L4 Team Draft Reagents and Their Stability

22 March 2012

L4: Reagents and their stability - Link with tiered approach Team members: Team lead •  Lindsay King – NA (EST)– lindsay.king@pfizer.com Other members •  Susanne Phil – EU (CET) •  Mark Ma– NA–(PST) •  Esme Farley – NA (EST) •  Priya Sriraman– NA (EST) •  Masood Khan-NA (CST) •  Jeannine Keefe-NA (EST) •  Mami Imazato-APAC (Japan)

In scope: LBA Critical Reagents •  What are critical reagents

•  Ab, peptides proteins, conjugates, Drug as reagent, ADA reagents including positive and negative control.

•  Reagent Documentation •  Reagent testing

•  Specificity testing •  What to do when you change critical

reagents •  Batch to batch testing

•  Stability of reagents •  Testing •  Reagent formulation

•  In-house vs. commercial reagents pros and cons •  Reagents and assay transfer

 

 

   

Out of scope: •  Reference Standards •  Internal Standards •  Cell Based PK assays •  Matrix •  Commercial Kits

   

 

   

Interdependencies with other teams – if any A3 Method Transfer: Ray Briggs A4 Reference Standards and Reagents: Joseph Bower A6 Stability: Nico van de Merbel L2 Large molecule specific assay operation: Lauren

Stevenson A8 Team Documentation: Tom Verhaeghe

 

 

   

27  April  2012  

Content reflects work in progress •  Have not addressed reagent in assay transfer yet •  Have identified some gaps •  Gaps need to be further refined

Still formulating draft recommendations •  Target May 25th completion date Key areas of focus •  Reagent lot testing •  Reagent stability testing •  In house vs Commercial •  Documentation related to these three areas

L4: Reagents and their stability Readout Status

27  April  2012  

L4: Critical Reagent Definition Reagent that are analyte specific are most often considered critical reagents; Ab, peptides, proteins, conjugates, Drug as reagent, ADA reagents including positive and negative control. Regulatory agencies have defined critical reagents for LBA and ADA controls

Literature expand this definition to include; peptides, such as receptors or ligands and fragments thereof and in some instances biological matrices Nowatzke and Woolf, 2007, Staak et. al., 2011, O’Hara et. al., 2012

General agreement in literature positive and negative controls need to be thoughtfully derived and well characterized. Shankar 2008, Mires-Sluis 2004  

General Agreement in Regulatory Guidance and Literature 26  March  2012  

L4: Critical Reagent Definition Additional considerations: rare critical reagents, difficult to replace, single source critical reagent

life cycle management “If there is only a single supplier of a critical reagent then it is advisable to consider some contractual relationship that warns the user about product changes or cessation of production.” Mire-Sluis 2004

“Successful lifecycle management of LBA critical reagents minimizes assay performance problems caused by declining reagent activity and can mitigate the risk of delays during preclinical and clinical studies. “O’Hara 2012

“To enable bioanalytical project support over the complete product life cycle, an appropriate long-term reagent supply is needed .”Staak 2011

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L4: Existing Guidance Critical Reagents    •  “Key reagents, such as antibody, tracer, reference standard and matrix should be

characterized appropriately and stored under defined conditions.” •  “Assay reoptimization or validation may be important when there are changes in

key reagents .” •  “..binding should be checked, Performance should be verified with standard curve and

QC’s, Key cross reactivity should be checked .” (FDA Guidance for BA Method Validation May 2001. )

•  “FDA believes positive control or QC samples are critical. The nature of the detection agent is also critical “ (FDA Guidance for Assay Development for Immunogenicity Dec 2009)

•  “Critical reagents ……have direct impact on the results of the assay and therefore their quality must be assured. “

•  “Conditions guaranteeing the maintenance of the stability of both non critical and critical reagents should be documented in order to ensure that the performance of the method is not affected over time. “

•  “When changing reagent batches during validation or sample analysis the analytical performance of the method must be verified to ensure that it is not altered …..”

(EMEA Guideline on BA method validation 2011)

 

     

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L4: Existing Guidance Critical Reagents •  “…binding reagents (e.g. binding proteins, aptamers, antibodies or conjugated

antibodies) and those containing enzymatic moieties have(ing) direct impact on the results of the assay…”

(EMEA Guideline on BA method validation 2011)

•  well characterized positive and negative controls. These reagents function as critical assay reagents” (EMA guideline on immunogenicity assessment emea/chmp/bmwp/14327/2006 )

•  “The analytical procedure refers to the way of performing the analysis. … describe in detail the steps necessary to perform each analytical test. This may include but is not limited to: the sample, the reference standard and the reagents preparations.” (ICH Q2(R1) 2005)

•  “…reagents….should be labeled to indicate identity (with concentration if appropriate),

expiry date and specific storage instructions”. (OECD ENV/MC/CHEM(98)17 1998)

•  Guidance documents from Japan, Brazil, China and India did not have any specific

guidance for LM-BA reagents

26  March  2012  

L4: White-Papers and Draft Guidance's Methods/ Criteria

•  Reagents used in assays need to qualified and acceptance specifications set, at least for those, which are most important. (EMA Guideline on Biotechnology-derived Therapeutic Proteins, 2010)

•  should demonstrate that the labeling of the detection antigen does not significantly obscure critical antigenic determinants. (Section 3.B.2: FDA (Draft) Guidance Assay Development for Immunogenicity Testing of Therapeutic Proteins)

•  ….should assess which conditions are likely to vary and design appropriate tests to

examine the parameters that are deemed critical. These may include changes in microtiter plate lots, reagent lots…... Study samples, or the positive control samples, can be used to test assay robustness. The use of acceptance criteria for ..… robustness validation (computed from the assay development….or validation control acceptance criteria) is recommended. (Shankar et. al 2008)

26  March  2012  

L4: White-Papers and Draft Guidance's Documentation

•  “Batch records documenting the preparation of ligand binding reagents including the source of the raw material, the method of expression, purification, final formulation buffer…” (Rup et. al. 2007)

•  “An appropriate, well-documented and reproducible purification protocol should be applied” (Staak et. al.20011)

•  “For ligand binding assays, the method of reagent qualification should be fully

described by the assay protocol or SOPs. Acceptance criteria should be defined a priori in an SOP.” (Nowatzke et. al. 2007)

•  “The actual conditions of use should be stated in documentation. .. further issues are

now addressed and details are provided herein (Table 1) to facilitate effective documentation.” (CC3 white paper :Vishwanathan et. al. 2007)

 

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L4: Agreement in Guidance

 

     

20  March  2012  

• Suggest a need to identify and understand critical reagents

• Suggest a need to assess impact of lot changes • Need to define Acceptance criteria

• Suggest a need to assess reagent stability • Need to define acceptance criteria

• Suggest a need to document

• Focus on PK and ADA • Include Biomarkers in thinking

L4: GAPS in Guidance •  What would be “sufficient characterization of key reagents” – for

“assurance of quality” ? What documentation needs to be available for each reagent? (CC3 documentation table does not address LM Critical reagents)

•  Under what conditions will a critical reagent change warrant a revalidation

(or partial validation) of an assay. Acceptance criteria need to be defined.

•  How lot change comparisons should be conducted or acceptance criteria set

•  The guidelines do not specify how reagent stability should be assigned and/or tested. No standardized acceptance criteria.

•  Where to document reagent stability

 

     

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L4: Reagent Documentation Gaps •  Regulations and regulatory guidelines do not provide specific documentation

for reagents and reagent stability. General documentation includes; SOPs, analytical procedures, labeling.

•  Literature provides recommendations for specific reagent documentation; “Batch records documenting the preparation..”

Rup et al., 2007 “ well-documented and reproducible purification protocol…”

Staak et al, 2011 “ reagent qualification should be fully described by the assay

protocol or SOPs. Nowatzke et al, 2007

             

•  Best practices for reagent documentation need to be better defined •  SOP can be used to define the need and process but actually

documentation may take many forms

26  March  2012  

L4: Critical Reagents Usage: Initiation Phase

1. Selection/Characterization: Selection of critical reagents based on: –  Analytical requirements and Assay

performance –  Sufficient characterization to enable

process control (FDA/GLP = identity, concentration, storage)

–  Recommend (as appropriate) - Identity, source, purity, concentration (or titer), MW, binding affinity, isotype (Mab), CDR, incorporation ratio, aggregation level

–  Also advisable to include availability as a consideration

 

2. Characterization/Qualification: -  Specificity (min. cross-reactivity

with structurally related compounds)

-  Selectivity (non-specific binding and/or interference

-  Assay performance data required, may be augmented with orthogonal testing

-  Stability discussed elsewhere

Guidance documents and whitepapers have broad expectations of critical reagents “appropriate characterization” but how to achieve commonality in this effort needs to be delineated.

26  March  2012  

L4: Critical Reagents Usage: Life Cycle Phase

3. Changing Critical Reagents (Tier 1) : If new reagent part # (original reagent no

longer available); new supplier; new animal ; new cell line etc;

–  Identify and characterize new reagent, including negative controls

–  Compare to original reagent (re-optimize if needed) in assay performance

–  Where possible testing in parallel with previous lots, multiple runs.

May need Re-validation for Regulated work

4. Changing Critical Reagents (Tier 2) : If New batch/lot # (original lot depleted or

expired); new purification or dilution from ‘mother’ lot;

If New labeling/conjugation –  Check of specific parameters that

have changed e.g. concentration for newly diluted prep., molar ratio for conjugate

–  Reagents may be changed in full assay batches or individually (more work).

Recommend tiered approach for control of critical reagents over time, and appropriate documentation to cover these activities:

267April  2012  

L4: Critical Reagents Usage: Life Cycle Phase

4. Changing Critical Reagents (Tier 2 continued) :

Test performance in assay as follows: –  Single parameter, with same CoA – 1

qualification run in parallel with original lot.

–  Single parameter, with same CoA, no overlap – 3 independent qual runs.

–  Multiple parameters - 3 independent runs or partial revalidation for GXP.

5. Changing Critical Reagents (Tier 2 continued) :

Failure of specifications in LBA test performance:

–  Check characterization –  Reoptimize assay –  Assay performance testing in multiple

runs  

Recommend failure of assay performance specifications after ‘minor’ changes loops over to Tier 1 activities:

Monitor performance with a priori acceptance criteria (as defined in method) for QCs + Calibrators as well as monitoring assay signal criteria; use both reagent lots in parallel; use control charting for monitoring through life cycle.

26  March  2012  

L4: Management of Reagent Stability

How to define the reagent stability Expiry or test/retest dates can be assigned based on experience with similar classes of reagents a) Approach conservatively at start and set short time period and then extend as get experience with reagent b)Assign very long and monitor performance and then revise when we see changes. This approach needs real time monitoring processes to allow this.  

Reagent stability and life cycle management The stability of critical reagents can be tested directly and either though real time or accelerated stability testing

Reagent life cycle management 1) Generation of large amounts of a

single lot a critical reagent reduces risk of lot changes

2) Reagents must be stored as recommended by the manufacturer. If not the may need to generate the appropriate storage stability data

3) Single use aliquots can be used to reduce risk of instability and contamination

Stability testing of reagents is not well defined in the guidance and is the area with the most gaps and range of practices

27  March  2012  

L4: Stability tests

•  Stability tests include but not limited to: Freeze/thaw cycles, storage times and temperatures

•  Nature of tests –  Assay performance off QC/

other specifications –  Reagent specific testing –  Real time vs Accelerated

Stability Testing –  Single reagent test

   

Need to define if will test regent stability and then how and against what criteria

Reagent  test/retest  is  typically  done  in  the  assay  in  which  the  reagent  is  used  and  acceptance  criteria  are  generally  associated  with  acceptable  runs  based  on  QCs      Control  chart  monitoring  of  assay  performance  characterisBcs  may  be  a  useful  tool.      

Reagent stability test runs using the assay needs to be defined as such to differentiate from other runs including reagent lot testing

26  March  2012  

L4: Stability Test Expiry Date Extension

Test Expiry Date Extension •  Trend analysis of assay

performance

•  Stability investigations for intended use –  Spiked samples (or QCs) –  Statistical analysis (optional)

•  Modification of the stability period or storage condition as necessary  

 

Need to define if will extend regent expiry data (test/retest date) and then how and against what criteria   Sources of instability and

prevention

Reagent Testing an SOP defined process for regulated studies

Bacterial Contamination can rapidly degrade reagents and several preservatives are available Formulation can improved reagent stability Prevent/reduce denaturation, aggregation, surface adsorption, deamidation, oxidation, isomerization, and fragmentation

26  March  2012  

L4: Commercial Vs In-House … continues

Commercial  Reagents  (Off  the  Shelf):  What is good? •  Ready availability / time saving •  A variety of vendors •  Relatively in-expensive / Wide variety to

select from •  Certificate of Analysis (CoA) / lot-release

specifications (limited availability) •  Pre-weighed calibrators (mass units, IU,

WHO Units, etc.) •  Solutions of Know Ab / Conjugate

concentrations available •  Technical support and vendor co-operation

in resolving reagent performance issues

LBA  Cri;cal  Reagents  •  Ab,  pepBdes  proteins,  conjugates,  Drug  as  reagent,  ADA  reagents    •  ADA  posiBve  and  negaBve  control-­‐  mostly  prepared  in-­‐house  using  commercial  components  

 What is Challenging? •  Lot-to-lot variability •  Change of lots (e.g. polyclonal or

monoclonal Abs, cognates) •  Time / resource commitment for feasibility

evaluation – for fit-for purpose use •  Concentration assignments for pre-

weighed calibrators may vary from vendor to vendor

•  CoA may not have all info that one need •  Stability data generally lacking •  Discontinuation of a critical reagent with

little or no prior notification •  Some vendors may not provide acceptable

technical support

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L4: Commercial Vs In-House

In-­‐House  Custom  Produc;on  /  Out-­‐sourced  Custom  Produc;on  

What is good? •  Can be produced in house or outsourced for

production per user’s specifications •  Tailored to needs of assay design •  Extensive characterization for intended use/

More complete CoA •  Lot-to-lot variability can be monitored &

controlled (e.g. labeled reagents) •  Better control on Life-Cycle of reagents-

availability of large amounts for extended period.

•  Internal technical expertise to monitor & resolve performance issues

LBA Critical Reagents  •  Ab,  pepBdes  proteins,  conjugates,  Drug  as  reagent,  ADA  reagents  •  ADA  posiBve  and  negaBve  control-­‐  mostly  prepared  in-­‐house  using  commercial  components  

   What is Challenging? •  In-house production requires advanced

planning, infra-structure & resources – expensive

•  Outsourced custom production as per specifications – requires advanced planning – relatively expensive

•  In-house expertise may be limited, may require dedicated personnel for reagent characterization

•  In early assay developmental stages – it may mot be cost effective

•  Over all time consuming & expensive.

Custom  Produc;on  through  Commercial  Companies  with  in-­‐house  oversight  of  Reagent-­‐Life  Cycle  Management  may  be  a  prac;cal  route  to  take  

26  March  2012  

L4: Summary • Content reflects work in progress

• Team plans to finalize draft recommendations for review by end of May 2012

• In interim can start incorporate comments on Key areas of focus • Reagent lot testing • Reagent stability testing • In house vs Commercial

• Include documentation and staging strategies

26  March  2012