**1. Introduction**

114 Analytical Chemistry

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Method validation has a long and productive history in the pharmaceutical and now, biopharmaceutical industries, but it is an evolving discipline which changes with the times. Though much has been written about method validation for conventional, small molecule (SM) pharmaceuticals, less has appeared providing an overview of its application for complex, high molecular weight (MW) biopharmaceuticals (or biotechnology) products. This appears to be satisfyingly changing with the times, and this particular chapter has been designed to address this area of method validation. We hope to address herein the important issues of where do analytical method validation guidelines and directives stand today for biopharmaceutical (protein or related) products. Due to the recognized differences and complexity of biopharmaceuticals relative to small molecule drugs, regulatory agencies have accepted that what is expected of all SM, single molecule entities (even enantiomers), cannot be required for complex protein biopharmaceuticals, such as antibodies. While it is quite a simple matter, in most instances, to characterize and validate methods for SM drug substances, this is not always the case for complex biopharmaceuticals. Biotechnology products will always be heterogeneous mixtures of product-related species.

While the chapter below focuses on the principles and practice of method validation for biopharmaceuticals in the biotechnology industry, some comments on the topic of "academic method validation," and if and how that differs from what is required by the industry, seem warranted. In general, academics are not required by any regulatory agency or governmental body to perform any degree of method validation. However, one instance where it might be appropriate to do acceptable (whatever that means) method validation is when a reviewer of a grant proposal or manuscript destined for publication demands that some validation be performed. At times, Journal/Book Editors may suggest that some degree of method validation be performed, but in the final analysis, this requirement is at

© 2012 Krull et al., licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2012 Krull et al., licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

the discretion of reviewers. It appears that for the most part, little to no method validation is performed in academic circumstances, but on occasion, attempts are made to validate methods in academic laboratories. However, even there, such efforts do not begin to approach what is expected by regulatory agencies for industrial methods used to release a product for clinical trials use.

Analytical Method Validation for Biopharmaceuticals 117

This reality, coupled with rapid technological advances and evolving regulatory expectations, impacts the ability of biotechnology companies to rapidly progress with

Method validation is a critical activity in biopharmaceutical product development which often causes confusion and, at times, consternation on the part of analytical development teams. Questions surrounding method validation abound: (1) when should we validate our analytical methods? (2) what are the requirements for achieving method validation in a manner that is compliant across multiple regulatory jurisdictions around the world? (3) how can I implement a validation strategy that fits my company's business infrastructure and provides for seamless method transfer activities to other QC organizations in the company

Prior to proceeding to a discussion of method validation, it is important to differentiate amongst the categories of analytical methods used in the biopharmaceutical industry for product evaluation. In general, the analytical methods used can be divided into three categories: (1) screening methods; (2) release and stability methods; and (3) characterization methods. Screening methods are used to guide discovery research and process development. These methods, which are often carried out in high-throughput format using automation due to the large volumes of samples tested, do not typically follow any validation guidance, since they are not intended for a QC environment. Nonetheless, it is important to understand the capabilities and limitations of these methods so that the results can be appropriately applied to making decisions during process and product development. This is generally achieved through experience with the method in the analytical development organization. The second class of methods, release and stability methods, are intended for use in a Quality Control environment for product disposition and formal stability studies. In addition, these methods are sometimes used in QC for in-process samples in the form of in-process controls, which are used in the overall control strategy to ensure product quality (and for which the validation strategy should mirror that used for the release and stability methods). Whether used for release, stability, or in-process control applications, these methods are generally validated prior to the validation (conformance) lots to demonstrate that they have acceptable performance according to regulatory guidance (discussed below). The third class of methods, characterization methods, are used to support product characterization studies during reference standard characterization, process characterization, comparability studies, and other product characterization activities, and data from these studies is often submitted to regulatory agencies. Industry practice has recently evolved to meet regulatory expectations that these methods will be qualified according to written company procedures, though no formal written guidance is available,

development of their pipeline candidates.

as well as contract QC organizations, when required?

and method validation is not expected for these analytical procedures.

In order to meet current compliance expectations, an analytical method used to support GMP activities must be suitable for its intended use, and appropriate experimental work must be documented that provides this assurance. The demonstration of method suitability can be divided into two sets of activities: qualification and validation. When methods are

At times, in the past, Editors have taken the time to list what is expected in future submissions related to some degree of method validation. However, it was never obvious or clear that a lack of such studies really has ended up in manuscript rejections. Again, to a very large degree, this has depended on the rigorousness of the reviewers, resulting in somewhat a "luck of the draw" approach. Some may view this as frustrating and unfortunate, because in the absence of method validation, there should be no reason to accept the method and its applications, prima facie, or its results/data. However, academics somehow don't believe that method validation is required in order to do "good science" or publish. This situation has been changing for the better, but it is not quite where it really should be today. It will change when all editors, reviewers and manuscript/proposal submitters agree on the importance of doing good science by doing thorough and complete analytical method validation studies. Clearly, practitioners in the pharmaceutical and biopharmaceutical industries have much to offer to academic scientists in this regard.

One publication, years ago, appeared to demonstrate in certain, newer capillary electrochromatography (CEC) studies, unusually high plate counts and efficiencies. However, when others attempted to reproduce such results and data, nobody could come even close to what was in that original publication. Eventually, it was admitted that in the original study, none of those astounding results were reproducible or even replicable in a single lab. The work was never repeatable in their own hands, something that they conveniently forgot to mention anywhere in their papers. How could that happen? Well, it happened because neither the editors nor reviewers were thorough and rigorous in their demand for analytical method validation. They did not ask to see some evidence of repeatability, intermediate precision and other performance characteristics, a situation that would not be permissible in the industrial world due to regulatory requirements for method validation

Method validation in the pharmaceutical and biopharmaceutical industries is designed to help ensure patient safety during clinical trials and later when the drug becomes commercialized. While this reasoning is not applicable to basic research, and basic research in the academic community has at least one self correcting mechanism, peer verification, the lack of a requirement to document the performance characteristics of the methods in the academic world can, at times, lead to the publication of analytical methodologies, as noted above, that may lack scientific integrity.

## **2. Method validation for the biotechnology industry**

The development of biotherapeutics is a complex, resource-intensive and time-consuming process, with approximately 10 years of effort from target validation to commercialization. This reality, coupled with rapid technological advances and evolving regulatory expectations, impacts the ability of biotechnology companies to rapidly progress with development of their pipeline candidates.

116 Analytical Chemistry

product for clinical trials use.

the discretion of reviewers. It appears that for the most part, little to no method validation is performed in academic circumstances, but on occasion, attempts are made to validate methods in academic laboratories. However, even there, such efforts do not begin to approach what is expected by regulatory agencies for industrial methods used to release a

At times, in the past, Editors have taken the time to list what is expected in future submissions related to some degree of method validation. However, it was never obvious or clear that a lack of such studies really has ended up in manuscript rejections. Again, to a very large degree, this has depended on the rigorousness of the reviewers, resulting in somewhat a "luck of the draw" approach. Some may view this as frustrating and unfortunate, because in the absence of method validation, there should be no reason to accept the method and its applications, prima facie, or its results/data. However, academics somehow don't believe that method validation is required in order to do "good science" or publish. This situation has been changing for the better, but it is not quite where it really should be today. It will change when all editors, reviewers and manuscript/proposal submitters agree on the importance of doing good science by doing thorough and complete analytical method validation studies. Clearly, practitioners in the pharmaceutical and

biopharmaceutical industries have much to offer to academic scientists in this regard.

world due to regulatory requirements for method validation

**2. Method validation for the biotechnology industry** 

above, that may lack scientific integrity.

One publication, years ago, appeared to demonstrate in certain, newer capillary electrochromatography (CEC) studies, unusually high plate counts and efficiencies. However, when others attempted to reproduce such results and data, nobody could come even close to what was in that original publication. Eventually, it was admitted that in the original study, none of those astounding results were reproducible or even replicable in a single lab. The work was never repeatable in their own hands, something that they conveniently forgot to mention anywhere in their papers. How could that happen? Well, it happened because neither the editors nor reviewers were thorough and rigorous in their demand for analytical method validation. They did not ask to see some evidence of repeatability, intermediate precision and other performance characteristics, a situation that would not be permissible in the industrial

Method validation in the pharmaceutical and biopharmaceutical industries is designed to help ensure patient safety during clinical trials and later when the drug becomes commercialized. While this reasoning is not applicable to basic research, and basic research in the academic community has at least one self correcting mechanism, peer verification, the lack of a requirement to document the performance characteristics of the methods in the academic world can, at times, lead to the publication of analytical methodologies, as noted

The development of biotherapeutics is a complex, resource-intensive and time-consuming process, with approximately 10 years of effort from target validation to commercialization. Method validation is a critical activity in biopharmaceutical product development which often causes confusion and, at times, consternation on the part of analytical development teams. Questions surrounding method validation abound: (1) when should we validate our analytical methods? (2) what are the requirements for achieving method validation in a manner that is compliant across multiple regulatory jurisdictions around the world? (3) how can I implement a validation strategy that fits my company's business infrastructure and provides for seamless method transfer activities to other QC organizations in the company as well as contract QC organizations, when required?

Prior to proceeding to a discussion of method validation, it is important to differentiate amongst the categories of analytical methods used in the biopharmaceutical industry for product evaluation. In general, the analytical methods used can be divided into three categories: (1) screening methods; (2) release and stability methods; and (3) characterization methods. Screening methods are used to guide discovery research and process development. These methods, which are often carried out in high-throughput format using automation due to the large volumes of samples tested, do not typically follow any validation guidance, since they are not intended for a QC environment. Nonetheless, it is important to understand the capabilities and limitations of these methods so that the results can be appropriately applied to making decisions during process and product development. This is generally achieved through experience with the method in the analytical development organization. The second class of methods, release and stability methods, are intended for use in a Quality Control environment for product disposition and formal stability studies. In addition, these methods are sometimes used in QC for in-process samples in the form of in-process controls, which are used in the overall control strategy to ensure product quality (and for which the validation strategy should mirror that used for the release and stability methods). Whether used for release, stability, or in-process control applications, these methods are generally validated prior to the validation (conformance) lots to demonstrate that they have acceptable performance according to regulatory guidance (discussed below). The third class of methods, characterization methods, are used to support product characterization studies during reference standard characterization, process characterization, comparability studies, and other product characterization activities, and data from these studies is often submitted to regulatory agencies. Industry practice has recently evolved to meet regulatory expectations that these methods will be qualified according to written company procedures, though no formal written guidance is available, and method validation is not expected for these analytical procedures.

In order to meet current compliance expectations, an analytical method used to support GMP activities must be suitable for its intended use, and appropriate experimental work must be documented that provides this assurance. The demonstration of method suitability can be divided into two sets of activities: qualification and validation. When methods are

new, under development, or subject to process or method changes, this activity is often called qualification, while more formal confirmation of method suitability for commercial applications is called validation (Ritter, Advant et al. 2004; Apostol and Kelner 2008; Apostol and Kelner 2008).

Analytical Method Validation for Biopharmaceuticals 119

associated activities relevant for method validation (Swartz and Krull 1997; Shabir 2003; Ritter, Advant et al. 2004; ICH 2006; Krull and Swartz 2006; Swartz and Krull 2006; Swartz and Krull 2009), the most frequently referenced document, is the ICH guideline Q2R1, "Validation of analytical methods: text and methodology"(ICH 2005), This document covers validation activities targeted at product registration; hence, this guidance is specifically applicable to commercial products. Method qualification has emerged as the typical means of filling the gap for assessing the suitability of analytical method

Characterization methods typically involve highly specialized technologies which are labor intensive and difficult to perform on a routine basis, which includes, for example, AUC, CD, FTIR, DSC, SEC-LS , and NMR. These methods are often used to supplement lot release methods to provide orthogonal detection/separation modes and/or to verify structural integrity (e.g. primary, secondary, tertiary structure). This is in contrast to Quality Control methods, which typically employ proven technologies to enable in-process controls, lot disposition and GMP stability assessment in the GMP laboratory setting, requiring stringent assessment of performance characteristics that follow ICH guidelines. Therefore, it is important to define an appropriate level of qualification for these complex and non-routine characterization methods. Industry practice has evolved multiple means of defining a

Ensuring that the equipment has a documented record of initial equipment

Ensuring that data are generated by scientists with appropriate technical skills

 Ensuring that all experiments are accompanied by proper controls to ensure that the method is capable of measuring the intended attributes of the product. Control experiments should be designed in such a way that the quantitative aspect of the measurement can be clearly demonstrated from the results of the experiments. Properly controlled experiments should be performed to address the

Recently Jiang et al. provided an excellent review of the qualification of the biophysical methods including AUC, CD, FTIR, DSC, SEC-LS, MFI and LO based methods. The authors describe how qualification of these methods enables better knowledge of the methods and objective interpretation of the results. The general considerations described there can be applied to other biophysical methods as appropriate as well (Jiang, Li et al. 2012). In most cases qualification of biophysical methods is focused on the determination of precision and demonstration that the methods are suitable for their intended applications. Successful qualification enables the understanding of the method capability and the consistent

qualification (IQ/OQ), preventative maintenance (PM), and/or calibration.

documented through training records and/or academic credentials.

performance at earlier stages of product development.

**3. Qualification of characterization methods** 

qualification path for characterization methods, including: Ensuring the adherence to written technical procedures

precision (repeatability) of the measurements.

determination of product attributes.

The strategy for method validation involves a continuum of activities that begins at the start of process and product development and carries through to the marketing application and beyond. Typically, analytical method development begins after the biological target has been identified and verified, the protein therapeutic has been defined (primary sequence), and the sponsor has made the decision to develop a manufacturing process that will enable human clinical trials. The initial demonstration that the method is suitable for its intended purpose for use as a release and stability method is generally carried out in the form of method qualification, an activity that generally takes place prior to the release of the material for first-in-human (Phase 1) clinical trials. At the later stage of product development, typically prior to the start of pivotal phase III clinical trials, method developers perform qualification studies which will enable method validation. Finally, method validation generally takes place prior to the release and stability testing of the validation manufacturing lots.

It should be noted that although method qualification, which evaluates the performance characteristics of the method against meaningful target expectations, is a critical development activity that establishes the suitability of the method for release of early to mid-phase clinical materials, this activity is not, to the best of our knowledge, clearly defined in regulatory guidance, which tends to focus on method validation. It is therefore difficult to define the scope of method qualification, though regulatory expectations and industry practices have evolved to define method qualification as a means to assure acceptable method performance during process and product development, prior to the formal validation exercise that occurs before the testing of the validation lots.

The necessity of method validation has been reinforced by a variety of national and international regulations (USP 1994; USP 1999; CDER 2001; ICH 2005) which are subject to user interpretation. For example, current GMP regulations, [21 CFR 211.194 (a)] require that methods used in testing of the samples meet proper standards of accuracy and repeatability. Validation provides assurance that this regulation is met. USP <1225> defines validation of analytical procedures as the process by which it is established by laboratory studies that the performance characteristics of the procedure meet the requirements of the intended analytical application. ICH guideline Q2R1 defines validation of analytical procedures as the demonstration that the method is suitable for its intended purpose. ICH guidance specifies that validation of analytical procedures needs to be included as part of the registration package submitted within the EU, Japan and USA. While the biotechnology industry, in a manner analogous to the pharmaceutical industry, is heavily regulated, the majority of the regulations are targeted at commercial products, leaving a significant gap in available regulatory guidance for earlier stages of product development. While numerous articles have been published to provide the scientific principles and exemplify the types of associated activities relevant for method validation (Swartz and Krull 1997; Shabir 2003; Ritter, Advant et al. 2004; ICH 2006; Krull and Swartz 2006; Swartz and Krull 2006; Swartz and Krull 2009), the most frequently referenced document, is the ICH guideline Q2R1, "Validation of analytical methods: text and methodology"(ICH 2005), This document covers validation activities targeted at product registration; hence, this guidance is specifically applicable to commercial products. Method qualification has emerged as the typical means of filling the gap for assessing the suitability of analytical method performance at earlier stages of product development.
