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.
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.
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-TNFα molecules exert their therapeutic properties by blocking the interaction of tumor necrosis factor alpha (TNFα) with the p55 and p75 cell surface receptors3, thus preventing signal pathways that finally lead to cellular apoptosis.
TNFα can produce inflammation in some chronic illnesses4. TNFα 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, TNFα deregulation is associated with the pathogenesis of these illnesses.Without control and under constant induction and cell stress, TNFα 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-TNFα 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-TNFα drug products when compared to a clinically tested reference substance.
1. Preparation of the Media and Solutions
2. Cell Culturing and Counting
3. Antibody Preparation and Dilutions
Plate 1 | Plate 2 | Plate 3 | |||
Wells | Sample | Wells | Sample | Wells | Sample |
B2:B11 | Reference Substance | B2:B11 | Control Sample | B2:B11 | Analytical Sample |
C2:C11 | C2:C11 | C2:C11 | |||
D2:D11 | Analytical Sample | D2:D11 | Reference Substance | D2:D11 | Control Sample |
E2:E11 | E2:E11 | E2:E11 | |||
F2:F11 | Control Sample | F2:F11 | Analytical Sample | F2:F11 | Reference Substance |
G2:G11 | G2:G11 | G2:G11 |
Plate Column | Volume of Assay culture medium (μL) | Volume of Reference Substance, Analytical Sample or Control Sample (uL) | Concentration in the Assay Plate (ng/mL) |
2 | 0 | 230 | 2000 |
3 | 150 | 150 from line 2 | 1000 |
4 | 75 | 75 from line 3 | 500 |
5 | 100 | 50 from line 3 | 333 |
6 | 75 | 75 from line 4 | 250 |
7 | 75 | 75 from line 5 | 166 |
8 | 75 | 75 from line 6 | 125 |
9 | 75 | 75 from line 7 | 83 |
10 | 75 | 75 from line 9 | 41 |
11 | 150 | 75 from line 10 | 13 |
4. Neutralization Assay with WEHI 164 Cells
5. Analysis of 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 receptor. This causes cell survival at higher mAb concentrations.
The controls used in the method were: cells with assay culture medium, cells plus TNFα plus assay culture medium, and assay culture mediumalone. Cells alone with assay culture medium under FBS starvation did not undergo to apoptosis, thus developing luminescence. Moreover, cells exposed to TNFα alone but cultivated with the culture medium did indeed survive.
Another important control is the assay culture medium alone. This controls helps with the understanding of molecular interference relating to the medium, specifically proteins that can digest the caspase substrate, thus indicating a false-positive luminescent signal. The EC50 and relative potency results are depicted in Table 3.
Sample | EC50 | Relative Potency (%) | Confidence Interval (%) | RDS (%) |
Reference substance | 241.5 | 100 | — | — |
243.6 | ||||
234.2 | ||||
Analytical Sample | 225.2 | 99.7 | 86.0-115.1 | 8.5 |
240.3 | ||||
258.8 | ||||
Control Sample | 230.5 | 97.1 | 86.5-108.7 | 6.9 |
264 | ||||
248.4 |
Table 3: Relative potency results. The analytical sample is compared to a known potency sample, described in the table as the reference, with a fixed 100% potency. The relation is calculated with the EC50 of each sample. The interval is calculated at a 95% confidence (α = 0.05). RSD: relative standard deviation.
Table 3 shows the relative potency, in percentage, between a reference mAb and a sample under investigation. Comparability is assumed within a range of 80-120% of the reference. However, sometimes the reference has large variability between batches; therefore, the range of acceptance can change. Hence, it is desired to select reference batches within a short period of manufacturing, tightening the physicochemical properties and acceptance interval.
This table shows that the reference has a potency of 100%, while the analytical sample has a potency of 99.7%. This result means that the TNFα neutralization capability by the sample is comparable to that of the reference. It is expected that illnesses related to the overexpression of the cytokine can be controlled by these mAbs.
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-TNFα molecules. Despite the common physicochemical method, this methodology is able to determine, through biological means, the potency and efficacy of a drug as a quality attribute, thus showing the complete and full significance of the effector functions.
Commonly, cell apoptosis responsiveness to TNFα can be a challenging task for researchers to perform. Troubleshooting can be conducted through the characterization of the cell bank that is used on a daily basis before standardizing this biological method. One example is the cell response variability within a time period relating to cell-line aging12. This problem is eliminated using a master cell bank frozen at -80 °C. The working cell bank must be large enough to cover the requirements of a DOE study performed by R&D or a one-year period for use in the quality-control laboratory. Also, this responsiveness can be fixed using temperature-stabilized solutions before adding them to cells at any step during this protocol. At least three passes must be performed before running a neutralization assay and restricting the length of time that cells are grown in continuous culture due to adaptation and population dynamics12,13.
TNFα molecule must be protected from freeze-thaw cycles, as the potency of this protein is substantially undermined if not stabilized. Formulations cited elsewhere14 for cytokine preparation are suggested when the reconstituted cytokine will be stored, as the concentration of TNFα is critical for protocol success. Responsiveness of a cell to a cytokine depends on the number of receptors per cell. Therefore, the optimal TNFα concentration depends on the cell line and density15,16. We diluted TNFα to a final concentration of 13.3 ng/mL and adjusted the cell density using a curve around 25,000 cells/well. Therefore, the cell density must be verified for each TNFα concentration.
Camacho-Villegas et al. reports a TNFα final concentration of 1.25 ng/mL; this group also uses actinomycin D as a stress cell factor9,17,18. We instead changed the FBS from 10% to 1% from culture medium to assay medium, giving a strong stress signal to induce apoptosis in WEHI 164 cells with TNFα alone, eliminating another variable from the protocol. Other groups report cell viability using MTT. Methodologies using a luminescence substrate sensitive to caspase 37, instead of the spectrophotometric method where the UV-Vis absorbance substances are present in the culture medium or the cells themselves (e.g., phenol red absorbed by cells), can interfere with the signal. Thus, the sensitivity of the assay was increased using this Ac-DEVD-pNA luminescentreactant, as the presence of luminescent substances (background) in the culture medium is not expected.
A limitation of this method is that it can only be applied to TNFα-sensitive cells; other cells lines must be tested and the cytokine concentration adjusted for an optimized cellular response. Furthermore, the absolute response cannot be measured, as we are not using a primary cell line or an in vivo assay; instead, orthogonal isothermal titration calorimetry (ITC) experiments are suggested for initial method adjustment. Information from ITC is helpful for establishing TNFα affinity with a new molecule under development and for specifying the initial conditions in the biological method.
This method is useful for testing new molecules when researchers have a reference substance for obtaining a basal relative response; thus, the evaluation of a bio-better or a molecular response relating to cell protection is recommended. It can be applied to other anti-TNFα proteins. For instance, it is applicable to evaluating the relative biological potency of Etanercept, Infliximab, Certolizumab, or Golimumab19. However, all these anti-TNFα molecules have different affinities for the cytokine; therefore, their concentrations must be adjusted individually. Another advantage of this method is the short time between the initiation of experiments and the achievement of results, making it easy to execute and inexpensive compared to animal models. Further, this method measures interaction of the mAb with a fully active TNFα molecule, suggesting that the mAb is recognizing the TNFα trimer and that the molecules were not modified during storage or laboratory manipulation. On the other hand, a pure physicochemical assay result is still dubious. For example, interaction between TNFα and a mAb using a conventional ELISA can be an artifact relating to structural changes due to cytokine chemical immobilization; therefore, the affinity could be affected and the measurements compromised. Moreover, during ITC analyses, dilution solutions can modify the structure of TNFα or the mAb, thus inhibiting TNFα trimer formation, mAb interaction, or artifactual epitope generation. The use of primary cells lines in this method could be demanding; nonetheless, protection against TNFα can give interesting results, mimicking in vivo responses.
We designed this method to be easy to follow and reproducible. As luminescence cannot be masked by phenol red or other UV-Vis absorbance substances, almost every culture medium additive can be used for cultivating cells. This modification aids researchers in the study of demanding cell lines, with a full detection response for viability after cytokine treatment. At least three cell passages and cell density adjustments must be conducted before executing the neutralization assay. Also, stabilizing the cytokine and its concentration, as well as warming and equilibrating the CO2 concentration in the culture medium and solutions are critical steps for assay success. Overall, this article shows the steps necessary to neutralize TNFα cytokine with a mAb using an in vitro biological test to compare a reference and a sample under development.
The authors have nothing to disclose.
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.
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 |