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Pharmaceutica Analytica Acta

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ISSN : 2153-2435
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Review Article Open Access
A Global GLP Approach to Formulation Analysis Method Validation and Sample Analysis
Monica Whitmire1*, Rebecca Ross1, Joy Mwalimu1, Lynann Porter2 and Melissa Whitsel2
1MPI Research, 54943 North Main Street, Mattawan, MI 49071, USA
2MPI Research, 3058 Research Drive, State College, PA 16801, USA
Corresponding Author : Monica Whitmire, MS, BS, BS, MT (ASCP)
Study Director; MPI Research
54943 North Main Street
Mattawan, MI 49071, USA
Tel: 1.269.668.3336, extn. 3138
E-mail: monica.whitmire@mpiresearch.com
Received June 02, 2011; Accepted September 08, 2011; Published September 10, 2011
Citation: Whitmire M, Ross R, Mwalimu J, Porter L, Whitsel M (2011) A Global GLP Approach to Formulation Analysis Method Validation and Sample Analysis. Pharm Anal Acta S2:001. doi: 10.4172/2153-2435.S2-001
Copyright: © 2011 Whitmire M, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
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Nonclinical pharmacokinetic (PK) and toxicokinetic (TK) toxicology safety studies are performed using good laboratory practice (GLP) regulations to ensure the availability of safe medicines. International GLP regulations uniformly require that dose concentration, homogeneity/uniformity and stability be known prior to administration. However, the United States Food and Drug Administration (US FDA) and the Organisation for Economic Cooperation and Development (OECD) both confirmed that GLPs do not apply to validation of analytical methods used to determine the concentration of GLP test article in drug dosage forms. It is our assertion that the outcome of nonclinical toxicology safety studies is inherently dependent upon accurate and precise dose formulations. In this paper, we attempt to provide supporting evidence as to why formulation method validation and sample analysis for supporting nonclinical toxicology studies should be consistently conducted under the framework of GLP principles across the globe. GLP studies are planned, performed, monitored, recorded, reported and archived according to a protocol, study plan or standard operating procedure (SOP) which is authorized prior to performing the experiments. All applicable experimental parameters and associated acceptance criteria are pre-defined. The FDA asked for responses to the Advance Notice of Proposed Rulemaking for 21 CFR Part 58 GLPs for Nonclinical Laboratory Studies [Docket No. FDA–2010–N–0548] on December 21, 2010. Several comments were received stating that guidance regarding the validation of formulation analysis methods and subsequent use for supporting GLP toxicology study sample analysis is warranted at this time and should be conducted consistently. Adherence to GLP principles for method validation and sample analysis would inherently improve the quality of nonclinical safety studies. Furthermore, the recently published White Paper titled, “Nonclinical dose formulation analysis method validation and sample analysis” should be the keystone of this effort.

GLP; Regulatory; Nonclinical; Pharmacokinetic; Toxicokinetic; Formulation; Formulation Method Validation; Formulation Sample Analysis; Acceptance Criteria
AAPS: American Association of Pharmaceutical Scientists; ADME: Adsorption Distribution Metabolism Excretion; API: Active Pharmaceutical Ingredient; AS: Autosampler (Post- Processed); BFG: Bioanalytical Focus Group; BT: Bench Top (Pre- Processed); cGMP: Current Good Manufacturing Practice; CMC: Chemical Manufacturing and Control; CoA: Certificate of Analysis; CVG: Canadian Calibration and Validation Group; EMEA: European Medicines Agency; EPA: Environmental Protection Agency; FDA: United States Food and Drug Administration; FIFRA: Federal Insecticide, Fungicide and Rodenticide Act; F/T: Freeze/Thaw; GBC: Global Bioanalysis Consortium; GCC: Global Contract Research Organization Council; GLP: Good Laboratory Practice; HPLC: High Pressure Liquid Chromatography; ICH: International Conference on Harmonisation; JMHW: Japanese Ministry of Health and Welfare; JMAFF: Japanese Ministry of Agriculture, Forestry, and Fisheries; k’: Capacity Factor; LC-MS: Liquid Chromatography Mass Spectrometry; LLOQ: Lower Limit of Quantification; MHRA: Medicines and Healthcare Products Regulatory Agency; MSDS: Material Safety Data Sheet; N: Theoretical Plates; OECD: Organisation for Economic Co-operation and Development; PK: Pharmacokinetic; PR: Percent Recovery; QC: Quality Control; R2: Coefficient of Determination; RSD: Relative Standard Deviation; SOP: Standard Operating Procedure; SST: System Suitability Test; STD: Standard; T: Tailing Factor; TK: Toxicokinetic; tR: Retention Time; TSCA: Toxic Substance Control Act UV: Ultraviolet
New pharmaceutical products require significant resources from concept to final market introduction. Every step along the way is paved with trials and tribulations. A successful new drug application requires a thorough nonclinical (also known as “preclinical”) toxicology safety package (e.g., adsorption, distribution, metabolism, and excretion (ADME), pharmacokinetic (PK) and toxicokinetic (TK)), a successful clinical program (Phases I, II, III and IV), and a comprehensive chemistry, manufacturing and controls (CMC) package. All toxicology safety packages are highly dependent upon accurate and precise analytical methods for quantifying the drug dosage formulations which are administered to the hosts. If the vehicle is free of interference, the dose formulations are carefully prepared according to the batch records, and the aliquots are stored appropriately, then the dose formulations should be at the correct concentrations when administered to the hosts. However, attention to detail alone is not enough to ensure a successful formulation or regulatory compliance. For Good Laboratory Practice (GLP) regulated studies, the formulation doses must be verified for concentration, uniformity (i.e. homogeneity) and stability [1-13]. Explicit regulatory guidance does not currently exist for nonclinical dose formulation analysis method validation or sample analysis. In fact, there has been minimal global regulatory or industry emphasis regarding how to conduct GLP formulation dose analysis in support of nonclinical studies. The White Paper titled “Nonclinical dose formulation analysis method validation and sample analysis” [1] was intended to provide a consensus opinion regarding method validation and sample analysis for nonclinical GLP regulated studies. The only GLP regulatory guidances published are in regards to bioanalytical method validation [14-21]. There has been pronounced emphasis on global harmonization of bioanalytical studies through the efforts of international organizations such as the American Association of Pharmaceutical Scientists (AAPS) Bioanalytical Focus Group (BFG), the Canadian Calibration and Validation Group (CVG), the Global Bioanalysis Consortium (GBC) and the Global Contract Research Organization Council (GCC) [22-38]. Undeniably, regulatory guidance for the validation of analytical procedures has been developed for final active pharmaceutical ingredients (also known as API or drug substances) and final drug product formulations (also known as drug products) testing under current Good Manufacturing Practice (cGMP) regulations [39-44]. There has been progress made towards harmonizing cGMP API and drug product method validations [45,46]. There are also some GLP / cGMP regulation comparative documents available [47,48]. However, this lack of GLP regulatory guidance results in nonclinical GLP formulation analysis laboratories relying on regulations which are neither fit for purpose or phase appropriate for conducting formulation method validations and subsequent sample analysis (for example, Bioanalytical GLP or cGMP).
The FDA recently requested responses to the Advance Notice of Proposed Rulemaking for 21 CFR Part 58 GLPs for Nonclinical Laboratory Studies [5]. Over 160 comments were received from the pharmaceutical and bioanalytical industry, contract research organizations, and others. Many of the respondents stated that consistency regarding the validation of formulation analysis methods and subsequent use for supporting GLP toxicology study sample analysis is warranted at this time. The White Paper titled, “Nonclinical dose formulation analysis method validation and sample analysis” [1] has been reviewed by well over one thousand readers, and several global organizations have begun aligning their SOPs with the fundamental recommendations. We believe that the time is ripe for a global approach for conducting formulation method validation and sample analysis.
Nonclinical PK and TK formulation method validation studies typically include the parameters of dose concentration range, system suitability, method linearity, accuracy and precision, specificity / selectivity, carryover, sensitivity, pre-processed stability (bench top (BT) stability), post-processed stability (autosampler (AS) stability), short term stability and long term stability. The acceptance criteria for each parameter are defined in study protocols, study plans, or standard operating procedures (SOPs) in advance of study execution. The dose formulation samples are not true “unknowns”, since nonclinical toxicology studies are performed at a target (nominal) dose concentration range. This paper attempts to provide recommendations of best practices on a global harmonized basis with proposed acceptance criteria for nonclinical dose formulation method validation and sample analysis. Like the White Paper [1], this paper will focus on small molecules and the use of high pressure liquid chromatography with ultraviolet or mass spectrometry detectors (HPLC-UV, LC-MS).
Present status of GLPs
All across the globe, industry in general is looking for a better solution to the work that needs done in today’s global economy. Formulation analysis assessments supporting nonclinical toxicology studies must be efficient, effective and compliant. As each calendar year passes by, the world seems to get a little smaller and more of us find ourselves asking the same question; “I can’t find any guidance on that, what do I do?” In the realm of GLP we are constantly reminded to refer to the basics, the predicate rules.
All GLP texts, regardless of their origin, stress the same five important common themes: resources (organization, personnel, training, facilities and equipment), rules (protocols and written procedures), characterization (test items and test systems), contemporaneous documentation (raw data, final report and archives), and quality assurance. The information provided in Table 1 lists pertinent definitions for key GLP regulatory aspects [1-13]. All GLPs apply to the chemical procedures used to characterize the test and control substances/items/articles - the pertinent information must be documented; for example using a relevant certificate of analysis (CoA; identity, strength, purity, stability, composition and uniformity, as applicable) and material safety data sheet (MSDS; safe handling, storage and disposal) documents. All GLP texts contain similar language regarding the mixtures of test and control substances/items/ articles with carriers/excipients/vehicles. No matter what semantic you prefer or require, the purpose is still the same; testing by an appropriate analytical method shall be conducted using a qualified, calibrated and maintained system, and conducted to determine the following properties of the mixture: uniformity (homogeneity), concentration and stability. Concentration assessment is required periodically, but not on every prepared formulation. Homogeneity assessment is required on suspensions, but is not required on true solutions. GLP is just a framework system to guide you through these five very important, yet common themes. Compliance with GLP principles is intended to assure the quality and integrity of the nonclinical toxicology safety data.
Discussion and Conclusions
Method validation
The White Paper titled “Nonclinical dose formulation analysis method validation and sample analysis” [1] presented a mechanism by which analytical methods used for dose formulation analysis could be validated in order to comply with multiple regulatory agencies. In order to accomplish this, the type of validation required must first be determined. The type of validation is based on the phase of the compound and status of any existing methods. Table 2 lists the experiments needed for each type of method validation.
An early phase compound is one being used in a study lasting ≤ 3 months and may be limited in availability. A full validation is performed when a method is being instituted for the first time for regulated analysis and is the most comprehensive validation type. A partial validation is performed when there is a change made to a validated method that may include changes to dose range or vehicle composition. A transfer validation is performed when a validated method is being performed by a second laboratory as written.
Quality control (QC) samples are prepared at three levels (low, mid and high) spanning the anticipated dosing levels of the in-life nonclinical toxicology safety study. The QC samples are prepared by adding a known amount of compound to the vehicle that is intended to be used in the study. Preparation of the QC samples are typically performed in class A volumetric flasks or by weight for solid vehicles. A QC batch run will typically contain triplicate preparations of each of the three QC levels. The stability of these solutions should be assessed at the anticipated storage condition for the in-life study. This is usually refrigerated or frozen. Stability should be assessed for the anticipated storage time from preparation to analysis, usually 2 weeks to 1 month. Recommended acceptance criteria are presented in Table 3.
The resulting analytical method document typically contains the following sections: method limitations (range of method, vehicle used in validation), reagents, preparation of solutions, preparation of standards, sample preparation / dilution, system suitability requirements, stability of solutions, instrument conditions, calculations, and safety indications. The method should be drafted prior to validation and finalized following successful validation. The validated method is then used for sample analysis.
Sample analysis
Following a successful method validation, dose formulation samples used in an in-life study may be analyzed using the method as written. Triplicate (≥ n=3) samples are analyzed in order to confirm the concentration of compound in the vehicle which was used to dose the subjects in an in-life study. Furthermore, dose formulations are assessed for homogeneity by analyzing triplicate samples from the top, middle and bottom of the bulk formulation. The homogeneity data is analyzed per strata (≥ n=3) as well as across the batch (≥ n=9). Stability of the actual dose formulations used in an in-life study may also be assessed by storing samples at the storage conditions used in the study and analyzing them for content. Recommended sample analysis parameters and acceptance criteria are presented in Table 4.
The White Paper is widely accepted as an industry standard representation of formulation validation and sample analysis parameters / activities. The mechanism of method validation and sample analysis presented there complies with all regulatory agencies’ expectations. There is no reason that the White Paper cannot be applied globally as a mechanism to validate analytical methods for formulations analysis and perform formulation sample analysis.
Tomasz Grabowski; Emily Mainstone; Anastasia Osredkar
The authors have no relevant affiliation or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. No writing assistance was utilized in the production of this manuscript.



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