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Recommendation and analysis of shedding studies by the FDA

Shedding assays require optimized extraction protocols tailored for each sample type and an accurate qPCR/dPCR assay with the necessary sensitivity and specificity. This ensures that shed products are detected and the risk of transmission to a third party is mitigated.

In 2015, the FDA published a non-binding recommendation on how and when to perform shedding studies during the preclinical and clinical development of VBGT products (virus or bacteria-based gene therapy products), oncolytic viruses, and bacteria. The document is titled “Design and Analysis of Shedding Studies for Virus or Bacteria-Based Gene Therapy and Oncolytic Products.” It uses the EMA’s 2009 scientific guideline, “ICH Considerations: General Principles to Address Virus and Vector Shedding,” as a reference. This is a summary of the text, including our notes as a provider of shedding services.

Shedding refers to the release of gene therapy products or oncolytic products from a patient through excreta (feces), secreta (urine, saliva, nasopharyngeal fluids, etc.), or through the skin (via pustules, sores, or wounds). A shedding study aims to determine whether the product is shed and whether the shed product is infectious. Unlike a biodistribution study, which evaluates how the product spreads within the body, a shedding study assesses how it is released from the patient’s body to monitor the potential transmission to untreated individuals.

Shedding data to enhance clinical safety

Depending on the shedding profile, information on shedding may be appropriate in the Investigator Brochure and the Informed Consent for Investigational New Drug (IND) studies. Shedding data may also be included in the package insert for licensed products. The shedding profile and transmission potential allow patients and physicians to take measures to prevent infection of untreated individuals.

Scope of FDA’s shedding recommendations

Virus or Bacteria-Based Gene Therapy Products (VBGT) and oncolytic products are derived from infectious bacteria and viruses. Although these drugs are less infectious or virulent than their parent strains, they may be shed and pose safety concerns for untreated individuals. Therefore, shedding studies are recommended in the patient population to understand and mitigate potential risks. Shedding events may also pose environmental risks, although this is discussed elsewhere.

Infection in third parties could occur if the product is shed intact but not when shedding occurs as viral or bacterial degradation products, such as nucleic acid fragments.

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Advanced sensitivity in PCR assays for shedding studies

At TATAA Biocenter, we design, optimize, qualify, validate, and run shedding studies in line with COU or regulatory requirements for preclinical and clinical studies. While PCR does not determine the infectious capability of a shedding product, it efficiently indicates whether more comprehensive growth-based assays are necessary to evaluate the infectious potential. A consistent increase in PCR signal over successive time points may suggest viral or bacterial replication.

Preclinical shedding studies

Collecting shedding data is an endpoint that can be integrated into other studies, such as safety and biodistribution assessments. The FDA recommends early engagement with the pharmacology/toxicology team during development to assess the need for collecting preclinical shedding data. While preclinical shedding studies are of value, they cannot replace clinical shedding studies, as the virulence and potential to infect may differ when using an animal model, particularly if the gene product is derived from a human-specific strain. Differences in immune status among animal models or species may also influence the shedding profile. Nonetheless, preclinical data help design measures to prevent transmission. Therefore, data gathered during preclinical studies should provide a clear and comprehensive understanding of the shedding profile to inform subsequent stages of development.

Clinical shedding studies

The clinical shedding study is typically integrated in the safety or efficacy trial. The collection of shedding data for replication-competent products is recommended to begin in Phase I trials due to their higher potential to release infectious viruses or bacteria. This collection should continue through Phases II and III to further characterize the shedding pattern.

For replication-incompetent or replication-deficient products, shedding data collection is advised to start in Phase II once a dosing regimen has been established.

The shedding profile may be influenced by several factors, including the dose, route, and frequency of product administration, additional treatments or medications that participants may be concurrently taking, any new treatments or procedures introduced before administration, or changes in the drug indication. Therefore, the shedding profile that supports the pivotal trial should closely mirror the conditions of the pivotal study regarding these parameters.

Replication potency and latency

Replication-competent viruses, which can infect a cell and produce new viruses, differ significantly in duration, presence, and shedding properties from replication-incompetent viruses, which can infect a cell but cannot produce new viruses. This principle also applies to dividing bacteria.
The stability of attenuation affects shedding, as a reversion to a replication-competent state is likely to increase shedding.
Viruses entering a latent phase can exhibit unpredictable behavior in the body, including their shedding profile.
For replication-incompetent or deficient products, such as most Adeno-associated viruses (AAVs), adenoviruses (Ads), and herpes simplex viruses (HSVs), quantitative PCR (qPCR) may be adequate due to their lower extent of shedding. However, the assay’s sensitivity must be exceptionally high to detect minimal quantities effectively.

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We recommend digital PCR (dPCR) for shedding assays.

While FDA mentions quantitative PCR (qPCR) as the standard method for shedding studies; dPCR offers several advantages. It is less sensitive to PCR inhibition, requires less dilution, and does not need a standard curve for absolute quantification. The standard curve can induce biases in the qPCR measurements.

Immunological effects

If a strong immune response is initiated, the product may be cleared more rapidly, potentially shortening the shedding period. Repeated administrations can also reduce the duration of shedding due to immune priming from initial doses. Conversely, immunosuppressed patients may experience persistent infections and shed the product for extended periods.

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Immunoprofiling

We use Olink’s immunological panels, for example Olink Target 48 Cytokine panels to monitor innate immune response activation through multiplex measurements of 42 key cytokines using only microliters of plasma and blood. The panel is available for mouse and human samples and is generally compatible with non-human primates (NHPs).

Shedding study design

The design of shedding studies begins with selecting the appropriate clinical samples, the collection frequency, and the duration of the monitoring period. The tropism of the therapeutic product dictates which samples should be collected for the assay. Furthermore, the route of administration determines the specific types of samples that should be collected. For example, the FDA recommends collecting skin swab samples from the injection site for intradermal routes and nasopharyngeal washes when an oncolytic virus is administered via inhalation or intranasally, respectively.
Sampling should start immediately after administration, with frequent collections during the initial weeks. It should continue until three consecutive data points are recorded below the assay’s limit of detection (LOD). If shedding levels never reach the LOD, they should at least stabilize, showing no significant fluctuations across three consecutive data points.

Assay recommendations

The assay should be capable of detecting the therapeutic product in clinical samples, whether it involves identifying nucleic acids or confirming the presence of infectious viral particles or dividing bacteria.

“Shedding assay(s) should be demonstrated to be specific, sensitive, reproducible and accurate,” FDA, 2015

At least one of the assays used should be quantitative, providing data in terms of the number of genome copies or infectious units. Quantitative PCR technologies are preferred for these measurements due to their ease of performance and standardization, high-throughput capabilities, rapid turnaround times, and exceptional sensitivity.

Assay validation

The assay should be capable of detecting the therapeutic product in clinical samples, whether it involves identifying nucleic acids or confirming the presence of infectious viral particles or dividing bacteria.

“Shedding assay(s) should be demonstrated to be specific, sensitive, reproducible and accurate,” FDA, 2015

At least one of the assays used should be quantitative, providing data in terms of the number of genome copies or infectious units. Quantitative PCR technologies are preferred for these measurements due to their ease of performance and standardization, high-throughput capabilities, rapid turnaround times, and exceptional sensitivity.

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A validated assay is an asset

PCR is easily performed and standardized for high-throughput sample analysis if the assay is properly validated. We validate the assay for COU with recovery from the matrix that meets acceptable criteria and with a sensitivity that provides acceptable LOD (Limit of Detection) and LOQ (Limit of Quantification). The LOD is determined using spiked blank samples, ensuring that at least 19 out of 20 samples detect the test item (= 95%).
The LOQ is defined by the concentration where the coefficient of variability is 35% or less.

The FDA does not require shedding assays to be fully validated; however, they must be qualified to meet minimal performance standards. Critical performance criteria for the assay include:

  • Reproducibility: The assay should be tested multiple times to ensure consistent results.
  • Specificity: Understanding the assay’s specificity is crucial to minimize the risk of false-positive or false-negative results.
  • Sensitivity: The assay’s sensitivity should be clearly defined, indicated by the limit of detection (LOD) and/or the limit of quantification (LOQ).
  • Selectivity: The assay must be selective enough to avoid detecting natural flora or environmentally circulating bacterial strains.
  • Matrix effects: The assay should be evaluated for matrix inhibition effects on the PCR process.
  • Reagent quality: The specificity and quality of the reagents should be controlled to ensure they are free from contaminants suitable for clinical shedding assays.
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Qualification vs. validation

The difference between qualification and validation lies in the number of times different parameters are tested. Both processes require that the assay performs consistently across different operators, instruments, days, reagent lots, and test item stocks. Validation is conducted on blank samples from untreated patients. Selectivity is often achieved using primers in non-natural regions to avoid detecting endogenous adenoviruses tested in untreated individuals representing the general population.

Matrix effects are tested by comparing equally spiked blank matrices and buffer, where PCR efficiency in the buffer is expected to be 100%, without inhibition. Reagent quality is constantly tested with non-template controls (NTC) to detect contamination in any reagent.

Sample handling, extraction and analysis

Shedding matrices are rich in organic matter, which can affect PCR performance. They also contain enzymes that degrade nucleic acids, potentially leading to an underestimation of shedding levels. Therefore, sampling, handling, shipping, and extraction processes should be evaluated for degradation. This can be done by spiking mock/donor samples soon after collection to determine recovery percentages and assess PCR performance. Collection, storage, shipping, extraction, and analysis should be conducted using the analyzed method. The sample can be diluted to overcome inhibition, leading to template dilution and requiring an even more sensitive PCR assay.

A positive shedding result

The shedding of a full-length genome suggests the presence of potentially infectious viruses or bacteria. For viral particles resistant to nucleases, PCR can be used to quantify the number of virus particles after nuclease treatment. Amplifying the complete viral genome using long PCR helps determine whether the genome is intact or fragmented. Regulatory agencies may consider even a fragment of the viral genome indicative of an infectious sample.
Infectivity is then compared to what is necessary to infect a third party. For example, the infective dose of adenovirus is reported to be greater than 150 PFU when administered intranasally, and it may be lower when aerosolized. This dose varies depending on the product, route of transmission, and other factors.

The shedding report

The FDA has provided a detailed list of what to include in the report; this is a summary:

Raw data: Present all the collected raw data.

Shedding analysis:

  • Report the percentage of patients shedding relative to the total number of patients, categorized by sample type, dosage, and regimen (number of doses) studied.
  • Document the duration of shedding, specifying the first and last day and the peak period for each sample type.
  • Identify the time point at which shedding ceases in most patients.
  • State which sample types and time points consistently showed shedding and which were consistently negative.

The assay description:

Sample preparation and nucleic acid extraction:

  • Detail the test sample preparation or nucleic acid extraction procedures, including any dilution factors and the specific amount of nucleic acid extracted per sample.

Quantitative PCR specifications:

  • For qPCR assays, specify the sample volume, amount of nucleic acid per reaction, number of PCR cycles, primers used, and the size of the DNA segment amplified.

Assay qualifications and sensitivity:

  • Provide a detailed description of the qualification studies, including standards and spike controls.
  • List the controls used, number of replicates, assay variability, and sensitivity, including the limit of detection (LOD) and the limit of quantification (LOQ).

Quantification of shed product:

  • The quantity of the shed product should be reported in terms of volume/mass (e.g., 10 PFU of virus per mL of urine, 10 CFU of bacteria per mL of urine, or 10 genome copies per microgram of stool), taking into account the stability of the product.

Shedding profile summary:

  • The report should also include a summary of the shedding profile for patients treated for a specific indication.

The FDA recommends against pooling shedding data from multiple trials for different indications to maintain clarity and specificity in the data presented.

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Reporting

We thoroughly describe how the extraction and PCR design are performed and validated. The Minimum Required Dilution (MDR) is the minimum dilution needed to ensure that the PCR is not inhibited. If the assay is validated for a specific reaction volume and template amount, increasing the template volume to enhance sensitivity requires a new validation to ensure consistent PCR performance, for example.

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