INTRODUCTION

Nonclinical studies intended to support applications for research or marketing permits are required to follow applicable federal regulations as specified in 21 CFR, Part 58 (Good Laboratory Practice (GLP) for Nonclinical Laboratory Studies) (1), the OECD “Principles of Good Laboratory Practice” (2), or Japanese MHLW no. 21 (3). The primary purpose of nonclinical studies is to establish safety margins that can then be extrapolated to clinical studies. Therefore, nonclinical dose formulation analysis (NCDFA) is required in all nonclinical regulated studies to verify the documented test article concentrations in formulations used to determine these safety margins (1–3). These analytical methods are used to assess the concentration of the test article in nonclinical formulations, formulation homogeneity, and formulation stability in support of regulated nonclinical studies (for example, safety, toxicokinetic, and pharmacokinetic studies). Recommended specifications according to the AAPS white paper for “Nonclinical dose formulation analysis method validation and sample analysis” are 100 ± 10 % recovery with ≤10 % relative standard deviation (RSD) for solutions, 100 ± 15 % recovery with ≤10 % RSD for suspensions, and 100 ± 20 % recovery with ≤20 % RSD for solids (4). Analytical samples that fall outside of this range are considered out of specification (OOS). When samples are OOS, an investigation should be conducted to verify if the result is OOS, to assess the root cause of the OOS, to determine the impact to the study, and to determine the path forward.

Presently, no regulatory authority has a guidance document for nonclinical dose formulation analysis out of specification (NCDFA-OOS) investigations. The U.S. Food and Drug Administration (FDA) has issued a guidance document for Good Manufacturing Practices (GMP) studies (U.S. Department of Health and Human Services, FDA, CDER, October 2006) on conducting OOS investigations (5). This document provides guidance on how to conduct an OOS investigation as it relates to GMP test results for pharmaceutical production, including the responsibility of the analyst and the laboratory supervisor, as well as the investigation of OOS test results, including review of production, additional laboratory testing, reporting testing results, and concluding the investigation. This GMP guidance is sometimes used as a basis for performing NCDFA-OOS investigations; however, it is not specific to nonclinical dose formulation sample test results. Its applicability is limited primarily to the analytical portion of the investigation and adopting it for NCDFA-OOS investigations, in the opinion of the authors, is not adequate. Nonclinical studies are typically short term (days to months) in nature where dose formulations are in early development stages and samples are limited. OOS investigations detailed in the GMP guidance would be time consuming, expensive, and would not necessarily provide any additional benefits since any impact of NCDFA-OOS results would be determined by the Study Director and Toxicologist.

Across the industry (contract research organizations, pharmaceutical companies, and biotechnology companies), dose formulation OOS investigations for nonclinical studies vary in regard to content, assessment, and impact statements. The amount of resources required for the investigation may vary depending upon the individual study and the study type. The opportunity exists to improve the consistency of these investigations by defining expectations of the NCDFA-OOS investigation and providing guidance in the areas of root cause assessment, impact to the study statements, and acceptable paths forward.

GOALS AND OBJECTIVES

A standard process for conducting an NCDFA-OOS investigation for nonclinical studies is proposed. This includes the following steps: review of data and methods (analytical and formulation), assessment of OOS root cause, assessment of the impact to the study, and determination of the path forward. The path forward may include reanalysis, re-testing, re-sampling, and/or re-preparation of the formulation (6).

ROLES AND RESPONSIBILITIES

The roles and responsibilities for conducting the OOS investigation need to be clearly defined. This may vary among companies in terms of specific responsibilities as defined by Standard Operating Procedures (SOPs); however, the process should be similar among companies. This should include the analytical lab, the formulation lab, quality assurance, and the Study Director (7). The analytical and formulation investigations should be conducted by the respective laboratories, and the results reviewed by management. Both Quality Assurance and the Study Director need to be involved in the investigation. The analytical and formulation laboratories are responsible for keeping the Study Director informed throughout the entire investigation. The Study Director is responsible for approving the conclusions of the investigation.

INVESTIGATION PROCEDURE

The primary objective of the investigation is to assess whether there is a specific identifiable reason for the OOS result (i.e., a root cause). The investigational procedure is divided into three sections: the “General Section” which covers items common to both analytical and dose formulation preparation, and the “Formulation Analysis” and “Dose Formulation Section”. The latter two sections include topics designed to address the unique aspects of analytical and formulation dose preparation investigations. The investigation for these sections may be conducted in parallel or sequentially. It is important to note that although these sections attempt to include the most probable areas for investigation/review, it is not possible to have an all-inclusive check list for every OOS investigational scenario.

If errors are found in any of the areas discussed, one must first determine whether these errors are indeed the root cause of the OOS or simply a deviation or contributing factor. It is necessary to evaluate the error in the context of the data. If, for example, a weight-check verification was not performed for a specific day, this would be a deviation; however, it may not be the root cause of the OOS result. As is the case with all laboratory investigations, sound science must be the top priority.

General Review

The “General Review” section of this White Paper deals with areas of the investigation that are common to both the analytical and formulation preparation laboratories. These points should be common to all GLP laboratories. Table I summarizes the OOS investigational components that have overlap between the two major branches of the investigation.

Table I General Review
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While these points are identified as overlaps between the “Analytical” and “Dose Formulations” sections, it is important to remember that other areas of overlap might also exist and should be considered, regardless of the stage of the investigation.

Formulation Analysis

When a dose formulation sample analysis result does not meet the stated acceptance criteria, the typical first reaction is to assume an error was made by the analyzing laboratory. This is not necessarily a correct assumption; however, review of the analytical procedure is a logical starting point of the investigation.

It is common for the analytical laboratory to be a separate test site from the test facility/study director, whether it is the pharmaceutical/biotech company's in-house laboratory or a third-party CRO. Therefore, it is essential that the analytical laboratory have its own SOPs for conducting and documenting the investigation as well as for communicating the investigation results to the Study Director.

There are various areas where errors may occur during the sample analysis procedure and for ease of discussion, investigation; they have been divided into three main categories: analytical data, analytical equipment, and supporting information. If the root cause is identified, no further investigation is required.

It should be noted that the NCDFA OOS investigation procedures outlined within are not required for instances where System Suitability Test (SST) samples fail to meet the acceptance criteria if the standards and study samples have not yet been analyzed, and should be handled according to internal SOPs.

Analytical Data

The investigation should include a thorough review of all analytical results, sample preparation, and analysis items associated with the OOS sample result. This review should attempt to determine if the dose formulation sample analysis was performed correctly by answering the following questions: Was the method followed and performed as written? Was the sample prepared as per the method? Did the instrument(s) perform as expected? Were there any calculation or dilution errors? Were any issues noted for the dose formulation sample itself? Items to check during the analytical data review are listed in Table II.

Table II Analytical Data Review
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Analytical Equipment

The analytical equipment review should evaluate whether the equipment used was operating properly and in accordance with the method and any associated SOPs. Dose formulation analysis is commonly performed using high-pressure liquid chromatography with ultraviolet (HPLC-UV) for separation and detection. The analytical equipment review has been written for HPLC-UV instrument methods, but the review procedure can be adapted to other methodologies, such as liquid chromatography–mass spectrometry (LC–MS), LC–MS/MS, gas chromatography (GC)-FID, GC-MS, and GC-MS/MS. Items to check during the analytical equipment review are listed in Table III.

Table III Analytical Equipment Review
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Analytical Supporting Information

The analytical supporting information review should include verification of all associated documentation that accompanies dose formulation analysis. This includes the review of protocols, SOPs, test methods, method validations, certificate of analysis (CoA), material safety data sheets, training records, and any other associated documents that may have an impact. Items to check during the analytical supporting information review are listed in Table IV.

Table IV Analytical Supporting Information Review
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If no assignable cause of the OOS result is identified after performing the analytical section investigation, the investigation should be expanded to the dose formulation preparation.

Dose Formulation Section

The “Dose Formulation Section” of the OOS investigation may be conducted in parallel to or sequentially after the analytical investigation. In many organizations, the dose formulation group may be notified while the analytical OOS investigation is being conducted, and the dose formulation investigation may be started. This portion of the OOS investigation should be conducted and approved by the dose formulation group. It is essential that the formulation laboratory have its own procedures or SOPs for conducting and documenting the investigation as well as communicating the investigation results to the Study Director.

In the dose formulation section of the OOS investigation for GLP studies, the two main sections are the “Formulation Equipment Review” and the “Formulation Preparation Review”. In the “Formulation Equipment Review” section, the equipment used in the preparation is reviewed and verified. In the “Formulation Preparation Review” section, supporting data/information and the formulation preparation procedure are reviewed and verified. If the root cause is identified, no further investigation is required.

Formulation Equipment Review

The formulation equipment review should systematically review the equipment used and assess whether there is a root cause for the OOS result. This review should attempt to determine if the formulation equipment used was appropriate by answering the following questions: Was all equipment used calibrated and correctly maintained? Were equipment verifications performed? Was the appropriate equipment used for the specific purpose? Suggested items to check during the formulation equipment review which is not all-inclusive are included in Table V.

Table V Formulation Equipment Review
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Formulation Preparation Review

This section systematically reviews the supporting data/information and the formulation preparation process to assess if there is a root cause for the OOS result. Questions asked here include: Were all study personnel appropriately trained, as documented in their training records? Was the correct factor applied to account for purity/activity as defined on the CoA or the study protocol? Did the grade of all materials used, including the active pharmaceutical ingredient (API), match that used for development and exploratory studies? Was the formulation in question prepared and stored as outlined in the protocol? Did the preparation instructions match the protocol and preliminary development methodology?

During the course of the investigation in this section, the cause may not be identified; however, it may be recognized that it might be necessary to further evaluate the formulation. This might be due to different lots of API or vehicle components or different formulation scale. In many instances, development work may be necessary during the nonclinical study to optimize the formulation preparation instructions. Suggested topics for review of the formulation preparation section of an OOS investigation are included in Table VI and are not an all-inclusive list.

Table VI Formulations Preparation Review
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The grade of the materials used must be evaluated to determine their appropriateness. The API lots used for formulation development and exploratory studies may be different lots and could have differences in particle size, morphology, and impurity levels between the lots. Some of these changes may influence suspendability and kinetic dissolution of the formulation. Therefore, even though the API may be the same chemically, there may be physical differences between lots, which could affect the formulation preparation. The excipients and vehicles used for the formulation preparation must also be evaluated to determine if the correct grade and viscosity (if applicable) for the study have been used. The expiry and storage conditions of the excipients should also be explored, as these might also contribute to an OOS result. If the wrong grade of excipient or vehicle has been used, they should be assessed to determine if this is the root cause of the OOS result. The recommended grade may not have been used; however, this may not be the root cause of the OOS result.

Formulations developed prior to nonclinical studies and/or for formulation stability studies are typically developed on a very small scale. The quantity of formulation prepared for formulation development purposes is “fit-for-purpose” and may not mimic the scale at which formulations are prepared for nonclinical studies. An assessment must be made to determine if the differences in scale (milliliter to liter ) may influence whether the formulations are within specification. Route of administration and physical state of the specific formulation must be taken into account. If there was a change in scale, it is important that the investigator evaluate whether the change in scale may have caused the OOS results. If a batch size has been changed by greater than 20 %, additional homogeneity analysis should be performed.

Compare the actual formulation preparation instructions to the protocol formulation instructions, and verify that the instructions match the proposed formulation instructions. The proposed formulation instructions may not match the actual formulation instructions. For example, a change in scale may cause the preparation conditions to require a change. Review the formulation records in the protocol, and assess if the formulations were prepared according to protocol instructions. Then compare these instructions to the formulation development work. This assessment will determine if the changes made to the procedure produced an equivalent formulation. Be sure to check not only that all reagents were added in the correct ratio, but also that they were added in the same order as originally planned. Variation in either of these could be a major contributor to the root cause.

Verify the procedure for sampling of the formulation for testing purposes. If data are available, verify the density of the sample taken against the density of the bulk formulation or the anticipated density of the sample. Make an assessment of sampling technique, for example taking an aliquot of the formulation to determine the technique, could influence the OOS result. If trituration is used with a mortar and pestle, the size of the mortar and pestle and the trituration time may affect content uniformity and particle size of the suspension.

If no assignable cause is identified, it is necessary to evaluate the data in context. Determine whether the OOS result is systematic or random. Are all of the values higher or lower than expected? The investigator must assess if it is an error with the formulation preparation method, the scale of the formulation, or the method for taking the analytical sample.

In addition to evaluating the data in context, it is sometimes necessary to observe the preparation of the formulation in order to assess the root cause of the OOS result. This allows the investigator to visually assess if there may be a potential cause for the OOS result.

Summary of Investigation

After the investigation is conducted, an out of specification report should be issued. It should include a summary of the analytical and/or the formulation investigations. The investigation summary should include an assessment of the root cause, the study impact, the path forward, and signatures from the appropriate parties. The root cause must be evaluated in both the analytical and formulation sections of the investigation summary. Although it would be expected to be an unusual occurrence, if after a thorough investigation a root cause is not identified, it should be noted that a root cause could not be determined and the impact on the study would be assessed by the study director. The study director will consult others as needed. When a root cause is identified, it must be taken into account when determining the path forward. A CAPA plan should be generated, when appropriate. If separate investigations were conducted for the analytical and formulation sections, reports should be issued by their respective groups. Reports will include a signed document for each of the individual investigations.

Within the investigation summary, study impact is assessed by the study director. The study director will rely upon the analytical data provided and utilize the expertise of the analytical and formulation investigation groups to determine what, if any, impact the OOS has on the study as a whole. The study director may try to correlate the dose formulation data to available pharmacokinetic data to determine if there were any changes to the expected exposure during the study.

As part of the investigation, the path forward must be identified. Possible paths include, but are not limited to, analyzing the sample duplicates (back-ups), analyzing additional samples (re-sampling), generating new dose formulations (re-preparation), reanalyzing the initial sample, and adjusting the reported dose level. The Study Director must be informed of and involved in the decisions made throughout this process. SOPs will typically determine how the process proceeds. Once an OOS investigation is concluded, the results of the OOS investigation may determine if new or additional investigative or study work will need to be completed.

CONCLUSIONS

This document provides an outline for performing NCDFA-OOS investigations of nonclinical studies in an attempt to overcome the current lack of regulatory guidance. A guidance document should be created to include review of data and methods (analytical and formulations), assessment of OOS root cause, assessment of the impact to the study, and the path forward (actions identified). This would benefit organizations that conduct regulated nonclinical studies.