Name: determination of the relative potency of an anti-tnf monoclonal antibody (mab) by neutralizing tnf using an in vitro bioanalytical method
Uploaded: 2017-09-16T14:00:00
Description: Watch this Scientific Journal Video about Determination of the Relative Potency of an Anti-TNF Monoclonal Antibody mAb by Neutralizing TNF Using an In Vitro Bioanalytical Method at JoVE.com

This work was supported by the National Council of Science and Technology (CONACYT), Mexico grant PEI CONACYT 2015 220333, without participation in the design of the study.

1. Preparation of the Media and Solutions
<ol>
	<li>Prepare the culture medium: RPMI-1640 with 10% FBS, pH 7.4.</li>
	<li>Prepare assay culture medium: RPMI-1640 without phenol red but with 1% FBS, pH 7.4.</li>
	<li>Prepare cell wash solution: DPBS Mg- and Ca-free solution with 0.02% EDTA, pH 7.4.</li>
	<li>Prepare cell detachment solution: 0.125% trypsin with 1 mM EDTA.
	<ol>
		<li>Thaw 100 mL of a 0.25% solution of trypsin-EDTA and transfer to...

<ol>
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G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+regulation+of+the+TRAIL+death+receptors+DR4+and+DR5+by+the+signal+recognition+particle.">Differential regulation of the TRAIL death receptors DR4 and DR5 by the signal recognition particle.</a> Mol Biol Cell. 15 (11), 5064-5074 (2004).</li><li>Sud, D., Bigbee, C., Flynn, J. L., Kirschner, D. 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W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lyophilized+formulations+of+recombinant+tumor+necrosis+factor.">Lyophilized formulations of recombinant tumor necrosis factor.</a> Pharm Res. 9 (1), 33-36 (1992).</li><li>Ponnappan, S., Ponnappan, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aging+and+immune+function:+molecular+mechanisms+to+interventions.">Aging and immune function: molecular mechanisms to interventions.</a> Antiox Redox Signal. 14 (8), 1551-1585 (2011).</li><li>Matsumaru, K., Ji, C., Kaplowitz, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+for+sensitization+to+TNF-induced+apoptosis+by+acute+glutathione+depletion+in+murine+hepatocytes.">Mechanisms for sensitization to TNF-induced apoptosis by acute glutathione depletion in murine hepatocytes.</a> Hepatology. 37 (6), 1425-1434 (2003).</li><li>Camacho-Villegas, T., Mata-Gonzalez, T., Paniagua-Solis, J., Sanchez, E., Licea, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+TNF+cytokine+neutralization+with+a+vNAR+from+Heterodontus+francisci+shark:+a+potential+therapeutic+use.">Human TNF cytokine neutralization with a vNAR from Heterodontus francisci shark: a potential therapeutic use.</a> mAbs. 5 (1), 80-85 (2013).</li><li>M&#228;nnel, D. 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This characterization helps to determine a priori the biological behavior of a molecule under development before expensive and time-consuming clinical trials are conducted. It is also useful for the batch-to-batch release of an approved drug product. It is worth mentioning that these assays are useful for determining if a molecule has an adequate biological effect regarding its mechanism of action. The bio-analytical method presented in this tutorial is critically important to the comparison of different anti-TN...

Biological potency is the quantitative measure of biological activity based on the assayed product attributes that are linked to the relevant biological properties, whereas quantity (expressed in mass) is a physicochemical measure of protein content. Potency tests, along with other analytical methodologies, are performed as part of product conformance, stability, and comparability studies. In this sense, potency measurements are used to demonstrate that product batches meet the critical quality attributes (CQAs) or acceptance criteria during all phases of clinical trials and after market approval.
Apoptosis is programmed cell death, naturally occurring when cells are infected with a virus or when the cells are stressed by an environmental factor that compromises cellular viability and function1,2. Among others, apoptosis inhibition, or biological neutralization, is one of the principally known therapeutic mechanisms of mAbs, particularly in the treatment of chronic diseases, such as immune-mediated inflammatory disorders. Anti-TNF&#945; molecules exert their therapeutic properties by blocking the interaction of tumor necrosis factor alpha (TNF&#945;) with the p55 and p75 cell surface receptors3, thus preventing signal pathways that finally lead to cellular apoptosis. 
TNF&#945; can produce inflammation in some chronic illnesses4. TNF&#945; is spuriously secreted into the extracellular milieu by macrophages, which are sentries of the innate immune system and the main actors in this kind of disease5. As a common path, TNF&#945; deregulation is associated with the pathogenesis of these illnesses.Without control and under constant induction and cell stress, TNF&#945; induces cell death and tissue degeneration, ultimately leading to rheumatoid arthritis, Crohn's disease, and other pathological profiles6.
TNF antagonists that block the interaction between TNF and its receptors have been increasingly used as an effective therapy to reduce symptomatology and hinder the progression of these diseases. Nowadays, anti-TNF&#945; drug products are widely used to control the systemic concentration of this cytokine, thus preventing further degeneration of involved tissues. In this sense, providing a reproducible and robust bioassay to describe the specific ability of a drug to achieve its biological effect is imperative. 
In this protocol, critical steps-identified during the development of a neutralization assay-for the successful measurement of biological potency are highlighted, with a particular emphasis on the skills needed to execute the bio-analytical method. This bio-analytical method provides useful comparability information between different batches or anti-TNF&#945; drug products when compared to a clinically tested reference substance.

<table border="0" cellpadding="0" cellspacing="0" width="904">
	<tbody>
		<tr height="22">
			<td height="22" style="height:22px;width:203px;">WEHI 164</td>
			<td style="width:111px;">ATCC</td>
			<td style="width:127px;">CRL-1751</td>
			<td style="width:463px;">Fibrosarcoma cells from Mus musculus</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:203px;">RPMI-1640 Medium</td>
			<td style="width:111px;">ATCC</td>
			<td style="width:127px;">30-2001</td>
			<td style="width:463px;">Store medium at 2 &#176;C to 8 &#176;C</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:203px;">RPMI 1640 Medium, no phenol red</td>
			<td style="width:111px;">GIBCO</td>
			<td style="width:127px;">11835-030</td>
			<td style="width:463px;">Store medium at 2 &#176;C to 8 &#176;C</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:203px;">Trypsin-EDTA(0.25%),phenol red</td>
			<td style="width:111px;">GIBCO</td>
			<td style="width:127px;">25200-056</td>
			<td style="width:463px;">Store medium at -10 &#176;C to -20 &#176;C</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:203px;">DPBS, no calcium, no magnesium</td>
			<td style="width:111px;">GIBCO</td>
			<td style="width:127px;">14190-136</td>
			<td style="width:463px;">Store medium at 2 &#176;C to 8 &#176;C</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:203px;">Recombinant Human TNF-alpha Protein</td>
			<td style="width:111px;">R&#38;D Systems</td>
			<td style="width:127px;">210-TA-020</td>
			<td style="width:463px;">Store at -20 &#176;C to -70 &#176;C</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:203px;">Fetal Bovine Serum (U.S), Super Low IgG</td>
			<td style="width:111px;">HyClone</td>
			<td style="width:127px;">SH3089803</td>
			<td style="width:463px;">Store at -10 &#176;C to -20 &#176;C</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:203px;">Fetal Bovine Serum (U.S.), Characterized</td>
			<td style="width:111px;">HyClone</td>
			<td style="width:127px;">SH3007103</td>
			<td style="width:463px;">Store at -10 &#176;C to -20 &#176;C</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:203px;">Caspase-Glo 3/7 Assay kit</td>
			<td style="width:111px;">Promega</td>
			<td style="width:127px;">G8093</td>
			<td style="width:463px;">Store the Caspase-Glo. 3/7 Substrate and Caspase-Glo. 3/7 Buffer at &#8211;20 &#186;C protected fromLight</td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:203px;">EDTA, Disodium Salt, Dihydrate, Crystal, A.C.S. Reagent</td>
			<td style="width:111px;">J.T.Baker</td>
			<td style="width:127px;">8993-01</td>
			<td style="width:463px;">--</td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:203px;">Sample mAb Adalimumab</td>
			<td style="width:111px;">Probiomed</td>
			<td style="width:127px;">NA</td>
			<td style="width:463px;">Final concentrations in the microplate are:&#160; 0.666, 0.333, 0.167, 0.111, 0.083, 0.056, 0.042, 0.028, 0.014 and 0.004 &#956;g/mL</td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:203px;">Reference and Control mAb Adalimumab</td>
			<td style="width:111px;">Abbvie</td>
			<td style="width:127px;">NA</td>
			<td style="width:463px;">Final concentrations in the microplate are:&#160; 0.666, 0.333, 0.167, 0.111, 0.083, 0.056, 0.042, 0.028, 0.014 and 0.004 &#956;g/mL</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:203px;">Microplate Reader</td>
			<td style="width:111px;">Molecular Devices</td>
			<td style="width:127px;">89429-536</td>
			<td style="width:463px;">SpectraMax M3 Multi-Mode</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:203px;">Microplate reader Software&#160;</td>
			<td style="width:111px;">Molecular Devices</td>
			<td style="width:127px;">--</td>
			<td style="width:463px;">SoftMax Pro 6.3 GxP</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:203px;">Incubator&#160;</td>
			<td>Revco&#160;</td>
			<td>30482</td>
			<td style="width:463px;">Revco RNW3000TABB Forced-Air CO2</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:203px;">Laminar Flow Hood&#160;</td>
			<td style="width:111px;">The Baker Company&#160;&#160;</td>
			<td>200256</td>
			<td style="width:463px;">Baker SG603A-HE | High Efficiency, Class II Type A2&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;</td>
		</tr>
	</tbody>
</table>

determination of the relative potency of an anti-tnf monoclonal antibody (mab) by neutralizing tnf using an in vitro bioanalytical method

This protocol shows the measurement of the apoptotic activity neutralization of TNF&#945; in a mouse fibroblast cell model (WEHI 164) using an anti-TNF&#945; mAb. In addition, this protocol can be used to evaluate other anti-TNF&#945; molecules, such as fusion proteins. The cellular model employed here is sensitive to TNF&#945;-mediated apoptosis when an additional stress factor is induced in cell culture conditions (e.g., serum deprivation). This procedure exemplifies how to execute this analytical assay, highlighting the key operations relating to the sample preparation, cell dilution, apoptosis induction, and spectrophotometric measurements that are critical to ensure successful results. This protocol reveals the best-performance conditions relating to apoptosis induction and efficient signal recording, leading to low uncertainty values.

Dose-response Graph (with Controls) 
Figure 1 represents the luminescence response versus mAb concentration. This sigmoidal function exemplifies caspase 3 and 7 release in the assay culture medium due to cell lysis. Cell death is enhanced by serum starvation plus TNF&#945; signaling induction. Therefore, the anti-TNF&#945; molecule (mAb) interacts with the cytokine, inhibiting (by steric hindrance) its interaction with the TNF cell recep...

Watch this Scientific Journal Video about Determination of the Relative Potency of an Anti-TNF Monoclonal Antibody mAb by Neutralizing TNF Using an In Vitro Bioanalytical Method at JoVE.com

Determination of the Relative Potency of an Anti-TNF Monoclonal Antibody mAb by Neutralizing TNF Using an In Vitro Bioanalytical Method

A protocol for the determination of the relative anti-apoptotic activity of an anti-TNF&#945; mAb using a neutralization mechanism with WEHI 164 cells is presented here. This protocol is useful for comparing the neutralization strength of different molecules with the same biological functionality.

Determination of the Relative Potency of an Anti-TNF Monoclonal Antibody (mAb) by Neutralizing TNF Using an In Vitro Bioanalytical Method

This protocol shows the measurement of the apoptotic activity neutralization of TNF&#945; in a mouse fibroblast cell model (WEHI 164) using an ...

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