Phil Borman, DSc, has served in multiple quality-related roles at GlaxoSmithKline; as of April 2021, he is senior fellow and director of product quality.

Phil Borman

For a robustly developed analytical method, a well-chosen replication strategy can effectively reduce the uncertainty. A statistically sound approach involves replicating the factors associated with large amounts of variation followed by an appropriate analysis. The amount of reduction in uncertainty needed is informed by the precision requirements of the analytical target profile (ATP), ensuring that risks of making inappropriate quality decisions are minimized.

In this article, the authors describe two case studies: the use of a high-performance liquid chromatography separation method for a small-molecule API and a bioassay for a biopharmaceutical or vaccine.

Phil Borman, DSc, is senior fellow and director of product quality, GlaxoSmithKline; Timothy Schofield is owner and consultant, CMC Sciences, LLC; and David Lansky, PhD, is president, Precision Bioassay, Inc.


Vol. 45, No. 4
April 2021
Pages: 46–56

When referring to this article, please cite it as P. Borman, T. Schofield, and D. Lansky, “Reducing Uncertainty of an Analytical Method through Efficient Use of Replication,” 

Articles

A well-chosen replication strategy can effectively reduce the uncertainty for an analytical method, enabling appropriate decisions regarding pharmaceutical quality. 

Reducing Uncertainty of an Analytical Method through Efficient Use of Replication

Validation of analytical procedures requires study of intermediate precision for within-laboratories variations; however, there is no global guidance on how to develop such designs. By applying science- and risk-based principles-aligned with quality by design (QbD)-to analytical procedures, studies can be designed to ensure the factors selected present the highest risk of impacting the performance of the analytical procedure. In addition, the number of independent analytical runs to include in these designs should be linked to the overall risk/complexity associated with the analytical procedure.

	Useful pre-QbD guidance (provided by the Japanese National Institute of Health Sciences) proposed the use of a generic design which utilizes six independent analytical runs. Examples are provided which instead link the design structure and the number of independent analytical runs to the risk.

, September 2019
	September 2019
	Pages: 12–22

	When referring to this article, please cite it as P.J. Borman, et al., “Risk-Based Intermediate Precision Studies for Analytical Procedure Validation," 

Pharmaceutical Technology Regulatory Sourcebook

Validation of analytical procedures require assessment of the impact of variations within laboratories; however, guidance to study intermediate precision has been lacking. Science and risk-based principles should be used in the design of intermediate precision studies. 

Risk-Based Intermediate Precision Studies for Analytical Procedure Validation

In 2010, the European Federation of Pharmaceutical Industries and Associations (EFPIA) and the Pharmaceutical Research and Manufacturers of America (PhRMA) groups focusing on analytical quality by design (QbD) published a joint paper to stimulate industry discussion and debate around the implications and opportunities of applying QbD principles to analytical measurements (1). This topic is now commonly referred to as an ‘enhanced approach for development and utilization of analytical procedures’. In this article, the terms ‘analytical procedure’ and ‘analytical method’ are used interchangeably. Since this publication, industry, regulators, and pharmacopeias have debated the concepts widely and, as with any new paradigm, the concepts have evolved considerably. Additionally, new regulatory concepts have been developed to support pharmaceutical product lifecycle management.  

	While the technical benefits of applying an enhanced approach to the lifecycle of an analytical procedure are clear, it can be helpful to describe how to apply the concepts and tools to show how these benefits can be realized. The purpose of this article is to propose definitions, exemplify the use of individual elements of this enhanced analytical lifecycle concept, and to identify areas where they could help to support emerging regulatory concepts and/or guidance.

	The lifecycle of an analytical procedure is generally understood to encompass all activities from development through validation, transfer, operational execution, and change control until final discontinuation. Application of the enhanced approach for the development and use of analytical procedures within the analytical lifecycle management concept aligns with one of the key quality risk management principles outlined in International Council for Harmonization (ICH) Q9: “The evaluation of the risk to quality should be based on scientific knowledge and ultimately link to the protection of the patient” (2). 

	The enhanced approach for analytical procedure lifecycle management focuses development effort on understanding sources of variability and controlling parameters that truly affect the output from the analytical procedure (i.e., the reportable result). This will result in more robust and rugged analytical procedures that are controlled within pre-determined operational parameter range(s) and/or region(s) so that they consistently deliver the output within predefined target performance criteria.

	The enhanced approach uses science and risk-based approaches that build on the concepts and tools described in ICH Q8 (3), Q9, Q10 (4), and Q11 (5), and certain associated process validation guidelines (6). It then applies these approaches to gain enhanced understanding of the analytical procedure through its lifecycle (see 

for an overview of the enhanced approach).

	The analytical controls for a pharmaceutical product comprises specifications-tests, references to procedures, and acceptance criteria-as described in ICH Q6A and B (7,8). Acceptance criteria are usually linked to defined quality attributes. In the enhanced approach, the measurement requirements for each quality attribute are defined in an analytical target profile (ATP), which can be used as a tool to aid analytical procedure development, qualification, verification, and continued improvement.

	The ATP for a measurement performs a similar role to the quality target product profile (QTPP) defined in ICH Q8 for a pharmaceutical product. Compendial and regulatory requirements, or consensus industry guidance, that include acceptance limits or ranges for specific quality attributes will aid understanding of accuracy and precision requirements and can therefore contribute to building the ATP (9). 

	Once defined, the ATP can be used as follows:

		To direct the selection of an appropriate analytical technique. 

		To support risk assessment and rigorous systematic evaluation of procedure variables. The ATP is used to develop a full understanding of how input parameters affect the reportable result leading to development of an analytical procedure.

		To serve as the focal point for continuous improvement and change control of the analytical procedure within the analytical lifecycle management concept.

	Enhanced understanding enables the definition of conditions (parameter set points and/or ranges) that provide a high degree of confidence that the procedure will consistently generate results that meet the requirements of the ATP. If procedure parameter ranges are determined and evaluated, these are referred to as a method operable design region (MODR). 

	The MODR is analogous to the design space concept applied to products and processes introduced in ICH Q8 and has been described (1) and exemplified extensively elsewhere (10–12). Univariate and/or multivariate experimental design approaches may be deployed to establish a MODR, so that an in-depth understanding of the interactions and criticality of procedure parameters, with respect to their impact on specific performance criteria, and the reportable result, can be achieved. The MODR constitutes a region within which changes can be made without impact on the reportable result, and therefore, its boundaries should not be close to any identified edges of failure. 

	The enhanced approach features a systematic assessment of inputs and how they impact robustness and ruggedness (13) of the procedure; this facilitates the definition and establishment of controls within the analytical procedure that ensure consistent operation. ICH Q8 defines the control strategy as a planned set of controls, derived from current product and process understanding, which ensures process performance and product quality. 

	In an analogous fashion, an analytical procedure could contain the following key elements:

		A system suitability test (SST) as described in ICH Q2 R1 (14) and the pharmacopeias. For an analytical procedure developed using the enhanced approach, the SST limits should ensure that the ATP criteria are consistently met and all parameters critical to procedure performance are appropriately controlled. SST criteria are traditionally selected to confirm measurement system performance prior to and/or during analysis and may include resolution, injection precision (for chromatographic methods), detection limit, linearity criteria, or reporting limits. Additional controls that verify the performance of the constituent operational units within a procedure (such as variability of standard or sample preparation, resolution of critical components, extraction efficiency, and analysis of control samples), and therefore act as additional confirmation of procedure performance, may further support the ATP as part of a performance-based approach to procedure change control.

		A detailed set of instructions that clearly specify parameters requiring control identified during risk assessment, development or validation (robustness) experiments. This may be a range or set point, or combination of both. These instructions allow the trained analyst to operate the procedure correctly and thus meet the criteria described in the ATP.

		A defined replication strategy (e.g., the number of injections and sample preparations) that define the reportable result. By increasing the number of replications, the precision of the mean can be improved, as required by the ATP (15).

		A quality system that supports an enhanced approach including written standard operating procedures, change management and facilities/equipment operation, control forms, and continual monitoring performance criteria.

		Continual monitoring of critical predefined criteria to identify when changes or adaptations are necessary during the analytical procedure lifecycle. The increased understanding that the enhanced approach delivers, aids in identifying the implications of any proposed change and informs any change assessment strategy.

	As a pharmaceutical product progresses through the development lifecycle, the associated ATPs for each of the measured quality attributes should be refined as needed to ensure the associated procedures fully support the evolving clinical and commercial specifications. If performance requirements or specifications change, ATPs can be revised accordingly, and the suitability of the methods re-assessed (if required). 

Examples of the performance criteria that could potentially be included in an ATP for three different types of measurement

 are provided in the online version of this article. Further exemplifications of ATPs can be found in the literature (16,17). 

	The benefits of applying the enhanced approach on validation and transfer

	The enhanced approach for the development and application of analytical procedures uses risk assessment and systematic experimental evaluation to gain enhanced understanding of the procedure parameters critical to the consistent delivery of fit-for-purpose reportable results.

	Such enhanced understanding leads to the development of analytical procedures whose performance criteria are based on the requirements of the reportable result throughout the analytical procedure lifecycle. This understanding further underpins knowledge of the impact to procedure performance when individual or combined critical inputs are changed. Consequently, there is increased understanding (and control) of the inherent variability associated with the reportable result through the procedure lifecycle, which ultimately facilitates greater understanding of true process variability.

	Furthermore, the enhanced operational robustness of analytical procedures strengthens the continuity of the supply chain by lowering the risk of procedure related problems and by enabling more efficient, robust out-of-specification and out-of-trend (OOS/OOT) investigations and root cause determination if problems are observed. 

	In an enhanced approach, performance qualification and verification are part of the lifecycle-the demonstration of an analytical procedure’s suitability is not a singular activity, but instead part of continued assurance that it remains fit-for-purpose throughout its deployment. This includes when any changes are made to the procedure parameters or its operating environment. 

	The analytical procedure lifecycle approach is aligned with the three sequential stages described in current process validation guidelines: procedure design (stage 1), procedure performance qualification (stage 2), and continued performance verification of the procedure (stage 3). An analytical procedure that is designed in stage 1 is qualified against the performance acceptance criteria derived from the ATP at stage 2 (analogous to a traditional method validation and transfer into a receiving site). During stage 3 (routine application) monitoring of critical performance attributes ensures the procedure continues to meet the requirements of the ATP. 

	If changes are made to the analytical procedure that impact the quality of the data produced, a further qualification exercise should be performed to confirm the procedure performance continues to meet the requirements of the ATP. Performance monitoring across the lifecycle, change management, and efficient knowledge transfer are facilitated by well-designed analytical controls that ensure the procedure delivers fit-for-purpose data throughout its lifecycle. 

	In summary, the benefits of the enhanced approach include reliable analytical procedures with performance criteria based on the requirements of the reportable result. Furthermore, these analytical procedures have less likelihood of ‘failure’ (which can better ensure product supply), lend themselves to more efficient investigations if OOS/OOT results are observed, and come with knowledge and understanding about how procedure performance is impacted when both individual and combined critical inputs are changed.

	The traditional versus the enhanced approach

	The traditional approach is typically an iterative and univariate process with emphasis on meeting predefined, and often generic, validation criteria and limited use of risk assessment and structured experimental design. The enhanced development approach fundamentally differs in its dual recognition of the need to i) systematically identify and understand the interconnected multivariate procedure parameters which have potential to influence the performance of the analytical procedure and ii) evaluate quality risks posed by these parameters based on their impact on the reportable result.

	This holistic understanding facilitates lifecycle activities such as the transfer and improvement of analytical procedures and support to any investigations required by providing a common knowledge base and baseline for procedure performance. For traditionally developed methods, these activities are often performed independently, with redundancies and duplication, leading to less efficient change management.

	An ATP could also be developed and applied retrospectively to a traditionally developed analytical procedure for the purposes of continual monitoring and improvement, if considered appropriate. For example, as a result of investigations on OOS/OOT results or, for a post-approval tightening of a specification limit it may be helpful to revisit or even define the required analytical performance for the first time. In the enhanced approach, the ATP is prospective and serves as focal point for the continuous improvement of the analytical procedure.

	The principles of the enhanced approach can be applied to any type of analytical technology and are not restricted to specific molecule classes or method types (e.g., the approach is applicable to in-line or at-line, as well as off-line analyses). The greatest value is gained from the application of the enhanced approach to measurements that present a significant risk of variation or inconsistency as a result of the complexity of the measurement or the nature of the analyte. Simple methods such as those resulting in a qualitative result, or simple pharmacopeial tests and limit tests, are less likely to benefit from adopting an enhanced approach.

	Sound knowledge management and quality risk management is recognized as an important enabler of the enhanced approach for development and application of analytical procedures. A company’s quality system should support the design, qualification/validation, and continued verification and improvement stages for analytical procedures. 

	Suitable processes or business practice may include how to generate an ATP, how to perform a risk analysis and define the analytical controls for analytical procedures, qualification/verification of analytical procedures and handling non-conformances with acceptance criteria predefined in qualification protocols and the ATP, internal and regulatory change control of analytical procedures and exchangeability of alternative procedures, and how to monitor and trend analytical procedure performance in a continued manner as well as handling unfavorable trends.

	An overall lifecycle concept for analytical procedures, including ATP definition and use as a development tool, has been described in a series of stimuli articles by expert working groups in the 

	A number of papers dating back to 2007 have considered how application of enhanced tools can be applied during the analytical procedure lifecycle, with particular focus on chromatographic technology platforms (21). These papers have cited the specific elements of the enhanced approach and outlined how statistical experimental design and handling of the data, risk assessment, categorization, and prioritization tools can all lead to greater understanding and controls to assure the requirements for the reportable result.

	A Parenteral Drug Association (PDA) workshop on the role of the analytical scientist in QbD recognized the challenges of harmonizing new approaches across multiple stakeholders as a result of the global nature of the pharmaceutical industry (22). Similarly, two USP workshops on the lifecycle approach to validation of analytical procedures have explored the statistical tools and provided examples of their application (23,24). 

	A more recent industry survey posed several questions about progress with analytical quality by design. Approximately half of the companies polled were implementing some aspects of the enhanced approach. The survey concluded that while the benefits are clear in terms of the development of more robust procedures, the desired streamlining of regulatory aspects of analytical procedure change processes have not been realized so far (25). 

guideline has reached Step 2 in the ICH process (26), with publication of the draft guideline (27) and requests for comment in a number of regions. The guideline may therefore undergo revision before it is finalized at Step 4 and then implemented in the ICH regions at step 5 in the ICH process. The Q12 guideline:

provides a framework to facilitate the management of post-approval CMC changes in a more predictable and efficient manner. It is also intended to demonstrate how increased product and process knowledge can contribute to a reduction in the number of regulatory submissions. Effective implementation of the tools and enablers described in this guideline should enhance industry’s ability to manage many CMC changes effectively under the firm’s Pharmaceutical Quality System (PQS) with less need for extensive regulatory oversight prior to implementation.”

	The Q12 Step 2 document includes a number of concepts/tools that may be relevant to analytical lifecycle management within the following chapters: 

		Chapter 3: Established Conditions (ECs)

		Chapter 4: Post-Approval Change Management Protocols (PACMPs)

		Chapter 8: Structured Approach to Analytical Procedure Changes

	Established Conditions are defined in Chapter 3 as follows:

		“ECs are legally binding information (or approved matters) considered necessary to assure product quality. As a consequence, any change to ECs necessitates a submission to the regulatory authority.”

		Within Chapter 3, there is a description of how to identify ECs for analytical procedures and a caution that the extent of ECs could vary depending on the method complexity, development, and control approaches. Two control approaches are noted:

		“Where the relationship between method parameters and method performance has not been fully studied at the time of submission, ECs will incorporate the details of operational parameters including system suitability.”

		“When there is an increased understanding of the relationship between method parameters and method performance defined by a systematic development approach including robustness studies, ECs are focused on method-specific performance criteria (e.g., specificity, accuracy, precision) rather than a detailed description of the analytical procedure”.

	It is important to note that a suitably detailed description of the analytical procedures is expected to be included in Module 3 of the Common Technical Document (CTD) whichever approach is used to identify ECs for analytical procedures. 

	The authors interpret the enhanced approach described in this paper to be fully aligned with the latter approach to identifying ECs, and therefore, ECs for an analytical procedure could be considered as analogous to an ATP (28). Furthermore, in many cases, it could be argued that procedures successfully validated according to the current ICH Q2 guidance could also have ECs described by their method-specific performance criteria.

	Chapter 3 also describes how changes to ECs for manufacturing processes may have different reporting categories proposed by the applicant (prior approval or notification) depending on the risk associated with the process change. A similar risk-based approach could be adapted for reporting categories associated with changes to ECs for analytical procedures. When changes to procedures remain within approved ECs these should be managed solely within an applicant’s pharmaceutical quality system.

	Following the initiation of Q12, FDA published a draft guidance that describes how the concept of ECs can be used to clarify the elements of a license application that constitute a regulatory commitment (29).

	In Chapter 4, PACMPs or comparability protocols are discussed. These are regulatory tools that exist in the European Union and United States, and the Pharmaceuticals and Medical Devices Agency (PMDA) has recently initiated a pilot program on PACMPs in Japan. While it is not required by Q12, the enhanced knowledge and understanding gained from applying an enhanced approach to analytical procedure development may be valuable in supporting proposals for ‘broader’ PACMPs (e.g., those concerned with one or more changes to analytical methods to be implemented across multiple products and/or multiple sites).

	The structured approach to analytical procedure changes described in Chapter 8 is not related to ECs for analytical procedures. It is intended to enable companies to follow this structured approach for changes to currently approved analytical procedures, whether they were developed using an enhanced approach or not, and without needing a prior regulatory submission before implementing the change to the analytical procedure. The approach incorporates good change management practices and ensures the revised analytical procedure is equivalent or better to the original. The scope of procedures where this approach may be used has some limitations, and a regulatory notification is required at the end of the change.

	The past few years have seen the emergence of regional guidance on analytical procedures, for example, from FDA (30), European Medicines Agency (31), Brazilian Health Regulatory Agency (32), and Ministry of Health, Labor, and Welfare (33), which adopt some of the newer risk based/lifecycle development concepts. In June 2018, the ICH Assembly agreed to initiate development of harmonized guidance(s) for analytical procedure development and revision of Q2(R1) analytical validation Q2(R2)/Q14 (34). The first task for the working group will be to develop a concept paper and work plan and the authors of this paper look forward to the development of this ICH topic and its relationship to the ICH Q8–Q12 guidelines. 

	The current ICH Q2 guidance on the validation of analytical procedures was first published in 1994 and the text and methodology combined into the current ICH Q2(R1) guideline in 2005. Although the concepts in Q2 have stood the test of time, the initiation of the Q14 topic provides the opportunity to include elements of lifecycle management of analytical procedures and extend the concepts to contemporary measurement technique applications, for example with process analytical technology (PAT) or methods using multivariate models.

	The publication of papers, stimuli articles, and case studies continue the active debate on the enhanced approach for development and application of analytical procedures. Recent concepts such as the analytical target profile and method operable design region are increasingly becoming established, with the ATP being a valuable tool to focus development of fit-for-purpose analytical controls and procedures (35). 

	Recent developments in the progression and initiation of ICH quality guidelines (ICH Q12, Q2 revision, and ICH Q14) show that the regulatory aspects of the development and lifecycle management of analytical procedures is likely to be of continuing interest in the coming years. Concepts associated with the enhanced approach, including the ATP concept and method control strategies, may provide useful input for consideration by the expert groups developing these harmonized global guidelines, and ultimately contribute to the development and supply of high-quality medicines for patients throughout the product lifecycle.

, Step 4 version (2005).
	3. ICH, Q8 (R2) 

Development and Manufacture of Drug Substances (Chemical Entities and Biotechnological/Biological Entities)

Guidance for Industry, Process Validation: General Principles and Practices, CGMP Revision 1

, (Rockville, MD, June 2012).
	7. ICH, Q6A 

Specifications: Test Procedures and Acceptance Criteria for New Drug Substances and New Drug Products: Chemical Substances

Specifications: Test Procedures and Acceptance Criteria for Biotechnological/Biological Products

, Step 4 version (1999).
	9. B. Harrington et al., 

., 38 (2), 52–57 (2018).
	10. M. Hanna-Brown et al., 

, 2, 12–20 (2010).
	11. G.L. Reid et al., 

., 16 (5), 49–59 (2013).
	12. J. De Sousa, D. Holt, and P.A. Butterworth, “Analytical Method Design, Development, and  Lifecycle Management: A Practical Approach in 

,” W.S. Schlindwein and M. Gibson (Eds); pp 257–279 (2018).
	13. P.J. Borman et al., 

, 703 (2), 101–113 (2011).
	14. ICH, Q2 (R1) 

Validation of Analytical Procedures: Text and Methodology

, Step 4 version (1994).
	15. J. Ermer and C. Agut, 

, 160, 73–79 (2018).
	17. P. Jackson et al., “Using the Analytical Target Profile to drive the Analytical Method Lifecycle,” 

, 42 (5), (2016).
	20. K. Barnett et al., 

, 42 (5), (2016).
	21. P.J. Borman et al., 

, 34 (2), 52–59 (2007).
	22. PDA Workshop on Analytical Science and QbD, Mar. 6–7 (Liverpool, UK, 2012).
	23. USP Workshop, Lifecycle Approach to Validation of Analytical Procedures with Related Statistical Tools (Rockville, MD, USA, 2014).
	24. Proceedings of the Workshop on lifecycle Approach of Analytical Procedures, 

, 8–9 43 (6) (2016).
	25. M Argentine et al., 

Technology and Regulatory Considerations for Pharmaceutical Product Lifecycle Management

, Step 2 Draft, (October 2017).
	28. J. Hoffmann, “Performance and Context Based Established Conditions for Analytical Procedures to Support ICH Q12,” IFPAC Annual Meeting, (2018).
	29. FDA, 

Draft Guidance for Industry, Established Conditions: Reportable CMC Changes for Approved Drug and Biologic Products

Guidance for Industry, Analytical Procedures and Methods Validation for Drugs and Biologics, FDA CDER/CBER Pharmaceutical Quality/CMC Guidance for Industry

Guideline on Bioanalytical Method Validation 

EMEA/CHMP/EWP/192217/2009 Rev. 1 Corr. 2, (2011).
	32. ANVISA, Agência Nacional de Vigilância Sanitária, 

Guide for Validation of Analytical and Bioanalytical Methods

, Resolution-RE n. 899 (2003).
	33. NIHS, 

Guideline on Bioanalytical Method Validation in Pharmaceutical Development 

(2013).
	34. ICH, “ICH Assembly, Kobe, Japan, June 2018,” 

Pilot Program for Parallel Assessment of Quality-by-Design Applications: Lessons Learnt and Q&A Resulting from the First Parallel Assessment


	Vol. 42, No. 12
	December 2018
	Pages: 18–23

	When referring to this article, please cite it as Andy Rignall, 

, “Analytical Procedure Lifecycle Management: Current Status and Opportunities," 

	The authors are members of the European Federation of Pharmaceutical Industries and Associations (EFPIA) Analytical Lifecycle Management Team. 

is product technical director at AstraZeneca; 

* is director, Product Development & Supply at GSK, phil.j.borman@gsk.com; 

is external technology & collaborations lead at Pfizer; 

is director, Collaborative Solutions at Elanco; 

is director, Analytical Sciences Biologicals at UCB; 

is senior scientist, New Biological Entities, Analytical R&D at AbbVie; 

is director, Attribute Sciences at Amgen; 

 is director, CMC Regulatory Compliance at Merck KGaA; 

is head of Analytical Lifecycle Management Chemistry Frankfurt at Sanofi; 

is laboratory head Analytical Development at Bayer; 

is Analytics Biochemistry site head at Roche; and 

is analytical project leader at Novartis.

	*To whom all correspondence should be addressed

Drawing on practical experience, the authors examine key questions and answers about various aspects relating to the enhanced approach for analytical procedure lifecycle management.


Analytical Procedure Lifecycle Management: Current Status and Opportunities

	Three hypothetical ATPs are provided in the following tables, reflecting the current thinking of the EFPIA Analytical Lifecycle Management Team. The ATPs are preceded by some background information to support the selected performance criteria and rationale. 

	Example 1: Measurement of potency using a cell-based potency assay for a biological product

	In addition to performing measurements of product CQAs, testing of potency is a unique feature of the release specification for biologics. Potency can be measured by 

 methodologies but is most often measured by 

	A company is using a cell-based assay to measure the relative potency of a recombinant protein. The method is based on the mode of action of the drug and is used to measure the relative potency at release and during stability studies. The assay compares a test sample with a designated standard to provide a quantitative measurement of the bioactivity of the test relative to the standard. Prior to the development of a cell-based assay, the activity of the molecule was measured using a binding enzyme-linked immunosorbent assay (ELISA) method. This method will then be replaced by a cell-based assay that is more representative of the mode of action for Phase III studies. The proposed ATP could be applied to both the binding assay (e.g., ELISA) and the cell-based assay. One of the key aspects of the cell-based assay method is its specificity towards the molecule of interest and of the corresponding mechanism of action (i.e., signaling pathway). 

	During development, forced degradation studies highlighted some modifications in the structure of the molecule as confirmed by physicochemical assays. The cell-based assay to be developed should be able to detect a change in the dose response curve parameters and/or a shift in potency upon forced degradation.

	All performance characteristics are linked to the reportable results generated using the method.
	

	Example 2: Measurement of high molecular weight species (HMWS) in a biological product

	For most biological products, HMWS in the form of dimers and/or oligomers constitute product related impurities, which need to be controlled. Consequently, the level of high molecular weight species in biomolecules need to be controlled as impurities at release and during stability studies of drug substance and drug product.

	The method is used to quantify the size variants relatively, especially HMWS compared to the monomer species. The ATP was defined for early phase development but could also be applied to later phases, where the history of the product would allow adapting the method range. During stability and forced degradation testing, the company has generated data that establishes upper and lower limits for the observed HMWS changes under intended and accelerated storage conditions.
	

	Example 3: Measurement of impurities in a small molecule drug product

	Impurity content is a critical attribute of a drug product (see ICH Q6A as an example).

	A company is using a purity method to monitor the stability of a small-molecule drug product. The method is used to quantify specified, known, and unknown degradation products in multiple quality control laboratories. Specified and known degradation products did not raise any specific safety concerns (e.g., genotoxicity) and limits, using the ICH Q3B thresholds, can be applied based on the maximum daily dose of the product not exceeding more than 1 g. 

	During stability and forced degradation testing, the company has generated data that indicates that one main degradation product (degradation product A) is formed under mild acidic conditions and could be present at levels up to 0.5% during the anticipated shelf life and storage condition. The shelf-life specification for degradation product A was not more than 0.5%. 

	Forced degradation studies also showed that basic conditions would form degradation product B. Degradation product B was not observed during accelerated and long-term stability studies. 

	Other degradation products were observed during the acidic and basic forced degradation studies, but at significantly lower levels. No additional degradation products or degradation mechanisms were observed under the other forced degradation conditions studied. The company decided to include impurity B along with any other unspecified degradation products with a specification of not more than 0.1% for each individual degradation product. 

	Although not a degradation product, impurity C is observed as a process related impurity regularly present in the API used to manufacture the product.

	The company has provided sufficient supportive regulatory information and data that the following statements are considered true:

		ICH thresholds are met (relevant guidelines are fulfilled e.g., ICH Q3B; ICH M7);

		API losses can be recovered as degradation products (i.e., mass balance can be demonstrated);

		Degradation pathways are known, which enables the discriminatory requirements to be established for the stability indicating method;

		The origin and influencing factors for process related impurities is understood.

	In the given example, method performance criteria could be set independent from the analytical principle chosen. However, a technology specific validation must be performed to demonstrate the suitability of the method (e.g., the precision/range requirement would translate into a linearity test and limit of quantitation test for a high-performance liquid chromatography method).
	

	36. USP <1033> Biological Assay Validation, May 2017.
	37. ICH, Q5C Quality of Biotechnological Products: Stability Testing of Biotechnological/Biological Products, Step 4 version, 30 November 1995.

Three hypothetical analytical target profiles (ATPs) are provided, reflecting the current thinking of the the European Federation of Pharmaceutical Industries and Associations Analytical Lifecycle Management Team.
