The role of immunoassays and bioassays in biopharmaceutical ...

11 September Issue 2005

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The role of immunoassays and bioassays in biopharmaceutical development: an overview for regulatory specialistsPaul Chamberlain

Director – Biopharmaceuticals, Drug Development Programs, MDS Pharma

Services, Paris, France

Focus on Biotechnology

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February Issue 2006 3

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1. IntroductionImmunoassays and bioassays are applied at many stages of biopharmaceutical development. The aim of this article is to provide an overview of the principles upon which these techniques are based, and to highlight the most important regulatory considerations. Immunoassays and bioassays are considered in the same article, since their application is often complementary.

Key Words

Biotechnology, chemistry, drug/pharmaceutical legislation, drug/pharmaceutical development, European Union, guidelines, ICH, legislation, US FDA

Abstract

Immunoassays and bioassays are applied at many stages of biopharmaceutical development. This article reviews of the EU, FDA and ICH regulatory guidelines applying to the use of these techniques and the control of reference standards. It also describes the application of immunoassay and bioassay techniques in product development, manufacture and quality control.

Immunoassays depend on the exquisite sensitivity of antibodies to probe the integrity of the conformation of the recognition sites (epitopes) in a target antigen. A bioassay will then be used to confirm that there is no impact on downstream signalling or effector function.

In the context of immunoassays, antigenicity is described as the ability of an antigen to bind to a specific antibody. In contrast, immunogenicity is the ability of an antigen to elicit an immune response. The latter effect may be harmful, eg, when cross-reacting antibodies are produced following replacement of endogenous human proteins, but may also be beneficial in the case of vaccines.

Various types of assay formats are listed and the factors applying to development and validation of potency assays discussed. A common problem is the failure to define the intended purpose of the method and then to establish acceptance criteria and validate accordingly.

There are particular regulatory issues associated with immunoassays and bioassays. Approaches applied to validate conventional physico-chemical methodologies may not always apply and the dose-response relationship may be non-linear. The sponsor is therefore obliged to justify the particular approach taken, including the refinement of the assay conditions to minimise bias and the validity of the data analysis and interpretation approaches. The choice of reference standards, critical reagents and control of assay performance is also discussed. The four main sources of spurious results that could be challenged by regulatory agencies for immunoassays and bioassays are also described.

2. Regulatory guidelines relevant to the design, validation and application of immunoassays and bioassaysGeneral principles for the validation and application of immunoassays and bioassays for biopharmaceutical analysis are incorporated within ICH topics Q2A/B and Q6B1-3. FDA has provided more specific guidance4 on the approach to be adopted for the validation of these methods for bioanalytical applications. There is a draft CHMP guideline5 that discusses application to the pharmacokinetic assessment of therapeutic proteins – which recommends the use of a combination of immunoassays and bioassays for clinical development. Common Technical Specifications6 define minimal requirements for the diagnostic sensitivity for in vitro diagnostic medical devices regulated under Annex IIA of Directive 98/79/EC.

In addition to this general guidance, regulatory guidelines for specific applications, eg, the registration of immunoassays as diagnostic tests, are also available7-9. Product-specific requirements for bioassay design, eg, for vaccines10 and monoclonal antibodies (mAbs)11,12, may also apply.

Pharmacopoeial standards13,14 apply to the design and data interpretation of bioassays used for product batch release.

Definitions

ImmunoassayAn immunoassay is a quantitative or qualitative analytical method that relies on the binding of an antibody to an antigen.

BioassayA bioassay is a quantitative analytical method that measures a defined biological effect in a living system, such as an intact cell.

4Regulatory Rapporteur

T O P R A – T H E O R G A N I S A T I O N F O R P R O F E S S I O N A L S I N R E G U L A T O R Y A F F A I R S

3. Other key reference documents of regulatory significanceIn addition to these guidance documents, a number of important publications are available that address key aspects of bioanalytical method design, validation and interpretation15-18, including application to the measurement of anti-product antibody responses for the non-clinical and clinical evaluation of immunogenicity19.

4. Applications in biopharmaceutical development and product quality controlImmunoassays and bioassays are used throughout the biopharmaceutical development cycle (Table 1). It is important to distinguish different modes of application: for example, the considerations for the design and validation of methods used for product quality control purposes are not the same as those used in bioanalytical (to measure analytes in physiological sample matrices for pharmacokinetic (PK) or immunogenicity) applications.

4.1 Immunoassays

Immunoassays are often developed during the research phase, and may be used in the selection of the lead candidate for entry into formal manufacturing process development. The most important attribute of these assays is the relatively low detection limit for a specific analyte that is conferred by the high affinity of an antibody for a cognate antigen; this high specificity enables measurements to be made in complex sample matrices without an intermediate purification process. Also, the suitability of microtitre plate-based formats coupled with inexpensive reagents enables a high-throughput approach using relatively small amounts of sample.

A “sandwich” ELISA format (Figure 1) will commonly be used to monitor the expression levels of the unpurified drug substance in the fermentation medium, to enable optimisation of the upstream process. The same assay may also be used to monitor the down-stream purification process, with respect to both the drug substance and to residual host cell protein.

Following purification, a critical quality parameter for a monoclonal antibody product will be its binding affinity for the target antigen: a relative measure may be determined using a competitive or displacement ELISA (Figure 2); the competitive format has the advantage of maintaining the analyte in the solution phase. If the product is a cytokine, the same principle could be used to measure binding to a soluble receptor domain.

Immunoassays are also applied to quality control, characterisation and stability testing of biopharmaceutical products, sometimes as “hybrid” assay formats in combination with a bioassay20. They may also be developed in their own right as marketed products, ie, as in vitro diagnostic devices.

4.2 Bioassays

Since the binding of a cytokine to its receptor, or a monoclonal antibody to a target antigen, may not necessarily correlate with biological potency, the bioassay represents an essential complementary tool for product characterisation and quality control for most therapeutic proteins. A bioassay will provide a composite response that depends not only on ligand-receptor binding, but also on signal transduction and the end response. In the case of many monoclonal antibody products, the molecule may actually have more than one biological effect: the variable region idiotope mediates antigen recognition, while the Fc domains may activate CDC and/or ADCC. The same is true of interferons, for which alternative bioassays options have been applied21.

Subtle conformational changes in the structure of a protein molecule may affect functional activity, eg, by reducing receptor binding affinity, in a manner that is difficult or impossible to detect using physicochemical techniques. Thus, a primary role of immunoassays is to use the exquisite sensitivity of antibody reagents to probe the integrity of the conformation of the recognition sites (epitopes) in a target antigen. A bioassay will then be used to confirm that there is no impact on downstream signalling or effector function. This is necessary because other elements of the protein structure, remote from the epitope recognised by an antibody, may impact on the biological activity. The differing effect of glycosylation on the in vitro and in

vivo biological activity of epoetin exemplifies this case22.

4.3 Antigenicity vs. immunogenicity

Immunoassays rely on the property of the antigenicity of a given molecule: this means the capacity of a molecule – the antigen – to bind to pre-formed antibody. A single antigen may contain multiple epitopes, enabling recognition by many different antibodies. Accordingly, a panel of antibodies could be used to probe different aspects of the structural integrity of a biopharmaceutical product. Some antibodies will be highly sensitive to the native state of the protein – they recognise “conformational” epitopes, which could consist of amino acids that are remote in the primary sequence, but which are brought together in close proximity in the 3-dimensional structure that exists in solution phase – whereas other antibodies will target an epitope that represents a linear sequence (typically 6 amino acids in length) in the primary structure; these latter antibodies may be useful for probing proteins in the denatured state – eg, using SDS-PAGE/Western blotting.

It is important to distinguish the term antigenicity from immunogenicity, which reflects the ability of an antigen to stimulate an immune response. An antigen, particularly if it is a small molecule (molecular weight less than 1000 Da), may not be immunogenic by itself; it may need to be combined with a larger molecule that facilitates the immune response to the “hapten”.

For biopharmaceutical products that represent a replacement for endogenous human proteins (eg, growth hormone, insulin, coagulation factors, epoetin, G-CSF) an important regulatory consideration will be the demonstration of the absence of undesired immunogenicity, which could blunt efficacy and, more seriously, give rise to antibodies that cross-react with the endogenous factor. On the other hand, for vaccine products, the most important quality control element will be the capacity to induce a protective or a therapeutic immune response. Consequently, the assessment of immunogenicity is an integral part of the quality, non-clinical and clinical dossiers for all biopharmaceutical products.

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In certain cases, antigenicity may correlate with immunogenicity – however, such a correlation must always be established during the development cycle. A definitive demonstration of this correlation often depends on clinical validation, using the results of a Phase III efficacy study to confirm that the method used to control the potency of the drug product truly reflects the desired clinical response.

5. Choice of assay formatWithin the broad definitions of “immunoassay” and “bioassay” given above, there is a wide diversity of methodological formats (Table 2). Usually, different assay formats will be compared during the initial part of the product development cycle, and the most suitable options may then be validated to support product registration.

It should be remembered that assays developed in the research environment might not necessarily be suitable for a quality control environment: a common problem is the failure to define the intended purpose of the method and then to establish acceptance criteria and validate accordingly.

The development of a potency assay for product Quality Control purposes often requires the parallel evaluation of different assay formats. If the biopharmaceutical product exerts its action through binding to a soluble target, eg, in the case of the neutralisation of a pro-inflammatory cytokine by a monoclonal antibody, the ICH Q6B guideline allows a ligand-binding approach (eg, ELISA or SPR) to be used in place of a bioassay. Nevertheless, the sponsor should try to demonstrate a correlation between the response parameter in the immunoassay with a biological activity as measured in a bioassay.

Because the ICH Q6B guideline defines potency in terms of a “quantitative measure of biological activity based on the attribute of the product which is linked to the relevant biological properties”, the sponsor will have to provide a plausible justification for the choice of the potency assay format. This places emphasis on building a structure-activity database commencing in the pre-clinical phase, to enable correlations to be drawn between physicochemical and biological attributes on

the one hand and pharmacology data on the other.

If the biopharmaceutical product has more than one biological activity, eg, in the case of a monoclonal antibody with an FC -mediated effector function, it will be necessary to measure all functions11. Measures of enzymic activity may substitute for a true bioassay to control the potency of certain types of products, such as blood coagulation factors23.

For the assessment of immunogenicity, a bioassay24 will be used in tandem with an immunoassay25,26 to enable measurement of the neutralising capacity of anti-product antibodies detected in the immunoassay that is used as the primary screening tool. Similarly, a potency assay for a vaccine product often comprises an ELISA to measure the anti-product response generated in a whole-animal bioassay.

6. Particular regulatory issues for immunoassays and bioassays

6.1 Assay validation

Regulatory guidance recognises that immunoassay and bioassay formats have particular features that increase the complexity of their design, validation and interpretation. For example, the ICH Q2B guideline acknowledges that:

“Due to their complex nature, analytical procedures for biological and biotechnological products in some cases may be approached differently than in this document."

“Some analytical procedures, such as immunoassays, do not demonstrate linearity after any transformation. In this case, the analytical response should be described by an appropriate function of the concentration (amount) of an analyte in a sample.”

This places more responsibility on the sponsor to justify the particular approach taken, including the refinement of the assay conditions to minimise bias and the validity of the data analysis and interpretation approaches.

Guidance for the validation of specific diagnostic test applications7,8 must be

followed if available but, in general, the evaluation should enable a detailed description of the characteristics listed in Table 3 – which will be used to define the Standard Operating Procedure (SOP).

Validation for bioanalytical applications should be approached in two phases5:

1. An examination of the assay performance relative to defined acceptance limits/validation criteria, including stability of analyte in relevant matrix, specificity, accuracy/dilutional linearity, precision, limit of quantitation, limit of detection;

2. Application to real samples in the relevant biological matrix, with appropriate calibration by reference standard(s) to enable quantitation and inclusion of control samples to confirm that system suitability criteria have been met for any given assay run.

Terms such as “sensitivity” and “specificity” have defined regulatory meanings in the case of diagnostic applications of immunoassays6:

Definitions – Diagnostic applications of immunoassays

SensitivityHow often the test is positive when the disease is present.

SpecificityHow often the test is negative when the disease is absent.



6.2 Reference standards

European Law (Directive 2003/63/EC, 3.2.2.1) specifies:

“Units of biological activity shall be used for substances which cannot be defined molecularly. Where an International Unit of biological activity has been defined by the World Health Organisation, this shall be used. Where no International Unit has been defined, the units of biological activity shall be expressed in such a way as to provide unambiguous information on the activity of the substances by using where applicable the European Pharmacopoeia Units.”

The guidance in ICH Q6B adopts a consistent position:

“The results of biological assays should be expressed in units of activity calibrated against an international or national reference standard, when available and appropriate for the assay utilised.

“Where no such reference standard exists, a characterised in-house reference material should be established and assay results of production lots reported as in-house units.”

A corresponding requirement applies to immunoassays, as indicated by the European Pharmacopoeia27:

“It is essential to standardise the components of an immunoassay and to use, wherever available, international reference preparations for immunoassays.”

The European Directorate for the Quality of Medicines (EDQM) supervises the Biological Standardisation programme that is facilitated by the network of Official Medicines Control Laboratories. The latter support collaborative studies to establish new reference standards using common methodology28,29, as well as conducting batch release testing and market surveillance. Where a sponsor is developing a novel biological entity, early contact with an Official Medicines Control Laboratory, eg, National Institute for Biological Standards and Control (NIBSC), will enable the development of appropriate reference standards.

There are situations where the analyte to be measured does not represent a defined substance, eg, a polyclonal anti-HIV antibody response. In this case, it will be necessary to

establish a detection limit using a representative panel of positive clinical samples.

6.3 Critical reagents

The EU GMP guide30 – in addition to the relevant ISO standard31 – requires critical reagents to be controlled rigorously. Particular factors for immunoassays and bioassays32 are summarised in Table 4.

Batch-to-batch variability of test kit reagents (including controls) may have a significant influence on results. It is the sponsor’s responsibility to confirm the validity of a test kit for a given purpose: selectivity and sensitivity, in particular, should be verified using the authentic sample matrix and assay conditions.

6.4 Data analysis

Immunoassay and bioassays are usually configured to include dilutions of the test article and the reference material so that their responses can be compared at several concentrations. Similar behaviour of the test and reference samples is confirmed by the absence of any departure from parallelism of the respective dose-response curves following mathematical transformation of the raw data to yield a linear plot

Immunoassays are usually calibrated with a reference preparation of the antigen whose concentration has been determined by a physicochemical method, so that one or more dilutions – with each dilution tested as three replicates – of the test sample can be assigned a concentration value based on mass units from the corresponding response obtained by interpolation of the data points for the reference material. A linear regression model can often be applied to immunoassays used in the Quality Control setting, whereas a 4-parameter logistic model (extended sigmoid dose-response curve) is more common for bioanalytical applications. This reflects the fact that many biological assays display linearity over a relatively limited concentration range.

Pharmacopoeial standards apply to the analysis of data generated by immunoassays and bioassays for use in drug product quality control13,14. Alternative data analysis models may be applied. For example, a 4-parameter logistic model may be used to examine

the displacement of the steep part of the concentration-effect curve relative to the reference material to determine the potency of the test article (Figure 3). Acceptance criteria for individual assay runs can be set for parameters such as the relative slope, asymptotes and variance from the fitted curve. Although data analysis software to support this type of analysis is commercially available33, a specialist biostatistician should be consulted to advise on the most valid approach for a given assay.

Acceptance limits should be specified both for the potency (eg, 80 – 125 % of stated value) of the product and for the 95% confidence interval that describe the uncertainty (error) associated with the measurement. In practice, replicate assays are usually performed to estimate the mean potency for any given batch of drug substance/product; the mean potency for test batch must fall within the specified limits and the error associated with the measurement (95% confidence interval) must also fall within the specified range (referred to as the fiducial limits, eg, 64-156 %, Figure 4).

Potency is always a relative measure, and the stated potency of a biopharmaceutical product that is determined in a bioassay refers to units of biological activity assigned to the reference standard. Some biopharmaceutical products (eg, monoclonal antibodies without effector function) may be controlled using an immunoassay rather than bioassay, based on an earlier demonstration of correlation between the activity and concentration parameters, in which case a mass unit could be used for product labelling. In the case of human insulin, potency may be determined via physicochemical techniques34.

Regulatory agencies will expect that the specification limits defined for the potency of a biopharmaceutical product are based on a multi-factorial empirical basis, including:

■ Actual performance of a validated assay applied under routine operating conditions; and

■ Batch-to-batch product variability; and

■ Sensitivity of the assay to detect changes in critical product quality attributes, ie, parameters that could impact on safety and/or efficacy.



Thus, the specification for potency may change as a function of the accumulated quality, non-clinical and clinical data.

6.5 Precision and accuracy

The higher variability of biological systems will generally render bioassays as being intrinsically less precise than physicochemical analytical systems. Immunoassays tend to have an intermediate level of variability, and may be subject to experimental bias (eg, positional effects on microtitre plates) that will need careful evaluation and control. A typical acceptance criterion for intra-assay precision of immunoassay quality control samples is ≤ 20%.

For immunoassays, it may be possible to determine the absolute/nominal concentration of an antigen using an independent method. Therefore, accuracy can be expressed relative to this nominal concentration.

On the other hand, since the read-out for a bioassay is relative to the activity of the reference standard, accuracy for a bioassay will tend to refer the dilutional linearity of

different amounts of the calibrant when spiked into the assay matrix. For example, the read-out at each dilution should correspond to 80 – 120 % of the expected value within the defined measurement range of the assay.

6.6 Quality control samples

These are samples of the analyte that have been prepared in the authentic assay matrix, ie, do not need any further manipulation prior to use in any given assay run, that are used to provide an assessment of the intra- and inter-assay variability. Typically, the analyte level represents three different points on the calibration curve: low, medium and high. The lowest value should be just above the lower limit of quantitation (LLOQ).

Quality control samples (3 replicates at 3 concentrations) must be included on each assay plate, and enable the performance of the assay to be assessed for proximity of the calculated analyte levels relative to historical values. This provides an alert for errors in preparing the calibration curve in any particular assay run, for systematic bias across assay plates or drift between assays due to

reagent instability. System suitability criteria relating to the precision (eg, intra-assay variability for at least 5 determinations of a low positive control in 1 run) and accuracy of the QC samples will be established to enable acceptance or rejection of the results of individual assay runs.

6.7 Qualitative immunoassays

Immunoassays may be applied in to produce qualitative data, eg, to distinguish between positive and negative samples in a clinical population7,8. A cut-off value must be established during assay validation that meets pre-specified criteria for detection of false positives and false negatives6.

7. Concluding remarks: limitations of immunoassays and bioassays

The regulatory scientist needs to be aware of potential sources of spurious results that could be challenged by regulatory agencies. My choice of the “top 4” points-to-consider for each assay mode are as follows:

3rd Annual TOPRA Symposium2–4 October 2006 Hilton, Amsterdam, The Netherlands

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For 2006 we are delighted that with the agreement of TOPRA Advisory Council member, Dr Aginus Kalis, the Dutch Medicines Evaluation Board (MEB) will be assisting with the organisation of the programme and this meeting will be an ideal opportunity to get to know this key agency better.

Also in 2006 the programme will be expanded to include sessions covering medical technologies and veterinary matters, amongst others.

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As always there will be a trade exhibition showcasing companies with products and services to assist the regulatory professional, and a social event for informal networking.

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Immunoassay issues■ Adsorption onto a solid phase, or

conjugation to a reporter moiety, could alter the binding affinity of an antibody for its cognate antigen – this should be tested using alternative assay formats.

■ Antibodies can cross-react with apparently unrelated factors present in a complex sample matrix – specificity

must be examined thoroughly during method development and validation, ideally using competitive assay formats.

■ Antibody-antigen complexes present in a sample may interfere with the measurement of the un-associated components – validation for bioanalytical applications should examine the reactivity of both the unbound and bound analyte.

■ Endogenous antigen may interfere with measurement of the drug product in biological samples – the assay cut-off value/LLOQ should be evaluated using a sufficiently large number of samples from untreated subjects and the analyte level in the pre-dose sample must be below a pre-specified threshold.

Acknowledgements

I would like to thank Dr Steve Holmes (Domantis Ltd and, formerly, SmithKline Beecham) who taught me how to use antibodies as analytical reagents. During a number of collaborations, Dr Jane Robinson (NIBSC) guided me through the regulatory principles of applying bioassays and Tony Mire-Sluis (Amgen Inc) brought clarity to the use of these assays to evaluate the immunogenicity of biopharmaceutical products.

Bioassay issues■ The response parameter (functional

activity) is highly sensitive to other factors present in the sample matrix and/or assay medium – specificity and robustness must be rigorously examined.

■ Sensitivity and/or precision are often inferior to immunoassays – suitability

for the intended purpose must be adequately justified.

■ The specific activity of a recombinant protein may be different from the native protein – specific activity should be determined using a combination of validated functional and physicochemical methods.

■ Data analysis is complex – seek advice from a specialist biostatistician to select the most appropriate model.

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49. Hammerling U, Henningsson AC & Sjodin L. Development and validation of a bioassay for interleukin-2; Journal of Pharmaceutical and Biomedical Analysis, 1992, 10 (8), 547 – 553.

50. Hammerling U, Kroon R & Sjodin L. In vitro assay with enhanced sensitivity for human granulocyte colony-stimulating factor; Journal of Pharmaceutical and Biomedical Analysis, 1995, 13 (1), 9 – 20.

51. Meager A. Quantification of interferons by anti-viral assays and their standardization; in ‘Lymphokines and Interferons – a practical approach’, Clemens MJ, Morris AG, Gearing AJH (Eds), Oxford, IRL Press, 1987, pp 129 – 147.

52. A simple technique for quantifying apoptosis in 96-well plates. Ribble D, Goldstein NB, Norris DA & Shellmann YG; BMC Biotechnology 2005, 5, 12. Available at: http://www.pubmedcentral.gov/picrender.fcgi?artid=1142306&blobtype=pdf

53. Jansen WTM, Gootjes, Zelle M, Verhoef J, Snippe H & Verheul AFM. Use of highly encapsulated Streptococcus pneumoniae strains in a flow-cytometric assay for assessment of the phagocytic capacity of serotype-specific antibodies; Clinical and Diagnostic Laboratory Immunology, 1998, 5 (5) 703 – 710.

54. Bergman I, Basse PH, Barmada MA, Griffin JA & Cheung NK. Comparison of in vitro antibody-targeted cytotoxicity using mouse, rat and human effectors; Cancer Immunology and Immunotherapy, 2000, 49 (4-5), 259 – 266.

55. European Pharmacopoeia Chapter 2.7.16: Assay of pertussis vaccine (acellular), 01/2005:20716.

56. Kowarski CR, Liaou MY, Kowarski D, Weizer J, Boynes D & Kowarski AA. Improved bioassay for glucagons by continuous monitoring; Journal of Pharmaceutical Science, 1984, 73 (9), 1298 – 1299.

57. European Pharmacopeia 5.3: Concentrated Erythropoietin Solution, 01/2006:1316.



Application Example

Selection of product development candidates

■ ELISA to measure binding affinity of a mAb

■ In vitro cell-based bioassay to biological activity of a cytokine

Process development and in-process controls

■ ELISA to monitor yield of specific protein in product stream during recovery and purification

■ Bioassay to confirm target activity in bulk intermediates

Quality control ■ In vitro or in vivo bioassay to determine the potency of drug substance/product

■ SPR for batch release of receptor binding protein

■ ELISA to confirm identity of drug substance/product

■ SDS-PAGE/Western blotting to detect product-related variants in drug substance/product

■ ELISA and SDS-PAGE/Western blotting analysis of residual host cell protein

■ Cytopathogenicity bioassays to demonstrate viral clearance capacity of downstream process

Product characterisation ■ SPR (eg, BIAcore) analysis of association and dissociation rates for specific product conformers

■ In vivo bioassay to confirm impact of post-translational modification (process-related product heterogeneity) on PK/PD

Drug substance/Product stability testing

■ ELISA and in vitro bioassay are typically included as stability-indicating tests during formulation development and on-going stability testing programme. SPR is also used for this purpose

Product comparability analysis post-manufacturing change

■ Sensitivity of immunoassays and bioassays makes them essential tools to evaluate impact of process-related heterogeneity on critical product quality attributes. Typically, include a panel of bioassays and immunoassays in a product comparability protocol

Bioanalysis ■ Pharmacokinetic assessment using ELISA

■ Immunogenicity evaluation (ELISA, RIP or SPR in combination with a bioassay)

■ Monitoring biomarkers of efficacy and safety, eg, ELISA to measure pro-inflammatory cytokines or flow cytometry to assess immune status/suppression

Diagnostic assays ■ ELISA to measure expression levels of tumour-associated antigen(s) to enable recruitment of patients most likely to derive clinical benefit

Table 1: Applications of immunoassays and bioassays to biopharmaceutical development

Dr.Paolo M. Biffignandi, MD, Ph.D, FTOPRARegulatory & Strategic Advice Preparations of Nonclinical & Clinical Overviews and Summaries, QOS

Tel: +39-011-00148.38 Fax: +39-011 -00148.37 Mobile: +39-348-5146.006email: [email protected]



www.topra.orgwww.topra.orgwww.topra.org

Fig 1: Sandwich ELISA format to measure antigen concentration in a complex sample matrix

Antigen (Analyte)

Detector antibody – reporter conjugate

signal

During sequential incubation steps, the capture antibody is passively absorbed on the surface of microtitre plate wells, followed by sample containing the cognate antigen (= analyte) and then a secondary antibody (recognising a different epitope from the coating antibody) conjugated to a reporter group, (eg, horse-radish peroxidase). The measured signal is directly proportional to antigen concentration.

Capture antibody

Fig 2: Competitive ELISA formats to compare binding of sample vs. reference antigen to a limiting number of antibody binding sites

The competitive ELISA enables a comparison of the relative affinity of a test antigen vs. reference antigen for binding to a specific antibody. Either the antigen or the antibody can be adsorbed onto the microtitre plates. The signal is inversely proportional to the competing antigen (analyte or reference) concentration.

signal

signal

Competing Antigen (Analyte or Reference)

Adsorbed Antigen

Detector antibody – reporter conjugate

Antigen – reporter conjugate

Capture antibody

Competing Antigen (Analyte or Reference)



Types Of Immunoassays

Enzyme-linked immunoassay (ELISA)35

Radioimmunoassay (RIA)36

Radioimmunoprecipitation assay (RIP)25

Fluorescent immunoassay (FLIA)37

Time-resolved fluorescence (TRF)38

Chemiluminescent immunoassay (CLIA)39

Enzyme-linked immunospot (ELISPOT)40

Surface Plasmon Resonance (SPR)41

Multi-analyte profiling (MAP)42

SDS-PAGE/Western blotting43

Flow cytometry44

Table 2: Examples of different immunoassay and bioassay formats

Types Of Bioassays

Receptor activation

■ Kinase Receptor Activation Assay (KIRA)45

■ Phosphospecific Antibody Cell-based ELISA (PACE)46

Signal transduction event

■ Intracellular cAMP (follicle stimulating hormone)47

■ STAT nuclear translocation (interferons)48

End response

■ Cell proliferation (growth factors)49, 50

■ Cytopathic effect reduction (interferons)51

■ Apoptosis (anti-tumour agents)52

■ Opsonisation/phagocytosis (anti-infectious agents)53

■ Complement-dependent cytotoxicity (CDC)12 and antibody-dependent cellular cytoxicity (ADCC)54 (monoclonal antibodies with functional Fc)

Whole-animal systems

■ Immunogenicity (vaccines)55

■ Hyperglycaemia (glucagon)56

■ Erythrogenesis (erythropoeitin)57

Table 3: Standard Operating Procedures for immunoassays and bioassays: Characteristics to be described

■ Storage and preparation procedure for samples, controls, reference standard and assay reagents, including instructions for freeze/thaw steps

■ Description of equipment (microtitre plate type, plate shaker/incubator, plate washer, plate reader, data analysis software etc)

■ Details of all incubation and intermediate washing procedures (including reagent volumes and tolerance limits for time and temperature)

■ Clear instructions on data analysis procedure

■ System suitability criteria for acceptance of the results of any given assay

■ Handling of outliers/re-testing criteriaYour contacts:Paula HARRY International Project Manager - Tel: +33 (0)1 39 45 60 03 - [email protected] Saholy RAKOTOMANGA Director, Regulatory Affairs - Tel: +33 (0)1 39 45 60 00 - [email protected] - Energy 4 - 34, avenue de l’Europe - BP 136 - 78148 Vélizy Cedex - FRANCE

Consultancy in regulatory affairs:• Information • General and strategic advice • Audit of MA fi les • Support in mutual recognition and decentralised procedures, etc.

Marketing authorisation application dossiers:• Evaluation, writing and/or formatting • Submission to authorities and follow-up

Coordination and tracking of studies:• Pharmaceutical and clinical development

Let us manage your regulatory affairs projects in France and in Europe

A French Regulatory Af fairs Consultancy Companywww.medibridge.fr

MB-TOPRA-2006b.indd 1 27/01/06 10:45:37



Immunoassays Bioassays

Coating/competing antigen

Primary antibodies

Secondary antibodies

Conjugated antibodies

Assay buffer components

Wash buffer components

Blocking reagents

Detection reagents

Commercial kit active components

Plastic cuvettes or microtitre plates

Source of cell substrate (homogeneity and viability)

Stage of cell culture (passage number and density)

Basal medium

Serum and other growth factors

Antimicrobial agents

Cell harvesting reagent (eg, trypsin)

Plastic flasks or plates

Table 4: Critical reagents to be controlled in immunoassays and bioassays [modified from Ritter & Wiebe32]

Your contacts:Paula HARRY International Project Manager - Tel: +33 (0)1 39 45 60 03 - [email protected] Saholy RAKOTOMANGA Director, Regulatory Affairs - Tel: +33 (0)1 39 45 60 00 - [email protected] - Energy 4 - 34, avenue de l’Europe - BP 136 - 78148 Vélizy Cedex - FRANCE

Consultancy in regulatory affairs:• Information • General and strategic advice • Audit of MA fi les • Support in mutual recognition and decentralised procedures, etc.

Marketing authorisation application dossiers:• Evaluation, writing and/or formatting • Submission to authorities and follow-up

Coordination and tracking of studies:• Pharmaceutical and clinical development

Let us manage your regulatory affairs projects in France and in Europe

A French Regulatory Af fairs Consultancy Companywww.medibridge.fr

MB-TOPRA-2006b.indd 1 27/01/06 10:45:37



Fig 3: Four-parameter logistic curve model for estimating potency

Fig 4: Acceptance limits for potency and fiducial limits for 95% confidence interval [adapted from Dr Jane Robinson, NIBSC]

Limits around stated potency

Stated Potency

mean ±

pass fail retest retestfail/ fail/

80%

64%

156%

125%

95% CI

Fiducial limits

ReferenceStandard

Response

Test Sample

1n concentration

Potency is a relative measure that is calculated from the displacement of parallel response curves for the test material relative to the reference standard.

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