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

Summary

A protocol for the determination of the relative anti-apoptotic activity of an anti-TNFα 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.

Abstract

This protocol shows the measurement of the apoptotic activity neutralization of TNFα in a mouse fibroblast cell model (WEHI 164) using an anti-TNFα mAb. In addition, this protocol can be used to evaluate other anti-TNFα molecules, such as fusion proteins. The cellular model employed here is sensitive to TNFα-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.

Introduction

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, natural....

Protocol

1. Preparation of the Media and Solutions

1. Prepare the culture medium: RPMI-1640 with 10% FBS, pH 7.4.
2. Prepare assay culture medium: RPMI-1640 without phenol red but with 1% FBS, pH 7.4.
3. Prepare cell wash solution: DPBS Mg- and Ca-free solution with 0.02% EDTA, pH 7.4.
4. Prepare cell detachment solution: 0.125% trypsin with 1 mM EDTA.
   1. Thaw 100 mL of a 0.25% solution of trypsin-EDTA and transfer to a sterile 500-mL flask.
   2. Mix with 100 mL of cell wash solution and dispense 15 mL aliquots into 15 mL sterile tubes. Store at -70 to -80 °C....

Results

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 TNFα signaling induction. Therefore, the anti-TNFα molecule (mAb) interacts with the cytokine, inhibiting (by steric hindrance) its interaction with the TNF cell recep.......

Discussion

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.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
WEHI 164	ATCC	CRL-1751	Fibrosarcoma cells from Mus musculus
RPMI-1640 Medium	ATCC	30-2001	Store medium at 2 °C to 8 °C
RPMI 1640 Medium, no phenol red	GIBCO	11835-030	Store medium at 2 °C to 8 °C
Trypsin-EDTA(0.25%),phenol red	GIBCO	25200-056	Store medium at -10 °C to -20 °C
DPBS, no calcium, no magnesium	GIBCO	14190-136	Store medium at 2 °C to 8 °C
Recombinant Human TNF-alpha Protein	R&D Systems	210-TA-020	Store at -20 °C to -70 °C
Fetal Bovine Serum (U.S), Super Low IgG	HyClone	SH3089803	Store at -10 °C to -20 °C
Fetal Bovine Serum (U.S.), Characterized	HyClone	SH3007103	Store at -10 °C to -20 °C
Caspase-Glo 3/7 Assay kit	Promega	G8093	Store the Caspase-Glo. 3/7 Substrate and Caspase-Glo. 3/7 Buffer at –20 ºC protected fromLight
EDTA, Disodium Salt, Dihydrate, Crystal, A.C.S. Reagent	J.T.Baker	8993-01	--
Sample mAb Adalimumab	Probiomed	NA	Final concentrations in the microplate are:  0.666, 0.333, 0.167, 0.111, 0.083, 0.056, 0.042, 0.028, 0.014 and 0.004 μg/mL
Reference and Control mAb Adalimumab	Abbvie	NA	Final concentrations in the microplate are:  0.666, 0.333, 0.167, 0.111, 0.083, 0.056, 0.042, 0.028, 0.014 and 0.004 μg/mL
Microplate Reader	Molecular Devices	89429-536	SpectraMax M3 Multi-Mode
Microplate reader Software	Molecular Devices	--	SoftMax Pro 6.3 GxP
Incubator	Revco	30482	Revco RNW3000TABB Forced-Air CO2
Laminar Flow Hood	The Baker Company	200256	Baker SG603A-HE | High Efficiency, Class II Type A2