glp-compliant assay validation studies: considerations for implementation of regulations and audit...

DOI: 10.1002/qaj.365

GLP-compliant Assay ValidationStudies: Considerations forImplementation of Regulations andAudit of Studies

Henry Li*, Garreth Sharpy, Connie Pilkington, Dominique Pifat and Steve Petteway

Talecris Biotherapeutics, 85 TW Alexander Drive, Research Triangle Park, NC 27709, USA

Summary

Bioanalytical methods used in nonclinical safety studies are validated to demonstratethat the methods are reliable and reproducible for the intended use. Analyticalmethod validation studies should be designed according to regulatory guidance onmethod validation. A cell-based assay is described to discuss fundamental validationparameters, such as repeatability, immediate precision, accuracy, and linearity. AGood Laboratory Practice (GLP) compliance program provides a useful platform forensuring data integrity in assay validation studies. A good understanding of themethod validation concepts and regulatory requirements will help Quality AssuranceUnit (QAU) auditors focus on key areas and be more effective when auditing assayvalidation protocols, final reports, and associated data. Copyright # 2006 John Wiley& Sons, Ltd.

Key Words: assay validation; audit/inspection; Good Laboratory Practice (GLP); QualityAssurance

Introduction

It is scientifically sound and often required to

validate the bioanalytical methods used in

support of Good Laboratory Practice (GLP)-

compliant pharmacology/toxicology studies,

other nonclinical safety studies such as pathogen

safety studies and testing of human samples to

support clinical trials. To ensure quality, such

validation studies are often conducted in com-

pliance with GLP principles, even though this is

not required by the regulations. GLP concepts,

such as the requirements for protocol, final

report, and conduct of a nonclinical laboratory

study can be directly implemented in an assay

validation study. However, there are challenges

that require careful interpretation of the regula-

tions.

The Quality Assurance Unit (QAU) is respon-

sible for monitoring the assay validation studies

for GLP compliance and conducting protocol,

‘in-life’, and final report audits. To effectively

approach these audits, it is helpful for auditors

to be familiar with assay procedures and

validation requirements. Data generated from

an assay validation study can be complex and

several critical compliance elements are unique

to assay validation studies. It is important not to

lose sight of key areas when conducting quality

audits.

*Correspondence to: Henry Li, Talecris Biotherapeutics,85 TW Alexander Drive, Research Triangle Park, NorthCarolina 27709, USA. E-mail: [email protected] address: Ethicon Inc., Somerville, NJ, USA.

Copyright r 2006 John Wiley & Sons, Ltd. Qual Assur J 2006; 10, 92–100.

In this paper, a validation study of a cell-based

biological assay (porcine parvovirus assay (PPV))

is used as an example to discuss experience in

implementation of GLP compliance in assay

validation studies. Basic assay validation con-

cepts and regulatory requirements are discussed.

Points for consideration for auditing assay

validation studies, including those conducted in

compliance with GLP, are presented.

Regulatory Expectations on AssayValidation

Although assay validation is not directly

addressed in GLP regulations, it appears that

the Food and Drug Administration (FDA)

encourages the use of validated methods. In a

recent Warning Letter issued on 21 December

2004 to Nelson Laboratories Inc. [1] by the

Center for Devices and Radiological Health

(CDRH), FDA cited that [t]he QA unit did not

adequately monitor each study, as required by

21 Code of Federal Regulations (CFR)

Part 58.35(a). For example, ‘‘[t]he current test

methods, which are described in SOP[. . .], have

not been fully validated’’. It is not clear whether

this finding signals a significant shift in the

regulatory interpretation of the GLP regulations;

however, this finding does suggest that validat-

ing analytical procedures used in GLP studies is

the right direction moving forward. It is also

worth pointing out that in this case the agency

put the burden on the QAU to ensure that test

methods are validated.

The FDA guideline ‘Guidance for Industry:

Bioanalytical Method Validation’ provides cur-

rent regulatory expectations for the compliance

level of bioanalytical testing laboratories [2]. It

states that the analytical laboratory conducting

pharmacology/toxicology and other preclinical

studies for regulatory submissions should adhere

to FDA’s GLP regulations (21 CFR Part 58) and

sound principles of quality assurance through-

out the testing process.

The development of biopharmaceuticals

requires accurate and reliable analytical proce-

dures. The extent to which assay validation is

required has been extensively discussed in the

industry. It is often expected that the analytical

procedures used under the auspices of GLP or

current Good Manufacturing Practice (cGMP)

are validated [3]. Recently, at the third CMC

Strategy Forum held on the campus of the

National Institute of Standards and Technolo-

gies (NIST) [4], which was attended by both the

industry and government representatives, it was

concluded that complete assay validation is

required for GLP study assays, lot release assays,

raw material testing, in process testing, excipient

testing, and stability methods for defining

expiration dates/hold times. It was emphasized

that some assays, especially those used for GLP

animal toxicology or related safety studies, are

expected to be validated, despite such studies

occurring very early on in the drug development

process [4].

Basic Assay Validation Concepts

Types of method validation studies

There are several regulatory guidelines on assay

validation [2, 5–7]. Depending on the intended

use of a particular bioanalytical method and the

stage of the method development, types and

levels of the validation required can be quite

different. The types of method validation studies

can be classified into three general categories.

* Full validation is often required when new

bioanalytical methods are developed; the

method is used for detecting a new drug

entity; or metabolites are added to an existing

method for quantification [2].* Partial validation is necessary when already

validated bioanalytical methods are modified.

The validation exercise, depending on the

intended use of the method, ‘‘can range from

as little as one intra-assay accuracy and

precision determination to a nearly full

validation’’ [2].* ‘‘Cross-validation is a comparison of valida-

tion parameters when two or more bioanaly-

tical methods are used to generate data within

the same study or across different studies’’ [2].

GLP-compliant Assay Validation Studies 93


Viral infectivity assay

Biological assays based on cell culture and

animals are performed in complex biological

systems. These assays are often cumbersome and

highly variable. The PPV infectivity assay is used

here as an example to discuss assay validation

parameters and requirements. This assay is used

in GLP-compliant viral clearance studies [8,9] to

validate the capability of biologics manufactur-

ing processes to remove or inactivate PPV. The

results obtained from these safety studies will be

used for Investigational New Drug application

(IND), Biological License Application (BLA)

and to support changes to commercial produc-

tion processes [10]. This typical, cell-based viral

infectivity assay is schematically illustrated in

Figure 1.

Fundamental assay parameters

The objective of assay validation is to demon-

strate the assay is suitable for the intended use.

The requirements for the performance of the

PPV infectivity assay used in viral clearance

studies are included in the regulatory guidelines

[8,9]. The approach taken to the validation of

PPV infectivity assay is based on the recommen-

dations contained in the regulatory guidelines on

assay validation including assay validation

design and other considerations [2, 5–7]. How-

ever, this biological assay possesses unique

characteristics. For example, the readout of the

assay, cytopathic effect as a result of lysis of

infected cells, requires interactions of virus with

live cells, including viral entry into cells and

subsequent replication of virus inside the cells,

leading to cell death. Some assay acceptance

criteria were pre-determined based on the

complexity of the assay and might not be

consistent with those stated for analytical

methods in the FDA guideline [2]. Several

critical assay validation parameters validated

are briefly presented below.

RepeatabilityThe closeness of the results between a series

of measurements of a single sample obtained

by a single analyst under the same operating

conditions over a short interval of time. Repeat-

ability is also called as intra-assay precision.

Repeatability is often mathematically expressed

Day 1• Make serial half-log dilutions of each virus containing sample• Add each dilution to 8 replicate wells (one row) of the indicator cell plate with an appropriate cell density (40-60% confluence)• Let virus adsorb to cells for about 1 hour at 37˚C• Remove inocula from plates; replace with appropriate medium• Incubate plates for specified time at 37° C

Day 7-10• Examine plates microscopically for cytopathic effect

Titer Calculation

Cell Control

SampleDilution

Adsorption to cell

8/88/88/88/88/85/84/82/81/8

0/80/8

0/8

Incubation

Dilution of sample

High

Low

NegativePositive

Figure 1. Schematic diagram of PPV infectivity assay

94 H Li et al.


as percent of Coefficient of Variation (% CV)

of a series of measurements and is figuratively

illustrated in Figure 2. The repeatability of

the PPV infectivity assay was evaluated at three

PPV concentrations, high (undiluted), mid

(1:100 dilution of the high concentration), and

low (1:100 dilution of the mid concentration)

levels of PPV. A single analyst titrated three

samples in triplicate and performed the repeat-

ability experiment totally three times on

three separate days (Week 1, 2, and Week 3)

to yield nine determinations for each concen-

tration.

Intermediate precisionIntermediate precision is assay-to-assay varia-

bility within laboratories such as different

analysts, different instruments and different

days. The intermediate precision is also illu-

strated in Figure 2. During the first two

repeatability experiments, an additional analyst

also independently performed the PPV assay on

three PPV concentrations (high, mid, and low).

The results generated by two analysts were

analysed and compared.

AccuracyAccuracy is the measure of the closeness or

agreement between the measured value and the

true value or an accepted reference value of an

analyte (e.g. PPV in this study). Accuracy is

often expressed as percent of true or actual value

(Figure 2). In this assay validation study, the

PPV stock was serially diluted (1:10) to generate

nine PPV concentrations. The concentrations of

diluted PPV samples were determined based on

the calculation using the original titer of the PPV

stock and represented the ‘true values’ of PPV.

Each sample was assayed and the experiment

was repeated on two separate days in two

weeks. The measured values and the true values

were used to calculate the accuracy of the PPV

assay. The results from these experiments were

also used to determine the linearity, quantifica-

tion limit, and detection limit.

LinearityThe linearity of a method is its ability within a

given range to elicit results that are directly, or

by a well-defined mathematical transformation,

proportional to concentration of an analyte (e.g.

PPV in this validation study) in the sample.

Linearity is generally reported as the variance of

the slope of the regression line. In Figure 2,

the linear regression coefficient R2 is used.

The experimental design is described in the

section for accuracy.

Not Precise Precise & Accurate

Intermediate Precision

Not Precise

Repeatability

Precise Accurate Not Accurate

Accuracy

0

1

2

3

4

5

6

7

8

Log

PP

V T

iter

of O

bser

ved

0 1 2 3 4 5 6 7 8

Log PPV Titer of Expected

Linearity

Analyst 2Analyst 1

Figure 2. Repeatability, intermediate precision, accuracy, and linearity



Quantification limitThis refers to the lowest level of an analyte (e.g.

PPV in this validation study) in a sample that

can be quantitatively determined with suitable

precision and accuracy. The experimental design

for determining quantification limit is described

in the section for accuracy.

Detection limitThe limit of detection is the lowest concentra-

tion of analyte (e.g. PPV in this validation study)

that can be reliably detected by the method and

is expressed in terms of the concentration of

analyte in a sample. The experimental design is

described in the section for accuracy.

SelectivitySelectivity is the ability of the assay to differ-

entiate and quantify the analyte (e.g. PPV) in the

presence of other components in the sample. If

the method is intended to quantify more than

one analyte, each analyte should be tested to

ensure that there is no interference. In this study,

selectivity is defined as the ability of the assay to

measure accurately PPV infectivity in the pre-

sence of another virus. Reovirus was chosen in

this study since it is a non-enveloped virus and is

propagated in a similar medium as PPV. The

PPV assay was conducted to measure PPV

concentrations in the presence and absence of

reovirus and the results were compared.

Matrix effect‘‘It may be important to consider the variability

of the matrix due to the physiological nature of

the sample’’ and ‘‘appropriate steps should be

taken to ensure the lack of matrix effects

throughout the application of the method’’ [2].

This assay is used in determining PPV titers in

samples of different matrices, depending on the

protein purification processes in production. It is

required that the matrix effect be determined

during specific virus clearance studies [8,9]. In

this study, the matrix effects of three general

matrices, HBSS (Hank’s Balanced Salt Solutions)

used to dilute PPV during titrations and

the media that are used to propagate PPV

(Dulbecco’s Modified Eagle Medium (DMEM)

plus 2% fetal bovine serum (FBS) and DMEM

plus 10% FBS) were determined.

Stability‘‘Drug stability in a biological fluid is a function

of the storage conditions, the chemical proper-

ties of the drug, the matrix, and the container

system’’ [2]. The stability of PPV in three

matrices tested in this study was not determined

in this study; rather it was included in a separate

stability study.

RobustnessRobustness is the measure of an assay’s capacity

to remain unaffected by small, but deliberate,

variations in the method parameters. The

robustness of a method provides a good indica-

tion of the reliability of the method under

normal assay conditions. The variations to be

applied to the PPV assay are reagents, FBS, cell

density, cell passage number, adsorption tem-

perature and time, and incubation temperature.

Viral titers obtained at the low and high limits of

the variations were analyzed to determine if

there were any significant differences between

the results.

Assay validation results

The actual validation results for repeatability,

intermediate precision, accuracy, and linearity

are shown in Table 1. This was a full assay

validation study and the assay validation results

demonstrated that this assay is precise, accurate,

and linear, and robust, meeting the intended use

in pathogen safety validation studies to support

IND, BLA and commercial production of

biological products [10].

General Considerations forImplementation of GLP Requirements

Although the GLP regulations were initially

promulgated primarily for toxicology studies,

they are broad and comprehensive enough to

provide a compliance framework to ensure the

integrity and validity of other types of studies

96 H Li et al.


[10]. For this reason, many companies choose to

implement GLP elements in their assay valida-

tion studies. GLP concepts, such as the require-

ments for protocol, final report, and conduct of

a nonclinical laboratory study including doc-

umentation practices can be directly applied to a

GLP-compliant assay validation study. For ex-

ample, the following compliance practices

should be implemented:

* A scientist or professional is designated as a

study director by management who is

responsible for ‘‘the technical conduct of the

study, as well as for the interpretation,

analysis, documentation, and reporting

of the results’’ as defined in 21 CFR Part

58.33.* All study personnel, including the study

director, should have appropriate training

and/or experience, which are supported by

training records.* A specific, detailed description of the bioana-

lytical method to be validated should be

written. In the Standard Operating Procedure

(SOP) for the PPV assay, the key assay

parameters were described. The operating

ranges of these parameters were clearly

defined, providing the basis for the robustness

assessment.* Applicable elements for a study protocol as

described in 21 CFR Part 58.120 should be

incorporated into the assay validation protocol.* There also should be SOPs for operation of

instruments. For example, SOPs were pre-

pared for biological cabinets, cell incubators,

and pipettes.* Good documentation practices should be

followed and sufficient records generated to

allow reconstruction of the experiments.

During the PPV assay validation, standard

forms were used to record the experimental

procedures.* Equipment/Computerized Systems: GLP reg-

ulations for equipment should be followed,

including validation of computerized data

collection systems.* All experiments used to make claims or

draw conclusions about the validity of the

method should be presented in the final

report.* Applicable requirements for a study report as

described in 21 CFR Part 58.185 should be

met when preparing the assay validation

study report.* After the completion of the assay validation

study, all raw data, the protocol, the final

report, and other documentation should be

archived.

The QAU fulfils its responsibilities as defined

in the GLP regulations in 21 CFR Part 58.35. To

monitor a study, the following audits are usually

performed: (1) protocol review; (2) critical

phase/‘in-life’ audits (required); and (3) final

report audit (required). The frequency of critical

phase inspection should be determined based on

the nature of the assay validation study. Some

points for consideration when conducting

quality auditing are discussed in detail in the

following sections.

However, there are challenges in implement-

ing GLP compliance practices in assay valida-

tion studies. The following are some scenarios

that require careful interpretation of the GLP

regulations:

* The definitions for test article and test system

are not directly applicable because the pur-

pose of this type of study is to validate an

assay and not to test the safety of a drug or a

biological product. When entering the study

Table 1. Assay validation results

Assay parameter Result Acceptance criteria

Repeatability (% CV) 7.5% 25%Intermediate precision (% CV) 6.5% 25%Accuracy (recovery) 105% 100�30%Linearity (R2) 0.99 >0.95



information on the master schedule, it should

be indicated as such.* Reference standards are critical to a validation

study. The purity of the reference standard

used to prepare spiked samples can affect

study data. Also, if possible, the reference

standard should be identical to the analyte.

When this is not possible, an established

chemical form (free base or acid, salt or ester)

of known purity can be used [2]. There are

three types of reference standards: ‘‘(1)

Certified reference standards (e.g., United

States Pharmacopoeia compendial standards);

(2) Commercially supplied reference stan-

dards (from a reputable commercial source);

(3) Other materials of documented purity

custom-synthesized by an analytical labora-

tory or other noncommercial establishment.

The source, lot number, expiration date,

certificate of analyses when available, or

internally or externally generated evidence of

identity and purity should be furnished for

each reference standard’’ [2]. In the PPV assay

validation study, an in-house PPV stock was

characterized and used.* A bioanalytical method should be validated

for the intended use or application. A

protocol should include the acceptance criter-

ia for method parameters, which are estab-

lished based upon the application of the

assay. Remedial conditions for not meeting

such validation standards or revalidation

requirements should be provided. For exam-

ple, if the method does not meet the pre-

determined acceptance criteria, the method

parameters need to be readjusted and revali-

dated or the application is limited. The results

of the PPV assay validation study demon-

strated that the assay is suitable for use in

viral clearance safety studies.

Points for Consideration for AuditingMethod Validation Studies

Expectations for QAU

To be proactive and effective, QAU auditors

should be familiar with assay validation guide-

lines and requirements. The QAU may be asked

to provide guidance on designing assay valida-

tion studies. This is especially the case when the

QAU is helping a new group in either an

industrial or academic setting to establish a

GLP compliance program. The analytical scien-

tists may not necessarily understand the method

validation requirements. The QAU not only

performs the function of monitoring the com-

pliance of studies with GLPs, but also provides

assistance in the implementation of necessary

practices for compliance with the GLP regula-

tions as well as with regulatory guidelines on

method validation.

A good understanding of assay procedures

and validation requirements is very helpful for

auditors to conduct inspections. According to

the OECD GLP requirements, ‘‘[I]ndividuals

appointed to QA functions should have the

ability to understand the basic concepts under-

lying the activities being monitored’’ [11]. This

understanding also helps QAU auditors, as a

partner in drug development, to take a ‘risk-

based’ approach to auditing, knowing which

elements are most critical to data accuracy [12].

One way to follow new regulatory develop-

ments is to collect and analyze assay validation

related citations in regulatory inspections such

as FDA 483 s, Establishment Inspection Reports

and Warning Letters. It is very helpful to identify

common pitfalls encountered during assay vali-

dation studies, which are key reminders for QA

auditors to keep an eye on these critical issues.

The following are a few examples of FDA 483

citations on assay validation studies. They

pointed to the importance of reference standard,

meeting acceptance criteria, and matrix effect.

* ‘‘ The accuracy and sensitivity of the X and Y

assays were not determined in that the purity

of the reference material had not been

determined.’’ [13]* ‘‘Assay runs were accepted even though they

failed one or more predetermined acceptance/

rejection criteria.’’ [13]* ‘‘ The matrix that the assay was validated

at the contract research organization was

98 H Li et al.


different from the matrix of the samples from

the Sponsor.’’ [13]

Reviewing protocol

Reviewing a draft assay validation protocol

provides the first opportunity for a QA auditor

to understand and evaluate the method, the

scope of validation study and the strategies that

the study director plans to take to complete the

method validation. The following is a list of

reminders for auditors, including some key areas

to focus on during an audit of an assay

validation protocol. This list is not intended to

be exclusive, and not all elements apply to every

study:

* Be familiar with the bioanalytical method and

the instruments involved* Be familiar with assay validation guidelines* Verify that the method and applicable equip-

ment SOPs are written* Ensure that computerized systems are vali-

dated* Verify the assay validation design against

regulatory guidelines* Ensure that reference standards are appro-

priate and properly documented* Verify that statistical terminology and calcu-

lations to be used are described in the

protocol* Verify there are the acceptance criteria for

each method parameter to be validated* Ensure that the acceptance criteria are satis-

factory to meet the intended use of the

method* Verify that the matrix effect is evaluated* Verify that stability of samples are assessed

Auditing final report

The QA unit is required to audit the final assay

validation report to ensure validity and quality

of the data. The following is a list of key areas

that warrant a close look during a final report

audit:

* Evaluate the execution of the protocol

including documentation

* Verify that experiments are conducted

according to the protocol* Verify number of experiments* Evaluate use and handling of reference

standards* Assess handling of reagents and samples* Check statistical calculations* Verify if the acceptance criteria meet the

intended use of the assay* Verify if the acceptance criteria are met* Check handling of out-of-specification results* Verify if repeats are justified* Ensure that matrix effects are evaluated* Verify if revalidation requirements are in-

cluded

Conclusions

GLP-compliant assay validation studies present

a unique challenge to QAU auditors. To be

effective in performing QA functions when

monitoring GLP-compliant method validation

studies, QA auditors should have a good under-

standing of analytical procedures and regulatory

requirements for method validation. This will

help QA auditors efficiently navigate in the large

amount of complex data and not lose sight of

key compliance areas when conducting audits.

Acknowledgements

Authors thank Kang Cai and Randal

Hartwell for reviewing the manuscript.

References

1. FDA Warning Letter to Mrs Lynda Nelson, President,

Nelson Laboratories Incorporated, Salt lake City,

Utah, USA. Available at: http://www.fda.gov/foi/

warning letters/g5131d.htm (accessed 27 February

2006).

2. Food and Drug Administration. Guidance for Indus-

try: Bioanalytical Method Validation, Center for Drug

Evaluation and Research, May 2001.

3. Lundblad RL. Editorial. Approaches to assay valida-

tion for the development of biopharmaceuticals.

Biotechnol Appl Biochem 2001; 34: 195–197.



4. Ritter N, Advant SJ, Hennessey J et al. What is test

method qualification? Proceedings of the WCBP CMC

Strategy Forum, 24 July 2003. BioProcess Interna-

tional, September 2004; 32–46.

5. International Conference of Harmonization (ICH).

Q2A: validation of analytical procedures. Federal

Register, 60, 1995.

6. ICH. Q2B: validation of analytical procedures: meth-

odology. Federal Register, 62, 1996.

7. The United States Pharmacopoeia. Validation of

compendial methods.

8. CPMP Note for Guidance on Virus Validation Studies:

The Design, Contribution and Interpretation of

Studies Validating the Inactivation and Removal of

Viruses, CPMP/BWP/268/95 Final Version 2, 1996.

9. ICH Harmonised Tripartite Guidance Q5A: Quality of

Biotechnological Products: Viral Safety Evaluation of

Biotechnology products Derived from Cell Lines of

Human or Animal Origin, 4 March 1997 (CPMP/ICH/

295/95).

10. Li H, Hawlk S, Renfrow H et al. Establishing a GLP

compliance program for non-toxicology safety stu-

dies. Qual Assur J 2004; 8: 94–101.

11. Organisation for Economic Co-operation and

Development. OECD Series on Principles of Good

Laboratory Practice and Compliance Monitoring

Number 4 (Revised). GLP Consensus Document:

Quality Assurance and GLP. Environment Directo-

rate. Organisation for Economic Co-operation and

Development, Paris, 1999.

12. Jones AB. Science and quality assurance: from dose

to overdose. Qual Assur J 2004; 8: 156–160.

13. Pifat DY. Assay Validation. Bayer Corporation Inter-

nal Training Material, 2001.

100 H Li et al.


glp-compliant assay validation studies: considerations for implementation of regulations and audit...

Documents