Vaccine Pre-clinical Toxicology Testing

Glossary

Adjuvant - an immune enhancer added to the active ingredient (i.e., antigen) of the vaccine to improve immune responses - e.g., aluminum hydroxide or phosphate, IFA, CpG motifs, cytokines. Currently, the only adjuvants in U.S. FDA licensed vaccines are aluminum-containing formulations.

Bioburden - this term is often used to indicate the "microbial limits" test promulgated in the U.S. Pharmacopeia [25 USP <61>]. This is a test to measure the number of micro-organisms present and to determine that none that are present are "objectionable." Generally parenteral products should be sterile, but if there is a terminal sterilization step, then they may contain controlled levels of bioburden upstream.

Bulk - the drug substance or the drug product which has not been filled into final containers for distribution. Final formulated bulk generally refers to drug product which is formulated and being stored or held prior to filling. Drug substance may be stored or held as "bulk" or "concentrated bulk" prior to formulation into drug product.

21 CFR - Title 21 of the Code of Federal Regulations gives FDA the authority to regulate biologics (like vaccines), drugs, devices, etc., and investigational new drugs (including investigational vaccines).

CMC - The Chemistry, Manufacturing, and Controls section of the IND is the section in which the sponsor describes the product: including, but not limited to, the cell bank(s) characterization, adventitious agent testing, manufacturing procedures for the drug substance, manufacturing procedures for the formulation and filling of the drug product, in-process controls, bulk and final container lot release controls and specifications, release test methods, product characterization, and any other relevant product information (e.g., excipients, raw materials, adjuvants, delivery devices). For guidance on the content and format of the CMC section of a vaccine IND, please refer to http://www.fda.gov/cber/gdlns/cmcvacc.pdf. While this guidance document is intended to provide applicants with information about what to submit in a Biologics License Application, the guidance may also be useful in preparing a vaccine IND. For further guidance on CMC section contents for a Phase 1 IND, please refer to http://www.fda.gov/cber/gdlns/qaind1.pdf. However, this latter document is not specific to vaccines. Finally, for guidance on the CMC and other aspects of an IND for DNA vaccines, please refer to http://www.fda.gov/cber/gdlns/plasmid.pdf.

COA - A certificate of analysis is the document which lists the tests, methods, specifications, and results of the in-process, bulk, and final lot release tests for each bulk and/or lot of drug, chemical, or vaccine manufactured. The COA should be accompanied by the raw data or data worksheets to constitute the lot release documents.

Drug Product - final formulated vaccine with all excipients and adjuvant (if any) at concentration or dosage to be used.

Drug Substance - unformulated or partially formulated active ingredient (i.e., antigen or immunogen), usually concentrated compared to final dosage to be used. There may be multiple drug substances in a final drug product, such as multiple strains (like poliovirus types 1, 2, and 3) or multiple different antigens (like tetanus and diphtheria toxoids). Often referred to as bulk or (monovalent) concentrate.

GLP - Good Laboratory Practices are like GMP for laboratory and pre-clinical (i.e., animal) studies and are defined in 21 CFR 58. These regulations are all about control and documentation. Most basic research laboratories do not naturally meet GLP standards. Highlights of the requirements include, but are not limited to the following:

• Requirement for a study director
• Requirement for a quality assurance unit
• Responsible for maintaining a Master Schedule Sheet of all studies conducted
• Responsible for inspecting the study during its conduct
• Determine no deviations
• Review final report for accuracy
• Maintain records
• Facilities designed to
• Permit segregation necessary to ensure study integrity including:
• Areas for preparation of test and control articles
• Routine and specialized analytical procedures
• Separation of animal species
• Animals in different studies
• Quarantine of new animals
• Equipment must be inspected, cleaned, and maintained, including calibration
• Written SOPs for these procedures
• Written records kept on these activities
• SOPs for routine procedures (listed examples in 21 CFR 58.81)
• Reagents appropriately labeled
• Animals healthy and specifically identified
• Test and control articles characterized for identity, strength (potency), purity, and composition
• Manufacturing of test article documented
• Stability determined before or during study according to written SOPs
• Uniformity of mixtures ensured, stability determined
• Study protocol prospectively written detailing specific study (as opposed to SOPs which apply generically)
• Results recorded and raw data documented
• Records (including raw data) maintained for specified time periods
• Final report written documenting procedures, results, statistical analyses

If this list seems extreme, you will understand why we might recommend that you contract GLP studies with an experienced GLP facility.

GMP - Good Manufacturing Practices or current GMP (cGMP) are defined by FDA in 21 CFR 210 and 211 and consist of regulations governing the procedures and practices under which manufacturing is controlled (SOPs, training, lot release, etc.) and quality of the manufacturing is assured (documentation, batch records, certificates of analysis, records keeping, etc.) GMPs are all about consistency, control, and documentation. GMPs "...assure that such drug¹³ meets the requirements of the [FD&C] act as to safety, and has the identity and strength [potency] and meets the quality and purity characteristics that it purports or is represented to possess" (21 CFR 210.1). GMPs ensure product reproducibility so the FDA and the public will know that the vaccine delivered in clinical practice is the same as that tested for licensure. Both the FDA (http://www.fda.gov/cber/gdlns/active.pdf) and the ICH (http://www.ich.org) have guidance documents on how to comply with cGMP. Highlights of the requirements that constitute GMP include, but are not limited to the following:

• requirement for a QC unit responsible for releasing or rejecting lots, procedures, specifications
• personnel should be trained (including in cGMP) and that training documented
• facilities should be suitably designed, cleaned, maintained
• design allows segregation and flow that prevents mix-ups and cross-contaminations
• floors, walls, ceilings must be smooth, hard, and easily cleanable
• HEPA-filtered air handling under positive pressure
• systems in place for cleaning, disinfecting, aseptic handling, including written procedures
• environmental controls
• freedom from vermin, including written procedures for use of suitable methods for this control
• equipment
• designed so surfaces contacting the drug are not reactive, additive, nor absorptive, nor alter the drug's specifications
• cleaned, maintained, and sanitized regularly, including written procedures for doing so and records (logs) kept
• calibrated and inspected, incl. written procedures and records kept (see the trend here!)
• computerized systems must be controlled so that records cannot be altered and must be validated for accuracy
• filters don't release fibers into the product
• containers and closures
• stored to prevent contamination
• clean, sterilized (if appropriate), and pyrogen-free
• not reactive, additive, nor adsorptive
• raw materials quarantined until tested and released or rejected, based on prospectively defined specifications
• segregation of components of different status (quarantined, released, rejected)
• rotation of stocks of components
• production and process controls
• written procedures (SOPs)
• deviations recorded and justified
• yield calculated and calculations verified
• equipment used per batch identified and documented
• in-process controls and tests done on representative samples
• time limits for steps established and deviations documented and justified
• environmental controls in place (microbiological contamination, dust, etc.)
• prospective SOPs for re-processing
• SOPs for labeling and packaging controls
• records kept for labels accepted or rejected
• controls to reconcile quantities
• segregation to prevent mix-ups and cross-contamination during labeling
• inspection to ensure accuracy
• expiration dating determined by stability testing and must appear on labels
• written stability plan, results determine expiration dating
• written SOPs for warehousing and distribution
• lab procedures, sampling plans, specifications
• written procedures
• deviations recorded and justified
• controls on
• components
• containers/closures
• labeling
• in-process testing
• sampling
• calibrating instruments
• batches must meet prospective specifications before being released
• test methods validated for accuracy, sensitivity, specificity, and reproducibility
• reserve samples must be retained for specific time-frames
• lab animal use is controlled and documented
• records maintained for specific time-frames, including raw data
• complaints, recalls, returns, investigations documented and reviewed according to written procedures
• Master production records to ensure uniformity batch to batch
• Batch records
• Prepared for each batch documenting
• Dates
• Equipment used
• Batches of components used
• Personnel
• Control results
• Yield
• Samples taken
• Deviations and investigations
• Records must be reviewed
• Lab records documenting methods, personnel, etc.
• Records maintained on
• deviations and reasons,
• standardization of reference standards,
• calibration of equipment
• stability
• Distribution records maintained

If this list seems extreme, you will understand why we might recommend that you contract GMP manufacturing with an experienced GMP facility.

GST - the general safety test. The methods of the GST are promulgated in 21 CFR 610.11. This final container test does not constitute nor substitute for toxicology studies or adventitious agent tests. It is a test for extraneous toxic contaminants which may have inadvertently been introduced during formulation, filling, or storage.

ICFs - Informed Consent Forms

ICH - International Conference on Harmonisation - a tripartite committee (EU, US, Japan) which has established harmonized guidances that the participating regulatory control authorities have agreed to follow. While the guidances do cover biotechnology products, they do not uniformly cover vaccines; however, OVRR staff do look to these guidances and follow them, if applicable. http://www.ich.org

IND - Investigational New Drug Application - an IND is required to be filed with the FDA for investigational products to be exempted from premarketing approval requirements needed for interstate commerce. As of the 1997 Food and Drug Modernization Act (FDAMA), there is a presumption of interstate commerce for virtually everything because almost nothing can be made completely intra-state. One gets one's culture media, flasks, pipettes, or other components used in the manufacture of a vaccine candidate through interstate commerce, therefore the investigational product is considered to be in interstate commerce and must be exempted by the filing of an IND in order to be studied in clinical trials. If not exempt, unlicensed products can be seized and legal penalties can be imposed on the investigators. For exact rules, reference 21 CFR 312 or contact FDA.

Investigators' Brochure (IB) - This document and its contents are specified in 21 CFR 312.23(a)(5). Essentially, all INDs require an IB except those conducted at a single site by a single investigator who is the sponsor of the IND (and who generally is also the manufacturer and responsible for the performance of all pre-clinical studies). The IB is an important tool for communicating information about the product, the pre-clinical studies, and the proposed clinical protocol to the clinical investigator(s).

Lot Release - Each lot of product must be controlled and only released for its intended use if it meets prospectively defined quality control criteria (specifications). Lots should be controlled at the levels of in-process tests, bulk(s), and final container. Final containers must be controlled for identity, purity, potency, sterility (parenteral products) or bioburden (non-parenterals), and the general safety test. Lot release documentation should include the COA and the raw data or data worksheets for all in-process, bulk, and final container testing.

MSDS - Material Safety Data Sheet - an OSHA requirement

NOAEL or NOEL - no observed adverse effects level or no observed effects level - highest dose at which no effect or adverse effect is seen in the animal studies - i.e., an "unquestionably" "safe" dose in that animal model. Generally FDA considers risk/benefit when reviewing safety issues. But, when no benefit has been shown (i.e., before any clinical studies have been performed), balancing risk and unproven benefit is difficult. Consequently, FDA may require starting studies at an unquestionably safe dose level and escalation from there. Often, for vaccines, the highest dose level tested turns out to be a NOAEL anyway (which is why this term is not usually used in describing vaccines), but in the event it's not, it's helpful to know what dosage is a NOAEL.

Pharmacology - for a vaccine, pharmacology essentially means immunogenicity. However, this term may also extend to actual drug pharmacology of an adjuvant, such as a cytokine. The pharmacology/toxicology section of an IND application should contain the results of all animal studies, including formal toxicology, biodistribution (if applicable), attenuation (if applicable), neurovirulence (if applicable), immunogenicity, other specialized product-specific safety assessments, and challenge/protection (if applicable). This section provides the justification for the clinical dose selection and the safety of the product to go into clinical trial(s).

Potency - Potency of a vaccine is a little bit like the strength of a drug, but more complex. It's not a factor proportional to the concentration of the active ingredient (antigen) but rather to the immunogenicity of the drug product (formulated antigen). So, just saying there's 300 mg of gp120 or 1 mg of DNA there in the vial isn't sufficient, the question is "is it immunogenic"? For FDA guidance on potency testing of vaccines, you may wish to read Habig, Veterinary Microbiology, 37:343-51:1993. It concludes that a potency test should be reasonably predicative of efficacy in humans, i.e., measure a parameter that correlates with efficacy. Another important aspect to remember is that it should be sufficiently quantitative, so that you can tell if your vaccine has lost 30%, 50%, or 70% potency, for example.

Pre-IND - One of the perks of PDUFA2 and PDUFA3 (the Prescription Drug User Fee Act Reenactments) is the assurance that FDA will hold a pre-IND meeting with you, if you meet your end of the bargain (i.e., by following the rules and by supplying them with sufficient information to evaluate your vaccine) and it is not too premature or there is not some other significant reason to deny it. The rules you need to follow (as well as the rules FDA must follow) can be obtained at this link: http://www.fda.gov/cber/genadmin/pdufago111297.pdf and scroll to section III on page 6 et seq. Section III.D. outlines what you, as a sponsor, must do to properly request a pre-IND meeting. The rest of Section III outlines what former DHHS Secretary Shalala committed FDA to do. A guidance document on these procedures can be obtained at http://www.fda.gov/cber/gdlns/mtpdufa.pdf. [Please note that some of the timelines have changed slightly with PDUFA3 - see http://www.fda.gov/oc/pdufa/PDUFAIIIGoals.html for the latest.] Pre-IND meetings are an excellent opportunity to find out what the FDA will require in order to permit a Phase 1 study to proceed with your specific product. If you do not hold the pre-IND meeting with them, if you do not provide them with sufficient information in the meeting materials to evaluate, and if you do not ask them specific questions in order to permit them to give you specific answers, you will miss this excellent opportunity and may waste considerable effort and resources (including time, if you are placed on clinical hold for not meeting their requirements).

SOP - Standard Operating Procedure - a written document detailing all steps of a procedure and which you actually follow without modification. Any deviations from the SOP should be thoroughly investigated and outcomes of the investigation documented (e.g., implementation of a new procedure).

Stability - For phase 1, stability data should be provided in the IND to support product quality through the length of the trial - what this means is that some (generally 3 months' worth) real-time stability data is provided in the Original Submission of the IND, along with a plan to be testing on a frequent basis through the conduct of the trial, so that if quality is lost, subjects can be spared from receipt of a substandard, impotent product. Aspects of stability that should be assessed are described in a guidance document (intended for data to support licensure, but useful to consider) which can be obtained at http://www.ich.org. Briefly, a stability plan or protocol should include assessments of critical parameters such as potency, purity, sterility or bioburden, pH, appearance, general safety, etc. Additionally, for phase 1, stability data under actual clinical use conditions should be provided to the IND to support the proposed clinical protocol practice. In other words, if you are going to reconstitute a lyophilized vaccine, thaw a frozen vaccine, or remove from refrigerated temperatures a refrigerated vaccine; draw up into a syringe or place into another vial or delivery device; and deliver to the clinic for use within 2 hours, you should provide FDA with data supporting that the quality and safety of the vaccine is not lost during this process.

Sterility - FDA expects a particular kind of sterility test to be performed and the methods are published in 21 CFR 610.12. Just any old sterility test will not do. Parenteral products generally are required to be sterile.

Validation - the term "validation" means very specific things to FDA and the ICH. For guidance on assay validation (including what the term and related terms mean), please try the following links: http://www.ich.org/ and http://www.ich.org/. Even if your specific PCR assay is not validated, FDA will expect that you have a handle on many of its performance characteristics (e.g., analytical "sensitivity" or limits of detection/quantitation - depending on whether it's quantitative or not) and that you have "verified" its suitability "under actual conditions of use," even if it is not (yet) fully validated per the regulations as explained in the above guidances. Assay parameter optimization is a separate process that precedes assay validation and/or may be iterative with it (i.e., assay validation may identify parameters for which the assay should have been optimized - after further optimization, the assay would need to be re-validated.)

Vehicle - the excipients or matrix in which the active ingredient (i.e., antigen) is prepared - e.g., PBS, sucrose-phosphate-glutamate (SPG), normal saline