An In Vitro Method to Determine the Anti-Apoptotic Activity of Antibodies against TNFα

1. Preparation of the Media and Solutions

1. Prepare the culture medium: RPMI-1640 with 10% fetal bovine serum (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: Dulbecco's phosphate-buffered saline (DPBS) Mg- and Ca-free solution with 0.02% EDTA, pH 7.4.
4. Prepare cell detachment solution: 0.125% trypsin with 1 mM ethylenediaminetetraacetic acid (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 until use.
   3. Filter these solutions through a 0.22-µm membrane and warm up to 37 °C for at least 30 min prior to use.
5. Prepare apoptosis-induction stock solution TNFα solution at 3.3 µg/mL.
   1. Dissolve 20 µg of TNFα with 500 µL of filter-sterilized water in its primary container and mix until complete dissolution.
   2. Transfer into a 15 mL sterile tube and add 5.5 mL of DPBS Mg- and Ca-free solution to this tube. Mix gently using a vortex mixer.
   3. Aliquot the solution into 70 µL portions. Dispense each aliquot into 0.5 mL microtubes and store at -80 °C.
6. Prepare apoptosis induction solution: TNFα solution at 40 ng/mL.
   1. Thaw an aliquot of the apoptosis induction stock solution, incubating it in a water bath at 25 ˚C for 10 min.
   2. Dilute the apoptosis induction stock solution to 40 ng/mL by adding 61 µL of 3.3 µg/mL TNFα solution to 4.939 mL of assay culture medium in a 15 mL sterile tube.
   3. Mix with a vortex mixer for 10 seconds; this solution must be prepared freshly before use.
   4. Warm up the solution to 37 °C for at least 30 min prior to use in the neutralization assay.
7. Prepare the substrate solution: caspase 3/7 Glo solution.
   1. Thaw the caspase buffer solution (caspase 3/7 Glo buffer) for 12 hours before use.
   2. Let the caspase buffer solution and the substrate (caspase 3/7 Glo substrate) sit separately at 25 ± 5 °C for 30 min prior to mixing.
   3. Transfer 10 mL of the caspase buffer solution to the substrate vial and mix by inversion.
   4. Keep at 25 ± 5 °C, light-protected until use.
      NOTE: The solution is stable for 6 h at room temperature.

2. Cell Culturing and Counting

1. Cell thawing and the first subculture.
   1. Remove one vial with WEHI 164 cells from a freezer at -80 ˚C and transfer them to an ice bath.
   2. Pipette up and down with 1 mL of pre-warmed culture medium until the frozen cells completely thaw.
   3. Dispense 9 mL of pre-warmed culture medium onto a 15 mL sterile tube.
   4. Transfer the cell suspension into the 15-mL sterile tube and mix gently five times by inversion.
   5. Centrifuge the cell suspension at 125 x g for 3 min. Discard the supernatant and disaggregate the cell pellet.
   6. Add 5 mL of culture medium to the tube. Mix until the cells are completely resuspended.
   7. For cell counting, transfer 50 µL of the cell suspension to a 500 µL microtube and mix with 50 µL of 0.4% trypan blue. Count the cells and adjust them to 0.5 x 10⁶ cells/mL. See step 2.2, below.
   8. Add 13 mL of pre-warmed culture medium to a 75 mL cell culture flask.
   9. Dispense enough cell suspension volume from step 2.1.6 to achieve 0.5 x 10⁶ cells/mL in the cell culture flask and incubate at 37 °C and 5% CO₂ overnight.
2. Cell counting.
   1. Using the solution from step 2.1.6, transfer 0.05 mL to a hemocytometer and determine the cell density under a microscope using trypan blue exclusion.
   2. Quantify the total number of cells and viable cells.
   3. Adjust the cell suspensions to 0.5 x 10⁶ cells/mL.
      V_{culture medium}(mL) =
      V_{culture medium}(mL) = (5mL – V_{cell suspension})
      V_{culture medium}(mL) = Adjusted volume of WEHI 164 cell suspension
      NVC = Number of viable WEHI 164 cells/mL
      V_{culture medium}(mL) = Assay culture medium volume added to the cell suspension to achieve 0.5 x 10⁶ cells/mL
      0.5 x 10⁶  = Target cell density
3. Cell detachment and the second and third subcultures.
   NOTE: A vacuum system can be used to remove the solutions from the flasks. Disposable or glass sterile pipettes can be used. If the pipette has a cotton clog at the top, it must be removed before use.
   1. Remove the culture medium from the cell culture T-flask using a 1 mL sterile pipette and a vacuum.
   2. Dispense 5 mL of cell wash solution into the culture T-flask, mix gently, and discard the solution. Repeat this step twice.
      NOTE: The complete removal of the culture medium is critical for efficient cell detachment.
   3. Add 15 mL of cell detachment solution to the T-flask and let stand for 3 min in an incubator at 37 °C and 5% CO₂.
   4. Verify the absence of attached cells in the flask's inner wall under the microscope. Remove the cells from the culture T-flask using a 20 mL sterile pipette and dispense them into a 50 mL sterile tube.
   5. Centrifuge the cell suspension at 125 x g for 3 min. Discard the supernatant and resuspend the pellet with another 5 mL of culture medium.
   6. Count the cells and add enough culture medium to reach the desired cell concentration according to Equation 1.
   7. Add this suspension to a 72 cm² T-flask and incubate overnight at 37 °C and 5% CO₂.
   8. Subculture the cells at least two times before using them in the neutralization assay. Repeat steps 2.3.1-2.3.8 for the next two days.
4. Assay cell suspension.
   1. Select a WEHI 164 subculture that has at least three passes. See step 2.1.
   2. Detach and count the cells according to steps 2.2 and 2.3 of this protocol.
   3. Dilute the cell suspension according to Equation 1 to 0.5 x 10⁶ cells/mL.
   4. Use this cellular suspension for the neutralization assay. Mix all cell suspensions with a vortex mixer prior to use.

3. Antibody Preparation and Dilutions

1. Quantitation of the mAbs.
   1. Determine the concentration of the reference substance, control sample, and analytical sample through UV absorption at 280 nm using their mass extinction coefficient (1.39).
      NOTE: Original concentrations could be taken from drug product labels. However, this must be verified by UV absorption.
2. mAb dilutions.
   1. Dilute all samples independently in triplicate, with DPBS Mg- and Ca-free solution in 2 mL microtubes, down to 2 mg/mL. Confirm this concentration by UV absorption in triplicate, using DPBS Mg- and Ca-free solution as the blank.
   2. Mix the stock protein solutions for 5 seconds using a vortex mixer.
   3. Dilute 100 µL of each 2 mg/mL mAb solution with 0.9 mL of the assay culture medium.
   4. Mix for 5 s with a vortex mixer.
      NOTE: These solutions have a concentration of 200 µg/mL. Dilutions must be done for each triplicate.
   5. Dilute 10 µL of each 200 µg/mL mAb solution with 0.99 mL of the assay culture medium. Mix for 5 s using a vortex mixer. These solutions have a concentration of 2 µg/mL. Perform serial dilutions for each triplicate before using them in the neutralization assay.
   6. Make anti-TNFα mAb dilutions in three independent microplates. Make a duplicate from each independent triplicate and dispense them in one microplate, as indicated in Table 1. Reference Substance
   7. Perform mAb dilutions of each reference, sample, or control, as shown in Table 2.
      Note: The anti-TNFα mAb concentrations described in this table are not the final concentrations in the neutralization assay.
   8. Keep the plates at 25 ± 5 °C until use.

4. Neutralization Assay with WEHI 164 Cells

1. Mix by vortexing all cell suspensions (0.5 x 10⁶ cells/mL) prior to dispensing at any step of this protocol.
   NOTE: In this section, warm each solution to 37 °C for 30 min prior to use.
2. Transfer 50 µL of the cell suspension to each of the 60 wells of the microplates, moving from column 2 to 11 and line B to G.
3. Transfer 50 µL of mAb reference, sample, and control dilutions into microplates. Follow the pattern depicted in Figure 1.
4. Add 50 µL of the apoptosis induction solution to each well.
5. Use cellular controls of 50 µL of WEHI 164 cells, dispensed in three wells. Bring each well to a final volume of 150 µL with an assay culture medium.
6. Use a cytotoxicity control of a mixture of 50 µL of WEHI 164 cells plus 50 µL of apoptosis induction solution. Bring each well to a final volume of 150 µL with an assay culture medium.
7. For the TNFα control, use 50 µL of the apoptosis induction solution and bring it to 150 µL with the assay culture medium.
8. For the blank, use 150 µL of the assay culture medium alone.
9. Fill the remaining wells with 150 µL of culture medium to avoid plate evaporation effects.
10. Repeat steps 4.1.1-4.1.9 twice in two other microplates.
    NOTE: The mAb 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.
11. Load the samples in microplates, as indicated in Figure 1
12. Incubate the three plates at 37 °C and 5% CO₂ for 16 ± 2 h.
13. Let the caspase 3/7 Glo reagent stand at 25 ± 5 °C for 30 min before use.
14. Add 100 µL of this reagent to all wells, including the samples and controls.
15. Shake the plates using a microplate vortex mixer for 3 min at 25 ± 5 °C immediately after dispensing into the wells.
16. Incubate the plates for 2.5 ± 0.5 h at 25 ± 5 °C, protected from light.
17. Insert the microplates into the luminometer and complete the next section.

5. Analysis of Results

1. Using software for luminescence detection, select the luminescence mode and endpoint function.
2. Select a 96-well clear-bottom plate and its 80 internal wells, excluding columns 1 and 12.
3. Select an integration time of 1,250 ms and 10 s for mixing the microplate before reading.
4. Select the wells where the reference substance, analytical substance, and control sample will be placed and identify them with their corresponding concentrations.
5. Read the samples placed in the microplates with the luminometer.
6. Use a fourth parameter equation for the analyses of results. Graph a dose-response curve, as depicted in Figure 2.
   NOTE: In the fourth parameter equation, C is the effective concentration 50 (EC50). This value will be used to compare the reference substance, analytical sample, and control sample by means of the effector function.
7. For calculating the relative potencies, fix the reference substance to 100% calculate the potencies of the sample, and control accordingly.
   NOTE: Those values are depicted in Figure 3.

Table 1: Microplate sample arrays. A complete neutralization assay must be run in three microplates within coordinates B2 to G11. Random dispensing of reference, analytical, and control samples allows researchers to verify any bias in the assay.

Table 2: Anti-TNFα mAb dilutions. Serial dilutions of anti-TNFα mAbs are demonstrated in this table. Final concentrations described in this table are not the concentrations in the assay, where anti-TNFα mAbs were diluted by a factor of 3 (mAb dilution + culture medium + cells suspension). Lines in bold represent dilutions coming from lines 3, 5, 7, 9, and 10; non-bolded lines represent dilution from lines 3, 4, and 6. These serial dilutions are done just before performing the neutralization assay. Care must be taken to mix by pipetting up and down three times before dispensing the dilutions.