glycine-HCl pH 2.7 (elution buffer). Adjust pH before filtering the buffer.</li>
		<li>Prepare 50 ml of 1 M Tris pH 9.0 (neutralization buffer).</li>
	</ol>
	</li>
	<li>Ensure all system power and communication connections with computer are made. Devices should be visible in the software &#39;System Control&#39; module. Ensure UV cell is set at 280 nm wavelength. Optional: Calibrate pH meter if connected and to be used.</li>
	<li>Immerse inlet tubes of A, B and sample pump in ultrapure water to wash the system. Purge the pumps with a syringe if there is air in the tubing or a suspicion of air in the system. Wash the system with water by manual operation via the system controller or via an automated method. Observe that pressure, conductivity, UV280 tracing and pH remain consistent and that the pressure limit for the column is not exceeded as per manufacturer&#39;s specification. 
	NOTE: Abnormalities may indicate air or blockage in the system that should be addressed before proceeding.</li>
	<li>In the system controller module, start the flowrate at 1 ml/min manually via pump A in either load (bypassing sample loop) or inject (through the sample loop) position to begin connecting the column. Disconnect system tubing at the column position inlet and remove the stopper connected to the column inlet.
	<ol>
		<li>Allow water to flow from the system tubing dropwise on top of the column then attach the system tubing to the column. Having the column inlet and inlet fitting overflowing with drops of water ensures a connection free of air bubbles.</li>
	</ol>
	</li>
	<li>Attach the column outlet at the downstream outlet and verify all fittings are tightly fastened. With the column now attached to the system, do not exceed limits for maximum pressure limit and flowrate as outlined in column specifications.</li>
	<li>Wash column with 5 column volumes (CV) of water. If necessary, continue column wash until UV280 tracing has stabilized.</li>
	<li>Immerse inlet tubes of A in Binding buffer, B in Elution buffer and sample pump in Binding buffer to equilibrate the system in correct buffers. Fill the inlet tubes with buffer using PumpWash by manual operation.</li>
	<li>In the &#39;Method Editor&#39; module of the software, use the method wizard to setup an affinity chromatography method for the column that is intended to be used. 
	NOTE: Automated FPLC systems come with methods pre-filled with the recommended settings (i.e. flowrate and pressure limit) and run steps based on the column (manufacturer, matrix and size) selected for purification.
	<ol>
		<li>Use the following run steps for Protein A affinity chromatography:
		<ol>
			<li>Equilibrate system with 5 CV of Binding buffer and collect flowthrough into waste container.</li>
			<li>Load sample onto the column (volume to be loaded is specified manually in the method) and collect flowthrough into a separate container.</li>
			<li>Wash system with 5 CV of Binding buffer and collect flowthrough into a separate container.</li>
			<li>Elute column with 5 CV isocratic fractionation using Elution buffer and collect purified antibody sample as fractions by fraction collector.</li>
			<li>Equilibrate system with 5 CV of Binding buffer and collect flowthrough into waste container.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Once the method has been setup, specify the volume of conditioned media to be applied to the column and save the method. 
	NOTE: The specified volume in the method should be 5-10 ml less than the actual volume to avoid introduction of air during the sample run. The specified volume used in this protocol is 90-120 ml.</li>
	<li>Submerge the sample pump tubing into the vessel containing the conditioned media.</li>
	<li>Prepare a separate container to collect sample flowthrough via the specified outlet tubing. 
	NOTE: It is important to collect the flowthrough in the case an error occurs during the run or the binding capacity of the column is exceeded requiring the flowthrough to be reapplied to the column or the purification repeated.</li>
	<li>Prepare collection tubes in the fraction collector. Add 100 &#181;l of neutralization buffer per 1 ml fraction volume. The FPLC system will automatically elute the fractions into the collection tubes.</li>
	<li>In the &#39;System Control&#39; module of the software, open the method to be run. 
	NOTE: The method run is initiated in a series of pages that include checking the variables of the method, fraction collector setup and defining result file name and storage location.</li>
	<li>Click START to initiate the run. The run can be monitored in the &#39;System Control&#39; module.</li>
	<li>At the completion of the purification run, check the resulting chromatogram in the &#39;Evaluation&#39; module in the system software.</li>
	<li>Combine all protein-containing fractions into a separate tube, buffer exchange and concentrate in PBS using a centrifugal filter device with 30 kDa molecular weight cut off. Perform centrifugation steps according to manufacturer&#39;s instructions.</li>
	<li>Measure antibody concentration using bicinchoninic acid assay (BCA) according to manufacturer&#39;s instructions.</li>
	<li>If another purification run is to be performed, prime the sample pump tubing in Binding buffer and repeat steps 4.9-4.14.</li>
	<li>At the completion of purification runs, immerse inlet tubes of A, B and sample pump in ultrapure water and wash the system and column as performed in step 3.</li>
	<li>Submerge A, B and sample pump tubing in 20% ethanol and repeat wash procedure for storage of the system and column.</li>
	<li>Disconnect the column from the downstream outlet and replace column stopper, then disconnect column at the inlet and replace stopper, reconnect tubing to the system. Store the column at 4 &#176;C.</li>
</ol>

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The authors declare that they have no competing financial interests.

This protocol details the transfection, stable expression and purification of a therapeutic antibody in HEK-293 cells. Stable expression of antibody genes is the first step in generating an antibody-producing cell line for the development and manufacture of a therapeutic antibody. While Chinese hamster ovary (CHO) cells remain the expression platform of choice for therapeutic proteins, the HEK-293 cell line is gaining prominence with the realization that proteins produced in these cells are a closer match to naturally oc...

The success of therapeutic antibodies continues to drive substantial investment into antibody development as a wave of next generation therapeutics begins. The antibody market is expected to be reshaped by antibody fragments 1, antibody-drug conjugates 2, bispecific antibodies 3 and engineered antibodies with favorable properties 4. Another class gaining pharmaceutical interest are biosimilars. Biosimilar antibodies are &#39;highly similar&#39; replicate products of a therapeutic antibody that has already received regulatory approval. A proposed biosimilar must be comparable with the originator antibody with respect to its structure, function, animal toxicity, clinical safety and effectiveness, human pharmacokinetics (PK), pharmacodynamics (PD) and immunogenicity 5,6.
The approval rates of biosimilar antibodies have been slow due to the strict constraints on the final quality of the product. The exact manufacturing processes such as specific cell lines and culturing conditions through to the final processing steps can remain proprietary. What is more, the manufacturing of antibodies inherently involves a degree of variability which can add to the challenge of producing a highly similar product. A comprehensive physiochemical and biophysical characterization and comparison is quite difficult, yet a number of studies demonstrating the characteristics of biosimilar antibodies are emerging in the literature 7,8,9.
Generating a therapeutic antibody begins with transfection of mammalian host cells with a vector carrying the genes for the respective antibody. Vector design, cell line and culture conditions are key considerations for setting up the expression system.
The DNA sequences of antibodies can be sourced from Drug Bank (www.drugbank.ca), IMGT (www.igmt.org) or research publications including patents. For example, the sequence of trastuzumab is available through Drug Bank (DB ID: DB00072). The amino acid sequence of the variable regions can undergo gene design and optimization for synthesis in the desired host species. It is important for a biosimilar antibody that no modification is made to the amino acid sequence. Once synthesized, antibody genes can be subcloned into the appropriate vector of choice.
Human IgG antibodies consist of two identical heavy chains and two identical light chains. Tightly regulated expression of both chains is essential for optimal production of heterologous IgG protein in mammalian cells 10. Intra- as well as inter-chain disulfide bonds have to be formed and a number of post-translational modifications have to be introduced during protein biosynthesis. A number of vectors are available that have been designed specifically to express antibody genes (refer to Table of Materials). These antibody-specific vectors usually express the constant regions for both heavy and light chains so only the variable regions of each chain require cloning.
Transfection of cells with two independent constructs (co-transfection) is the most common approach for delivering heavy and light chain-encoding genes. That is, each gene is driven by its own promoter and transcribed as separate antibody chains before being assembled in the endoplasmic reticulum. On the other hand, multi-cistronic vectors have internal ribosome entry site (IRES) elements incorporated that allow expression of multiple genes as a single mRNA transcript with translation permitted from internal regions of the mRNA 11. In this instance, the heavy and light chain-encoding genes are coupled in an arrangement to achieve co-expression of both antibody chains 10,12.
While transiently transfected cells yield sufficient protein to perform a limited number of experiments, stably transfected cell lines that have undergone selection for genome integration can deliver higher yields. Higher protein amounts allow for assay development relating to in vitro characterization and can provide an indication of antibody quality in consideration for downstream applications such as clonal cell line and lead candidate selection.
The goal of this article is to describe the stable expression and purification of a therapeutic antibody produced in a mammalian expression system. Indeed, this method can be applied to the expression of a biosimilar antibody. The method can be used for the initial characterization of antibodies before proceeding on to the critical, albeit time-consuming steps of identifying a desirable clone for larger scale manufacturing. Moreover, this method can be used to express other proteins and not just antibodies.
The following detailed protocol describes the expression of therapeutic antibody trastuzumab. This consists of preparation of vector DNA followed by stable transfection in HEK-293 cell line and purification of antibody protein by an automated chromatographic method.

<table><tbody><tr><td>pFUSE vector series</td><td>InvivoGen</td><td>N/A</td><td>Heavy and light antibody genes expressed in separate vectors that require co-transfection.</td></tr><tr><td>mAbXpress vector series</td><td>ACYTE Biotech Pty Ltd.</td><td></td><td>Heavy and light antibody genes expressed in separate vectors that require co-transfection. Refer to: Jones, M. L. et al. A method for rapid, ligation-independent reformatting of recombinant monoclonal antibodies. J Immunol Methods. 354 (1-2), 85-90, doi:10.1016/j.jim.2010.02.001, (2010).</td></tr><tr><td>pVITRO1 vector</td><td>N/A</td><td>N/A</td><td>Heavy and light antibody genes are each driven by a separate promoter in a single vector.&amp;nbsp; Refer to: Dodev, T. S. &lt;em&gt;et al.&lt;/em&gt; A tool kit for rapid cloning and expression of recombinant antibodies. Sci Rep. 4 5885, doi:10.1038/srep05885, (2014).</td></tr><tr><td>GS vector series</td><td>Lonza</td><td></td><td>Multi-cistronic vector with heavy and light antibody genes co-expressed and translated as single transcript.</td></tr><tr><td>Multi-cistronic vector series 1</td><td>N/A</td><td>N/A</td><td>Multi-cistronic vector with heavy and light antibody genes co-expressed and translated as single transcript. Refer to: Li, J. &lt;em&gt;et al.&lt;/em&gt; A comparative study of different vector designs for the mammalian expression of recombinant IgG antibodies. J Immunol Methods. 318 (1-2), 113-124, doi:10.1016/j.jim.2006.10.010, (2007).</td></tr><tr><td>Multi-cistronic vector series 2</td><td>N/A</td><td>N/A</td><td>Multi-cistronic vector with heavy and light antibody genes co-expressed and translated as single transcript. Refer to: Ho, S. C. &lt;em&gt;et al.&lt;/em&gt; IRES-mediated Tricistronic vectors for enhancing generation of high monoclonal antibody expressing CHO cell lines. J Biotechnol. 157 (1), 130-139, doi:10.1016/j.jbiotec.2011.09.023, (2012).</td></tr><tr><td>pVITRO1-Trastuzumab-IgG1/&amp;kappa;</td><td>Addgene</td><td>61883</td><td>Mammalian expression vector containing trastuzumab antibody genes with hygromycin resistance gene; pVITRO1-Trastuzumab-IgG1/&amp;kappa; was a gift from Andrew Beavil.</td></tr><tr><td>Fast-Media Hygro Agar</td><td>Jomar Life Research</td><td>fas-hg-s</td><td>Used to prepare low salt LB agar containing 75 &amp;micro;g/ml hygromycin.</td></tr><tr><td>Fast-Media Hygro TB</td><td>Jomar Life Research</td><td>fas-hg-l</td><td>Used to prepare low salt TB broth containing 75 &amp;micro;g/ml hygromycin.&amp;nbsp;</td></tr><tr><td>Glycerol, BioXtra, &amp;ge;99%</td><td>Sigma-Aldrich</td><td>G6279</td><td>Prepare to 80% with water and autoclave. Store at room temperature.</td></tr><tr><td>Jestar 2.0/LFU Plasmid Maxi Kit</td><td>Astral Scientific</td><td>G221020</td><td>Plasmid Maxi Prep Kit; elute or resuspend DNA in water (pH 7.0-8.5).</td></tr><tr><td>FreeStyle 293-F Cells</td><td>Life Technologies</td><td>R790-07</td><td>HEK-293 cell line adapted to suspension culture in serum-free media.</td></tr><tr><td>FreeStyle 293 Expression Medium</td><td>Life Technologies</td><td>12338-018</td><td>Serum-free media specially formulated for maintaing 293-F cell line and high protein expression.</td></tr><tr><td>Kolliphor P188</td><td>Sigma-Aldrich</td><td>K4894</td><td>Non-ionic surfactant; pluronic F-68; prepare to 10% in water and filter-sterilize using 0.22 &amp;mu;m filter. Store at 4 &amp;deg;C.</td></tr><tr><td>DMEM, high glucose&amp;nbsp;</td><td>Life Technologies</td><td>11995-065</td><td></td></tr><tr><td>Heat-Inactivated Foetal Bovine Serum</td><td>Life Technologies</td><td>10082-147</td><td></td></tr><tr><td>Polyethylenimine, Linear, MW 25,000&amp;nbsp;</td><td>Polysciences, Inc.</td><td>23966</td><td>Prepare to 1 mg/ml in water. Adjust to pH 7.0 with 1 M HCl (solution becomes clear) and filter-sterilize using 0.22 &amp;mu;m filter. Store at -80 &amp;deg;C until use.</td></tr><tr><td>OptiPro SFM</td><td>Life Technologies</td><td>12309-050</td><td>Transfection formulated serum-free media</td></tr><tr><td>Hygromycin B Solution</td><td>Jomar Life Research</td><td>ant-hg-1</td><td></td></tr><tr><td>Dimethylsulphoxide (DMSO)</td><td>Thermo Fisher Scientific</td><td>AJA2225</td><td></td></tr><tr><td>Tryptone (casein peptone)</td><td>Thermo Fisher Scientific</td><td>LP0042B</td><td>Prepare to 20% in PBS and filter-sterilize using 0.22 &amp;mu;m filter. Store at 4 &amp;deg;C.&amp;nbsp;</td></tr><tr><td>Phosphate Buffered Saline (PBS) Tablets, pH 7.4, 100 ml</td><td>Astral Scientific</td><td>09-2051-100</td><td></td></tr><tr><td>HiTrap Protein A High Performance, 1 x 5 ml column</td><td>Sigma-Aldrich</td><td>GE17-0403-01</td><td></td></tr><tr><td>AKTApurifier 100</td><td>GE Healthcare</td><td>28406266</td><td>Automated FPLC system, which can include a P-960 sample pump and Frac-920 fraction collector.</td></tr><tr><td>Glycine-HCl</td><td>Sigma-Aldrich</td><td>G2879</td><td></td></tr><tr><td>Citric Acid, monohydrate</td><td>Astral Scientific</td><td>BIOC2123</td><td></td></tr><tr><td>Sodium Citrate, trisodium salt dihydrate&amp;nbsp;</td><td>Astral Scientific</td><td>BIOCB0035</td><td></td></tr><tr><td>1 M Tris-HCl solution pH 9.0</td><td>Astral Scientific</td><td>BIOSD8146</td><td></td></tr><tr><td>Amicon Ultra Centrifugal Filters (30 MWCO)</td><td>Merck Millipore</td><td>UFC803008/UFC903008</td><td>Used to buffer exchange and concentrate purified protein.</td></tr><tr><td>Pierce Bicinchoninic Acid (BCA) Assay Kit</td><td>Thermo Fisher Scientific</td><td>23227</td><td></td></tr><tr><td>BLItz System</td><td>fort&amp;eacute;BIO</td><td>45-5000</td><td>Instrument used for bio-layer interferometry (BLI) measurements.</td></tr><tr><td>Protein A biosensors</td><td>fort&amp;eacute;BIO</td><td>18-5010</td><td></td></tr><tr><td>Acrylamide/Bisacrylamide (37.5:1), 40% solution</td><td>Astral Scientific</td><td>786-502</td><td></td></tr><tr><td>Ammonium Persulfate (APS)</td><td>Astral Scientific</td><td>AM0486</td><td></td></tr><tr><td>TEMED</td><td>Astral Scientific</td><td>AM0761</td><td></td></tr><tr><td>Coomassie Brilliant Blue R-250</td><td>Astral Scientific</td><td>786-498</td><td></td></tr><tr><td>Precision Plus Dual-Color Protein Standard</td><td>Bio-Rad</td><td>1610374</td><td></td></tr></tbody></table>

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.

Stable production of trastuzumab by transfected HEK-293 cells was confirmed using bio-layer interferometry (BLI) as presented in Figure 1. An IgG standard curve was generated by measuring the binding rate between an IgG antibody standard and Protein A biosensor (Figure 1A). The crude supernatant sample was similarly measured, then its concentration interpolated from the standard curve (Figure 1B). The supernatant concentration was measure...

Watch this Scientific Journal Video about Laboratory Scale Production and Purification of a Therapeutic Antibody at JoVE.com

Laboratory Scale Production and Purification of a Therapeutic Antibody

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 ...

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Research

JoVE Journal

Biochemistry

17.3K Views.  University of Sydney.  Dit protocol beschrijft de productie van een therapeutisch antilichaam in een zoogdier expressiesysteem. De beschreven werkwijzen omvatten bereiding van vector-DNA, transfectie en stabiele serumvrije adaptatie van humane embryonische nier 293 cellijn opgezet van grootschalige kweken en zuiveren met behulp van affiniteitschromatografie. 

Video: Laboratory Scale Production and Purification of a Therapeutic Antibody

A High-throughput Automated Platform for the Development of Manufacturing Cell Lines for Protein Therapeutics

The fast-growing biopharmaceutical industry demands speedy development of highly efficient and reliable production systems to meet the increasing requirement for drug supplies. The generation of production cell lines has traditionally involved manual operations that are labor-intensive, low-throughput and vulnerable to human errors. We report here an integrated high-throughput and automated platform for development of manufacturing cell lines for the production of protein therapeutics.
The combination of BD FACS Aria Cell Sorter, CloneSelect Imager and TECAN Freedom EVO liquid handling system has enabled a high-throughput and more efficient cell line development process. In this operation, production host cells are first transfected with an expression vector carrying the gene of interest 1, followed by the treatment with a selection agent. The stably-transfected cells are then stained with fluorescence-labeled anti-human IgG antibody, and are subsequently subject to flow cytometry analysis 2-4. Highly productive cells are selected based on fluorescence intensity and are isolated by single-cell sorting on a BD FACSAria. Colony formation from single-cell stage was detected microscopically and a series of time-laps digital images are taken by CloneSelect Imager for the documentation of cell line history. After single clones have formed, these clones were screened for productivity by ELISA performed on a TECAN Freedom EVO liquid handling system. Approximately 2,000 - 10,000 clones can be screened per operation cycle with the current system setup.
This integrated approach has been used to generate high producing Chinese hamster ovary (CHO) cell lines for the production of therapeutic monoclonal antibody (mAb) as well as their fusion proteins. With the aid of different types of detecting probes, the method can be used for developing other protein therapeutics or be applied to other production host systems. Comparing to the traditional manual procedure, this automated platform demonstrated advantages of significantly increased capacity, ensured clonality, traceability in cell line history with electronic documentation and much reduced opportunity in operator error.

A high-throughput, automated platform of manufacturing cell line development for producing protein therapeutics is described. Implementation of BD FACS Aria Cell Sorter, CloneSelect Imager and TECAN Freedom EVO liquid handling system has demonstrated significantly increased processing capacity in cell line development with improved cell line quality and high reproducibility.

The fast-growing biopharmaceutical industry demands speedy development of highly efficient and reliable production systems to meet the increasing ...

Medicine

Experimental Protocol for Detecting Cyanobacteria in Liquid and Solid Samples with an Antibody Microarray Chip

Global warming and eutrophication make some aquatic ecosystems behave as true bioreactors that trigger rapid and massive cyanobacterial growth; this has relevant health and economic consequences. Many cyanobacterial strains are toxin producers, and only a few cells are necessary to induce irreparable damage to the environment. Therefore, water-body authorities and administrations require rapid and efficient early-warning systems providing reliable data to support their preventive or curative decisions. This manuscript reports an experimental protocol for the in-field detection of toxin-producing cyanobacterial strains by using an antibody microarray chip with 17 antibodies (Abs) with taxonomic resolution (CYANOCHIP). Here, a multiplex fluorescent sandwich microarray immunoassay (FSMI) for the simultaneous monitoring of 17 cyanobacterial strains frequently found blooming in freshwater ecosystems, some of them toxin producers, is described. A microarray with multiple identical replicates (up to 24) of the CYANOCHIP was printed onto a single microscope slide to simultaneously test a similar number of samples. Liquid samples can be tested either by direct incubation with the antibodies (Abs) or after cell concentration by filtration through a 1- to 3-&#956;m filter. Solid samples, such as sediments and ground rocks, are first homogenized and dispersed by a hand-held ultrasonicator in an incubation buffer. They are then filtered (5 - 20 &#956;m) to remove the coarse material, and the filtrate is incubated with Abs. Immunoreactions are revealed by a final incubation with a mixture of the 17 fluorescence-labeled Abs and are read by a portable fluorescence detector. The whole process takes around 3 h, most of it corresponding to two 1-h periods of incubation. The output is an image, where bright spots correspond to the positive detection of cyanobacterial markers.

Experimental Protocol for Detecting Cyanobacteria in Liquid and Solid Samples with an Antibody Microarray Chip

The presence of cyanobacterial toxins in fresh water reservoirs for human consumption is a major concern for water management authorities. To evaluate the risk of water contamination, this article describes an protocol for the in-field detection of cyanobacterial strains in liquid and solid samples by using an antibody microarray chip.

Global warming and eutrophication make some aquatic ecosystems behave as true bioreactors that trigger rapid and massive cyanobacterial growth; this has ...

Environment

Analyzing Tumor and Tissue Distribution of Target Antigen Specific Therapeutic Antibody

Monoclonal antibodies are high affinity multifunctional drugs that work by variable independent mechanisms to eliminate cancer cells. Over the last few decades, the field of antibody-drug conjugates, bispecific antibodies, chimeric antigen receptors (CAR) and cancer immunotherapy has emerged as the most promising area of basic and therapeutic investigations. With numerous successful human trials targeting immune checkpoint receptors and CAR-T cells in leukemia and melanoma at a breakthrough pace, it is highly exciting times for oncologic therapeutics derived from variations of antibody engineering. Regrettably, a significantly large numbers of antibody and CAR based therapeutics have also proven disappointing in human trials of solid cancers because of the limited availability of immune effector cells in the tumor bed. Importantly, nonspecific distribution of therapeutic antibodies in tissues other than tumors also contribute to the lack of clinical efficacy, associated toxicity and clinical failure. As faithful translation of preclinical studies into human clinical trails are highly relied on mice tumor xenograft efficacy and safety studies, here we highlight a method to test the tumor and general tissue distribution of therapeutic antibodies. This is achieved by labeling the protein-A purified antibody with near Infrared fluorescent dye followed by live imaging of tumor bearing mice.

Here we present a protocol to study the in vivo localization of antibodies in mice tumor xenograft models.

Monoclonal antibodies are high affinity multifunctional drugs that work by variable independent mechanisms to eliminate cancer cells. Over the last few ...

Cancer Research

Use of High-Throughput Automated Microbioreactor System for Production of Model IgG1 in CHO Cells

Automated microscale bioreactors (15 mL) can be a useful tool for cell culture engineers. They facilitate the simultaneous execution of a wide variety of experimental conditions while minimizing potential process variability. Applications of this approach include: clone screening, temperature and pH shifts,&#160;media and supplement optimization. Furthermore, the small reactor volumes are conducive to large Design of Experiments that investigate a wide range of conditions. This allows upstream processes to be significantly optimized before scale-up where experimentation is more limited in scope due to time and economic constraints. Automated microscale bioreactor systems offer various advantages over traditional small scale cell culture units, such as shake flasks or spinner flasks. However, during pilot scale process development&#160;significant care must be taken to ensure that these advantages are realized. When run with care, the system can enable high level automation, can be programmed to run DOE&#39;s with a higher number of variables&#160;and can reduce sampling time when integrated with a nutrient analyzer or cell counter. Integration of the expert-derived heuristics presented here, with current automated microscale bioreactor experiments can minimize common pitfalls that hinder meaningful results. In the extreme, failure to adhere to the principles laid out here can lead to equipment damage that requires expensive repairs. Furthermore, the microbioreactor systems have small culture volumes&#160;making characterization of cell culture conditions difficult. The number and amount of samples taken in-process in batch mode culture is limited as operating volumes cannot fall below 10 mL. This method will discuss the benefits and drawbacks of microscale bioreactor systems.

A detailed protocol for the concurrent operation of 48 parallel cell cultures under varied conditions in a microbioreactor system is presented. Cell culture process, harvest and subsequent antibody titer analysis are described.

Automated microscale bioreactors (15 mL) can be a useful tool for cell culture engineers. They facilitate the simultaneous execution of a wide variety of ...

Bioengineering

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 ...

Antibody Generation: Producing Monoclonal Antibodies Using Hybridomas

Source: Frances V. Sjaastad1,2, Whitney Swanson2,3, and Thomas S. Griffith1,2,3,4 
1 Microbiology, Immunology, and Cancer Biology Graduate Program, University of Minnesota, Minneapolis, MN 55455 
2 Center for Immunology, University of Minnesota, Minneapolis, MN 55455 
3 Department of Urology, University of Minnesota, Minneapolis, MN 55455 
4 Masonic Cancer Center, University of Minnesota, Minneapolis, MN 55455
Polyclonal antibodies are defined as a collection of antibodies directed against different antigenic determinants of an antigen or several antigens (1). While polyclonal antibodies are powerful tools for identifying biological molecules, there is one important limitation - they are unable to distinguish between antigens that share antigenic determinants. For example, when bovine serum albumin is used to immunize an animal, B cells with different surface Ig will respond to different antigenic determinants on bovine serum albumin. The result is a mixture of antibodies in the antiserum. Because bovine serum albumin shares some epitopes with human serum albumin in evolutionarily conserved regions of the protein, this anti-bovine serum albumin antiserum will also react with human serum albumin. Therefore, this antiserum will not be useful for distinguishing between bovine and human serum albumins.
Several approaches have been taken to overcome the specificity issue of polyclonal antisera. One is by absorbing the unwanted antibodies by passing the antiserum through a chromatography column of immobilized antigens (2). This method is tedious and frequently unable to completely remove the unwanted antibodies. Another approach is to isolate individual antibody-producing B cells and expand them in culture. However, like most normal untransformed cells, B cells do not survive in long-term culture.
To overcome the inability of B cells to survive in culture, one approach is to prepare a myeloma-B cell hybridoma. In 1847, Henry Bence-Jones discovered that patients with multiple myeloma, a lymphoid tumor, produced a large quantity of antibodies (3). B cells in these patients have become malignant and grow uncontrollably. Since the malignant B cells are derived from a single clone, they are identical and produce only a single type of antibody (i.e., a monoclonal antibody, or mAb). However, most of these myeloma cells produce antibodies of unknown specificities. In 1975, by fusing a myeloma cell to a B cell, Cesar Milstein and Georges Kohler succeeded in producing a hybridoma that can be cultured indefinitely in vitro and produces an unlimited number of monoclonal antibodies of known antigenic specificity (4). The rationale behind their approach is to combine the immortal properties of the myeloma cell and the antibody producing properties of the B cell. Their technique revolutionized antibody production and provides a powerful means for identification and purification of biological molecules using monoclonal antibodies.
Generally, preparing a monoclonal antibody requires several months. The general procedure includes the following steps:
<ol>
	<li>Immunization and screening of antibody titer</li>
	<li>Fusion of antibody-producing B cells and myeloma cells</li>
	<li>Selective growth of the hybridoma</li>
	<li>Screening the hybridomas for producing the desired monoclonal antibody</li>
	<li>Cloning by limiting dilution - a process whereby cells are diluted to a concentration to statistically allow for less than 1 cell to be added to the wells of a 96-well plate. Some wells will end up with 0 cells and some will have 1 cell. The wells with seeded with 1 cell will eventually grow into a monoclonal population of cells.</li>
	<li>Growth of the hybridoma and preparation of monoclonal antibody</li>
</ol>
This protocol focuses on the last step - growth of the hybridoma and preparation of the monoclonal antibody. The antibody is purified from the culture supernatant by ammonium sulfate precipitation (often referred to as salting out) - a commonly used method of removing proteins from a solution. Proteins in solution form hydrogen bonds, along with other hydrophilic interactions, with water through their exposed polar and ionic groups. When concentrations of small, highly charged ions (such as ammonium or sulfate) are added, these groups compete with the proteins for binding to water. This removes the water molecules from the protein and decreases its solubility, resulting in precipitation of the protein.

Producing Monoclonal Antibodies Using Hybridomas

Source: Frances V. Sjaastad1,2, Whitney Swanson2,3, and Thomas S. Griffith1,2,3,4
1 Microbiology, Immunology, and Cancer Biology Graduate Program, ...

Education

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Advanced Biology

Immunology

Production and Purification of Recombinant Antibodies from Mammalian Cells

Source: Shivange, Gururaj, et al. <a href="https://app.jove.com/v/60727">Analyzing Tumor and Tissue Distribution of Target Antigen-Specific Therapeutic Antibody.</a> J. Vis. Exp.(2020).
The video explains the process of producing antibodies in mammalian host cells, from gene expression to secretion. It involves transcription, translation, signal recognition, correct folding, and eventual release into the extracellular medium via secretory vesicles.

1. Expression and purification of antibodies

	Maintenance of CHO cells
	
		Grow CHO cells in FreeStyle CHO Media supplemented with commercially available ...

JoVE Encyclopedia of Experiments

Antibody-Based Technologies

Production and Purification

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.

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 ...

Antibody Production from a Batch Overgrowth Culture of Transfected Mammalian Cells

Source: Elgundi, Z. et al., <a href="https://app.jove.com/v/55153">Laboratory Scale Production and Purification of a Therapeutic Antibody.</a>&#160;J. Vis. Exp.&#160;(2017)
The video demonstrates a method for producing therapeutic antibodies using stably transfected mammalian cells. Tryptone supplementation stimulates cell division, resulting in overgrowth and supporting a higher antibody yield. The antibody-containing medium is harvested, filtered, and stored for further use.

NOTE: A suitable mammalian expression vector must be used for this protocol. Here, a single construct containing two expression cassettes is used ...

An Affinity Chromatography Technique for the Purification of Monoclonal Antibodies

Source: Elgundi, Z., et al. <a href="https://app.jove.com/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 ...

Laboratory Scale Production and Purification of a Therapeutic Antibody

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