Protocol for Evaluation of Virus Inactivation Using Low-pH Treatment

Preface

Biopharmaceutical processes typically incorporate a dedicated procedure to inactivate retroviruses and other enveloped viruses susceptible to inactivation. A common inactivation procedure is low-pH hold, in which the product pool is titrated to pH <4.0 with acid and held for a specific amount of time. Worse-case conditions for low-pH inactivation of retroviruses are now well known and include the upper limit of the target pH, lower limit of manufacturing temperature, and shortest hold duration. This protocol assembles current industry knowledge of considerations for performing well-controlled studies for assessment of virus inactivation by low-pH inactivation, and includes guidance on modeling worse-case conditions based on manufacturing ranges. An example calculation of the log reduction value (LRV) using output virus titration data is also provided.

1. Purpose

   The purpose of this protocol is to outline the equipment, procedures, and controls for assessing virus inactivation by low-pH treatment. The kinetics of inactivation will be determined by evaluating the virus titer at the start and end of the low-pH hold, and at one or more intermediate timepoints.

2. Scope

   The scope of this document applies to monoclonal antibody products and other therapeutic protein products where virus inactivation is performed using a low-pH treatment. Viruses used for the virus inactivation assessment include murine leukemia viruses and other enveloped viruses (e.g., pseudorabies virus) that are susceptible to this form of inactivation. In addition, the principles of ICH Q5A guideline are applied.

3. Definitions

   1. MuLV: murine leukemia virus, a gamma retrovirus. Types typically used are amphotrophic, ecotropic, or xenotropic.

   2. Process pool: pool from the process that contains product

   3. LRV: log reduction value = log₁₀ (virus in/virus out)

4. General Considerations

   1. Study design

      Assessment of viral inactivation should use protocols and procedures that are consistent with the manufacturing-scale viral inactivation step that is being modeled. This includes using representative product process pools with a representative product concentration, and comparable pH titration and measurement procedures. Additionally, virus inactivation should be assessed using conditions that represent the worst-case manufacturing conditions for inactivation. These include targeting the upper end of the low-pH target range and the shortest hold time used in manufacturing. Also, the assessment should be performed at the lower end of the manufacturing temperature range because the rate of inactivation decreases at lower temperatures. Table I provides an example of manufacturing conditions and worst-case conditions for virus inactivation.

   2. Virus assay

      A validated virus assay that measures virus infectivity should be used to determine the titer of virus in the samples generated during the assessment. Any cytotoxicity or interference of the samples with the selected assay should be mitigated by dilution (see Section 7).

   3. Virus spike percentage

      The virus will be spiked into the process pool, typically at no more than 10% of pool volume. See Section 7 for considerations for the spiked process pool.

   4. Documentation

      For virus inactivation studies intended for health authority submission, protocols should be prepared for the virus assay and the low-pH treatment operation. All study documentation prepared by the virus testing lab, which includes virus assay protocols and results, should have quality unit oversight, under a written agreement that outlines critical quality expectations (quality agreement) if conducted by a third party. Operating protocols for the low-pH treatment operation should have some form of quality oversight, depending on the product development stage. For early-stage development, operating protocols may be reviewed by the process sciences group and a quality unit (or equivalent function) with an appropriate science background, whereas late-stage development operating protocols (phase 3, process validation, etc.), should have quality unit oversight (1). In addition, a process record or other form of documentation should be available to record data and other information associated with the study. Origin and composition of the process intermediate used in the virus study should be documented.

   5. Precautions

      Follow universal precautions and applicable biosafety level when handling infectious virus and samples containing infectious virus.

5. Responsibility

   The product sponsor company is responsible for all procedures performed by their personnel and by contract testing company personnel.

6. Equipment and Materials

   1. Equipment

      1. pH meter

         Because the low-pH inactivation study will be performed at a temperature lower than 25 °C, the pH meter used should have the ability to compensate for temperature. Depending on the meter, the temperature can be determined using a pH probe containing temperature probe or a separate temperature probe that plugs into the meter. Alternatively, some meters allow for a temperature to be entered in, and some have pre-programmed calibration settings. The pH meter should allow enough time for the probe to equilibrate in the solution before displaying the pH reading. A meter set up with a gel-filled pH probe will need a longer time to equilibrate than a liquid-filled pH probe. The pH meter should be on a regular calibration and maintenance schedule.

         Note: Automatic temperature compensation (ATC) on the pH meter corrects only for the temperature effect on the pH probe itself. ATC does not adjust for the actual shift in pH resulting from temperature changes within a given solution.

      2. pH probe:

         The pH probe can be gel-filled or liquid-filled, and should be Tris buffer–compatible, as appropriate. The health of the probe can have a significant impact on the pH measurement. Contact with protein solutions over time can foul the glass pores of the probe and slow the passage of ions, leading to inaccurate measurement. The percent slope is not always a good indicator of poor probe health because an inaccurate probe can still have an acceptable slope. An alternative measure of pH probe health is mV offset, which denotes the difference in mV between the reference electrode and the measuring electrode in pH 7.0 standard solution, which under ideal conditions should be zero. Significant deviation can indicate that the probe is fouled. If a probe is used for several studies, probe maintenance, calibration, and lifetime should be prospectively defined. Alternatively, a new probe can be used for each study.

      3. Circulating water bath or equivalent equipment with temperature control set at the target temperature

      4. Calibrated thermometer or thermocouple, to verify temperature of water bath

      5. Balance and traceable weights, if measuring by weight

      6. Pipettors, calibrated and within calibration expiry

      7. Timer

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      Table I

      Example of Manufacturing Conditions and Worst-Case Conditions for the Virus Inactivation Study

   2. Materials

      1. pH standard solutions: Standards at pH 7, pH 4, and others as appropriate. It is preferable to bracket the pH range or use a 3 point calibration wherein the third buffer has a pH value as close as possible to the target pH of the sample. Standard solutions should be stored according to vendor recommendations and be within their expiry.

      2. Virus stock:

         Information regarding the preparation of the virus stock is expected to be provided or described to the study sponsor. The virus stock should be prepared from a controlled stock or source as outlined in PDA Technical Report 47. Ideally, master and working virus banks are established, if using a two-tiered banking system. Methods of preparation are according to a standard operating procedure. Tests for virus banks may be more extensive than those for virus production lots used in the spiking study. As outlined in PDA Technical Report 47, virus production lots are tested for identity, sterility, and potency; however, other tests may be applied. For example, purity tests can be useful to understand potential variability in inactivation when working at or near pH 3.9 where inactivation may be dependent on the matrix. The composition of the process intermediate should not significantly be influenced by the virus spike; this should be considered when using stabilizers in the virus spike preparations and/or in the virus spike percentage.

         Virus production lot testing:

         1. Potency; titer

         2. Purity; can include such attributes as aggregation status, protein content, host cell DNA/RNA, and particle-to-infectivity ratio. Recommended, as appropriate

         3. Safety; sterility or bioburden

         4. Identity

         5. Formulation

      3. Process pool:

         The process pool is the starting material for the virus inactivation assessment. The process pool is provided by the product sponsor and should be representative of the manufacturing process. Prior to study start, the pool should be thawed (if required) and equilibrated to the testing temperature.

      4. Acid titrant:

         The acid titrant (e.g., acetic acid, citric acid, phosphoric acid) is used to lower the pH of the process pool to the target. The acid titrant should be the same acid as that used in the manufacturing process and should be formulated according to the manufacturer's procedure. For some situations, an option may be to use a higher molarity acid titrant to lower the pH.

      5. Base titrant:

         The base titrant is used to neutralize low-pH material (e.g., 1 M HEPES, 1 M Tris base) in order to stop the inactivation reaction at the desired time prior to virus assay. It is good practice to prevent pH overshoots, that is, where the sample becomes strongly basic, by using a weaker or dilute base. The base titration is not intended to model the manufacturing process. An alternative neutralization procedure is dilution of the low-pH process pool using cell culture medium followed by addition of base titrant, if necessary.

      6. Cell culture medium or other negative control medium (optional):

         The spiked cell culture medium control can be used as an indication that the infectivity assay is performing as expected. Additionally, it may indicate whether the process pool was incorrectly neutralized or there is inherent instability of the spiking virus in the neutralized process pool from a non-pH-related interaction between the virus and process fluid.

7. Preparation for the Virus Inactivation Study

   1. Determine amount of acid titrant needed to reach low-pH target

      Given that virus preparations are formulated at neutral pH, the virus spike may increase the pH of the process pool, depending on the percent virus spike used and the properties of the pool. Thus, the amount of acid titrant required to titrate the process pool to the low-pH target in the presence of the virus spike must be determined in advance of the study. During the inactivation study, the pool will be titrated to a setpoint that, upon addition of the virus spike, results in the correct pH for the low-pH hold. The virus formulation buffer (without virus) may be used as a replacement when handling of virus is prohibited.

   2. Determine amount of base titrant needed to neutralize low-pH pool

      Prior to initiating the cytotoxicity and virus interference study (Section 7c), the amount of base titrant should be determined to return the low-pH process pool to a neutral pH suitable for cell culture; typically this is in the pH range of 6.5–8.0. In some situations, the product or antibody at high concentration may not be soluble at neutral pH, and dilution in cell culture medium is used for neutralization. As noted, an alternative neutralization procedure is dilution of the low-pH process pool using cell culture medium followed by addition of base titrant, if necessary.

   3. Cytotoxicity and virus interference testing

      The neutralized, low-pH process pool must be evaluated for potential impact on the virus assay. The pool can affect the assay by either being cytotoxic to the indicator cells or by interfering with virus infection of the cells. If diluted, the lowest dilution of the test article that does not result in cytotoxicity or interference should be used for the inactivation assessment study. Cytotoxicity is typically evaluated as a percentage of the cell culture affected (e.g., 25%, 50%, etc).

      Viral interference may be determined by adding a fixed amount of virus to the neutralized low-pH process pool and calculating the recovery versus a medium control spiked with the same fixed amount of virus. Viral interference can also be determined by using dilutions of the neutralized low-pH pool to titer the virus. Assay interference is determined by comparing the virus titer using the dilutions of the neutralized low-pH pool to the titer of the spiking virus control (titration assay positive control; refer to Section 8b). Due to the inherent variation of virus titration assays, significant effects, in the range of 0.5 log₁₀ or more, are required to conclusively indicate the presence of viral interference. Also, consider that multiple serial dilutions of a high titer virus stock may introduce variability in the titer calculation.

8. Virus Inactivation Study Procedure

   The low-pH inactivation study is expected to be performed in two independent runs according to the following procedure. Each step will be initialed and dated as required in the applicable process record. The virus will be spiked into the process pool, typically at 1% or 5%, but not more than 10% of pool volume. Controls will be used to monitor stability of the virus in the process conditions at neutral pH. See attachment 1 for a flow chart and summary of samples. It is recommended to monitor the pH at the beginning (optional) and at the end of the inactivation hold time with a pH probe to ensure that the correct target pH has been reached upon the addition of virus, and remains at that pH for the duration of the inactivation. A timeline of the inactivation procedure is provided in Figure 1.

   1. pH probe calibration

      Calibrate pH probe using pH standard buffers per the pH meter manufacturer's instructions. Best practice is to equilibrate the probe and buffers at the testing temperature prior to calibration.

   2. Control preparations for the virus inactivation study

      1. Spiking virus control (sample number 1 in Attachment 1):

         As appropriate, pool vials of spiking virus into a common vessel. Aliquot a portion of the pooled virus and label as spiking virus control. Place on ice until titration.

      2. Process pool control (sample numbers 3 and 9 in Attachment 1):

         Equilibrate the process pool at the treatment temperature. Record temperature.

         Add acid titrant to the process pool as required to reach target pH (Section 7a), then neutralize for the cell culture assay as outlined in Section 7b. Add spiking virus into the neutralized low-pH process pool and mix. Record time and temperature. Remove the T = 0 sample and place on ice until titration. This is sample number 3 in Attachment 1.

         Incubate the remaining spiked neutralized process pool until T = end time of treatment at the treatment temperature. Record time and temperature. At that time, place samples on ice until titration. This is sample number 9 in Attachment 1.

      3. Cell culture medium control, optional (sample numbers 2 and 8 in Attachment 1):

         Equilibrate cell culture medium at the treatment temperature. Record temperature.

         Add spiking virus into cell culture medium and mix. Record time. After spiking, remove the T = 0 sample and place on ice until titration. This is sample number 2 in Attachment 1.

         Incubate the remaining spiked cell culture medium until T = end time of treatment at the treatment temperature. Record time and temperature. At that time, remove sample and place samples on ice until titration. This is sample number 8 in Attachment 1.

   3. Inactivation treatment of the process pool (sample numbers 4, 5, 6, and 7 in Attachment 1)

      Note: The following is an example of an inactivation procedure; alternative approaches are acceptable.

      1. Equilibrate process pool and acid titrant at the appropriate temperature in water bath. Record temperature. Ensure there is sufficient pool material for all samples and sample pH measurements.

      2. If appropriate, adjust pH to target pH using acid titrant, taking into account the impact of the subsequent virus spike on the final pH. Measure pH. Record pH and sample volume or weight.

      3. Add spiking virus and mix. Measure pH and correct pH if needed to lower pH to target.

         Note: If the pH measurement of the spiked low-pH process pool is below the target pH, the study is invalid and should be re-started.

         Record pH, pool volume or weight, volume of spiking virus added and time; this is T = 0.

         Immediately remove samples, neutralize and label as T = 0 (sample number 4 in Attachment 1). Record pH, sample volume, and time.

         Neutralize per procedure (Section 7b).

         Place samples on ice until titration. This is sample number 4 in Attachment 1.

      4. Incubate virus-spiked process pool at target temperature. Record temperature.

      5. At T = intermediate time 1, 2, and T = end time:

         Remove sample. Record pH, sample volume, temperature, and time.

         Neutralize per procedure (Section 7b).

         Place samples on ice until titration. These are sample numbers 5, 6, and 7 in Attachment 1.

   4. Analysis

      The selected virus infectivity assay is used to determine the virus titer of samples and controls. The LRVs are calculated using virus titer of the spiked (neutralized) process pool control after treatment time, T = end (sample number 9 in Attachment 1) as the load or virus in. If this titer is significantly different from the virus titer of the spiked (neutralized) process pool control at T = 0 (sample number 3 in Attachment 1), this should be considered in the interpretation of the test results; it can invalidate the assessment. The virus titer at each timepoint (virus out samples) should be used to determine the inactivation kinetics and LRV. Typically, the lower LRV of the two independent low-pH runs is reported for the inactivation procedure LRV for the overall process viral clearance.

      The following is an example of an LRV calculation for a low-pH inactivation study without a statistical calculation for confidence interval. Because there is no change in volume of the low-pH inactivation step during manufacturing, there should not be a contribution to the LRV from a change in processing volume from virus in to virus out. The example uses a virus infectivity assay with results reported as focus forming units (FFU):

      1. Preparation of low-pH process pool:

         40 mL process pool + 4.0 mL acid titrant = 44 mL total low-pH process pool

      2. Load or virus in:

         Neutralized low-pH process pool spiked with virus at T = end time (sample number 9 in Attachment 1)

         Volume = 3 mL low-pH process pool + 0.6 mL base titrant + 0.3 mL virus

         = 3.9 mL total for titration

         Titer = 1 × 10⁶ FFU/mL

         Load or virus in = 1 × 10⁶ FFU/mL × 3.9 mL (total pool) ÷ 3.3 mL (low-pH process pool plus virus) x (Sample dilution, if performed, in this example = 1)

         = 1.2 × 10⁶ FFU/mL

      3. Virus out:

         Low-pH pool spiked with virus at T = intermediate time 1 (sample number 5 in Attachment 1)

         Volume of spiked pool = 30 mL low-pH process pool + 3 mL virus

         = 33 mL total (extra volume to allow for pH measurements)

         Remove 3 mL for titration and add 0.6 mL base titrant = 3.6 mL total for titration

         Titer = <1 x 10¹ FFU/mL

         Virus out = <1 × 10¹ FFU/mL × 3.6 mL ÷ 3 mL (to correct for dilution for adding base titrant) × (Sample dilution, if performed, in this example = 1)

         <1.2 × 10¹ FFU/mL

      4. LRV calculation:

         LRV = log₁₀ (virus in/virus out)

         = log₁₀ (1.2 × 10⁶ FFU/mL ÷ <1.2 × 10¹ FFU/mL)

         ≥ 5.0

   5. Acceptance criteria are as follows:

      1. There is no minimum acceptable viral clearance

      2. The virus spiking assessment is expected to be performed in two independent runs and should provide similar inactivation kinetics

      3. The medium control, if performed, and process pool control should show no more than 1. 0 log decrease from T = 0 to T = end time, unless justified

      4. The positive and negative control acceptance criteria for the virus titration assay must be valid

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Figure 1

Timeline of the low-pH inactivation procedure. The sample numbers in the timeline refer to the samples outlined in Attachment 1. Although four low-pH timepoints are presented in this example, multiple samples may be taken to demonstrate the inactivation kinetics and/or to establish the lower and upper limits for the low-pH inactivation procedure.