L4: Reagents and their stability Link with tiered approachbioanalysisforum.jp/images/2012_3rdJBFS/3rd_JBF_Sympo_L4.pdf · 6/22/2012 · L4: Reagents and their stability - Link with

L4: Reagents and their stability - Link with tiered approach •  Team members: •  Team lead •  Lindsay King – NA (EST)– [email protected] •  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

20 May 2012

Cri,cal Reagents used in PK, Immuogenicity and biomarker LBA Key areas of focus

•  Reagent lot tesQng •  Reagent stability tesQng •  In house vs Commercial •  DocumentaQon related to these three areas

•  Have not addressed reagent in assay transfer yet •  Have idenQfied some gaps and some best pracQces

•  Best prac,ces need to be further refined with feedback from the BA community

Please send comments to Lindsay King [email protected]

L4: Reagents and their stability Draft Recommendations For Comment

22 June 2012

1) Defining Cri,cal Reagents While Guidance does define criQcal reagents and the literature/white papers have expanded on

this. We recommend the following: Reagent that are analyte specific are most oKen considered cri,cal reagents; Ab, pep,des, proteins, conjugates, Drug as reagent, ADA reagents including posi,ve and nega,ve control.

While any component of an assay may be criQcal to its performance under certain circumstances, criQcal regents for any specific assay need to be flagged as such and then require assay life cycle planning/ risk management.

Regulatory guidelines (EMEA bioanalyQcal method validaQon 2011) define criQcal reagents as “.binding reagents (e.g. binding proteins, aptamers, anQbodies or conjugated anQbodies) and those containing enzymaQc moieQes have(ing) direct impact on the results of the assay…”. The bioanalyQcal community, as evidenced in the literature, agree and expand on this definiQon to include; pepQdes, 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)).


22 June 2012

2) Documenta,on Recommend an SOP or similar document that defines reagent requirements including how,

what, where and when. Recognize that documenta,on may take many forms. Documenta,on is required to ensure a consistent and reproducible process.

•  Reagent tesQng documentaQon is required for regulated studies •  DocumentaQon would include as appropriate to specific reagent:

–  DescripQon of selected criQcal reagent (e.g. cross reacQvity). –  COA (see secQon 3) –  AddiQonal reagent characterizaQon documents (eg, affinity. Conjugate molar raQo) (see secQon 3). –  DocumentaQon of the cell line ( e.g. method of expression). This may reside with provider. –  PurificaQon and/or conjugaQon methods used to produce criQcal reagents. This may reside with provider. Need to be

able to link to groups/vendors that provide criQcal reagent and this documentaQon if needed. –  Reagent formulaQon. –  Storage condiQons and history. –  Assay performance specificaQons that were derived from the use of the assay (during validaQon and/or sample

analysis) and that may be used to define acceptance criteria for new lots of reagents and stability retest. –  DefiniQon of how new Lots would be tested and accepted. Need to define where this data would be stored (see

secQon 5). –  Stability test/retest methods, acceptance criteria and results (see secQon 6-‐7)


22 June 2012

3) Characteriza,on of selected cri,cal reagents •  SelecQon of criQcal reagents is usually based on AnalyQcal requirements and Assay

performance. Recommend sufficient characteriza,on to enable consistency/process control. Recommend) -‐

Iden,ty, source, purity, concentra,on (or ,ter), binding affinity, isotype (Mab/polyclonal), MW, specificity, incorpora,on ra,o, aggrega,on level, storage)

–  IdenQty may include; vendor, part number, lot/unique idenQfier number. Also advisable to include availability as a consideraQon

Feedback requested


22 June 2012

4) Reagent Characteriza,on/Qualifica,on Based on Assay Performance: Recommend that specificity (eg min. cross-‐reac,vity with structurally related compounds is

tested/defined as per guidance Recommend that selec,vity (non-‐specific binding and/or interference is tested as per guidance •  Assay performance data required, may be augmented with orthogonal tesQng


22 June 2012

5) Changing Cri,cal Reagents: Acceptance criteria based solely on QCs does not address the potenQal for significant difference in maximum response despite acceptable back calculated QC values . Thus we have included the recommendaQon that acceptance criteria for both new lots of reagents and for stability tesQng include maximum response. When using signal control charts some consideraQon needs to be paid to the plahorm used to generate the signal and ability to compare signal obtained over Qme from the same instruments and between different instruments . Using signal may be more straighto forward for colorimetric assay (eg Max signal must be greater than 1.5 DD) than for other plahorms. The team is looking for comments on both the use of maximum signal and the applicability to mulQple plahorms.


22 June 2012

5) Changing Cri,cal Reagents: Tier 1 For a new reagent/part # (original reagent no longer available from supplier); new supplier, new animal, new cell line, etc. (EG rhIL6 from new supplier/ new MoAb Clone) need to IdenQfy and characterize new reagent, including negaQve control Recommend compare to original reagent (re-‐op,mize assay if needed) in assay based on assay acceptance criteria including maximum signal. Recommend where possible tes,ng in parallel with current/original reagent over mul,ple runs. Recommend test in reagent qualifica,on runs and then decide on degree of valida,on required Would likely require at least parQal validaQon for Regulated work

Feedback Requested: What is a qualificaQon run? GAP idenQfied regarding details here as to what to do when.


22 June 2012

5) Changing Cri,cal Reagents: Tier 2: For a new lot # derived in some way from old lot ( eg new purificaQon of same clone of

MoAb; new labeling/conjugaQon of same lot of protein) •  Need to IdenQfy specific parameters that have changed (e.g. concentraQon for newly diluted

prep., molar raQo for conjugate) •  Reagents may be changed individually or mulQple criQcal reagents may need to be changed

at once (eg when assay has not been used for many years (more work). Recommend test performance in assay as follows: •  Single CriQcal Reagent Change – min 1 reagent qualificaQon run in parallel with current/

original lot. Include 5 QC levels and max response as an acceptance criteria. •  Single CriQcal Reagent Change and no overlap with current /original lot – min 3 independent

reagent qualificaQon runs. Include 5 QC levels and max response as an acceptance criteria. •  Eg second bioQnylaQon of a monoclonal from same lot of purified Ab as use iniQally •  MulQple CriQcal Reagent Changed – min 3 independent runs. May require some degree of

re-‐validaQon for Regulated work. Include 5 QC levels and max signal as acceptance criteria.

Failure in Tier 2 tesQng would lead to assay re-‐opQmizaQon and back to Tier 1


22 June 2012

5) Changing Cri,cal Reagents: If fail to meet specificaQons in LBA test performance: unable to qualify a new lot of reagent Recommend failure of assay performance specifica,ons aKer ‘minor’ changes loops back to

Tier1 Recommend: •  Review new reagent characterizaQon; •  If acceptable, re-‐opQmize assay •  Redefine Assay performance in mulQple runs •  New assay validaQon may be required

Recommend control char,ng for monitoring assay performance Feedback Requested Any manipulaQon we would treat as a new lot #. We have included the ‘parent’ lot as well on

labels and in SOPs to make this link.


22 June 2012

6) Reagent Stability: within the assigned stability period Recommend assign test/retest rather that expiry dates to define cri,cal reagent stability

ini,ally. Recommend cri,cal reagents be stored long term at -‐20 or colder unless there are specific

known stability issues. Reagents must be stored as recommended by the provider/vendor. If not then need to generate

the appropriate storage stability data. Can be obtained from running the assay and would require documentaQon

–  IniQal dates can be assigned based on experience with similar classes of reagents Can assign very long and monitor performance and then revise when changes in reagent

performance idenQfied. –  Approach conservaQvely at start and set short Qme period and then extend as get

experience with reagent. The stability of criQcal reagents can be tested though real Qme or accelerated stability tesQng


22 June 2012

6) Reagent Stability: within the assigned stability period Recommend define how to test regent stability, who will do what test, where the data will be

stored, what criteria will be used , where to document this work. Applies to In house regents. Also applies to and commercial reagents when a) no stability informaQon provided or b) reagents is stored outside of vendor recommendaQons

Reagent stability tests can include but are not limited to freeze/thaw cycles, storage Qmes and

temperatures •  Nature of tests: •  May include Reagent specific tesQng (eg HRP funcQonal test) •  May include Real Qme vs Accelerated Stability TesQng •  May include single physical reagent test (eg aggregates)


22 June 2012

6) Reagent Stability: within the assigned stability period

Reagents stability tesQng runs may be treated the same way as Method Development runs ie

they are non-‐regulated runs . IniQal reagent stability tesQng would be done before method validaQon. Acceptable method validaQon qualifies reagent used during validaQon for regulated study support. Aner validaQon addiQonal specific regent stability tesQng may be required (e.g. long term reagent stability at a specific storage temperature, 1 month at 4°C) separate from sample tesQng.

Recommend regents stability specific runs be documented in a separate folder from the

valida,on . Op,on to link to method(s). Define these stability test runs using the assay as stability runs to differen,ate from other runs.


22 June 2012

6) Reagent Stability: within the assigned stability period Recommend acceptance criteria include back calculated concentra,ons of 5 QC levels.

Recommend include max signal monitoring through the use of control charts from all acceptable runs with the assay (valida,on and sample analysis) un,l enough data is obtained to include max signal as acceptance criteria.

•  Trend analysis of max signal and QC signal may provide the best and iniQal indicaQon that

one or more reagents are failing. Sample analysis can provide assay performance data to inform trend analysis.

Recommend define process for addressing stability related failures to allow a scien,fically

defendable ra,onal for either repea,ng the stability test or concluding that the reagent is no longer stable.

•  Failure of a reagent within the original assigned stability window can inform the assignment

of dates for future lots but reagent handling must also be considered


22 June 2012

7) Stability Test/Retest Expiry Date Extension. Beyond the assigned stability period •  Manufactures data may set expiry dates with no more data than that used to set test/ retest

dates for in house reagents. AddiQonally reagent stability is in part linked to its handling and storage. Thus extension of manufactures assigned storage dates may be jusQfied based on appropriate data. For regulated work it is criQcal that the process and acceptance criteria used to allow the use of a reagent beyond it original test/retest and/or manufacturers expiry date be defined a priori.

•  For an expired reagent can use the data generated prior to expiraQon date to define the new stability duraQon.

Recommend define if will extend regent expiry data a priori (test/retest date) and then define

how and against what criteria. Recommend that extension on be based on data and there are a number of poten,ally ways d

to obtain this. It is always important to monitor assay performance and use a scien,fically defendable ra,onal for decisions


22 June 2012

7) Stability Test/Retest Expiry Date Extension. Beyond the assigned stability period PotenQal route to set new Test/ Retest Dates include: •  Test/retest extension based on past performance

–  Trend analysis/StaQsQcal analysis can used to set new test/ retest dates •  Eg 2 years of past data then can extend by 2 yrs •  Does not address catastrophic reagent failure

•  Test/retest extension based on Current performance –  Test in assay alone

•  Eg MoAb at -‐70°C extend 6 months •  Accelerated stability tesQng •  Eg x days at 37°C projects y months at 4°C (Add Ref) Consider assessment of stability outside beyond assigned period as separate from sample

analysis with two runs with full set of five QCs (LLOQ to ULOQ) as a best pracQce for seqng stability extension based on current performance


22 June 2012

8) Reagent life cycle management Recommend regardless of the approach use to address reagent stability and test/retest date

assay performance must be monitored for both in house and commercial reagents. •  Reagents may start to degrade slowly and monitoring (as described in secQons 5-‐7) will

inform assignment of future test/retest dates. •  Best PracQces: ConQnued feedback needed from community on PotenQal Best PracQces •  History of reagent performance in the assay, subsequent characterizaQon or orthogonal

tesQng data •  1) GeneraQon of large amounts of a single lot a criQcal reagent reduces risk of lot changes. •  2) Single use aliquots can be used to reduce risk of instability and contaminaQon Sources of instability and preven,on •  Bacterial ContaminaQon can rapidly degrade reagents and several preservaQves are available •  FormulaQon can improved reagent stability •  Prevent/reduce denaturaQon, aggregaQon, surface adsorpQon, deamidaQon, oxidaQon, isomerizaQon,

and fragmentaQon


22 June 2012

9) Cri,cal Reagents from Commercial Sources Establish close relaQonship with vendors. This will help in geqng technical support for trouble

shooQng and resolving reagents/ kits related issues; obtaining Qmely informaQon about reagent lot changes; and securing supply of specific lots of reagents/kits for a longer period of Qme. Test/Retest or Expiry data is onen provided by commercial and can be used.

Proposed Best Prac,ces/ Points to Consider •  Secure bulk lots when possible. •  Recombinant proteins and pepQde with idenQcal concentraQon unit assignments from

different vendors may behave differently in your assay system. Therefore, select and retain sufficient quanQQes of this material for your long term needs. Specific acQvity of conjugates may also vary within a wide range from lot to lot


22 June 2012

9) Cri,cal Reagents from Commercial Sources Proposed Best Prac,ces/ Points to Consider •  Learn about vendors Quality Control/Quality assurance processes. Perform QA audit of their

facility if necessary. •  Obtain relevant documentaQon related to provider reagent characterizaQon, qualificaQon

and stability etc. •  Pre-‐coated ELISA plates should be considered as criQcal reagents. Establish internal

processes to assure uniform coaQng of plates especially where strips are used. Consider implementaQon of in house lot acceptance test for pre-‐coated plates

•  Where pracQcal for in house biomarker assays built with commercial reagents use pooled biological matrix (e.g. serum / plasma) containing endogenous analyte (preferably at two levels, low and high) to monitor lot-‐to-‐lot performance of commercial reagent.

•  Invest sufficient Qme in selecQng/qualifying a criQcal reagent vendor. <It will save Qme and money on long range


22 June 2012

L4: Reagents and their stability Draft Decision Trees For Comment

22 June 2012

Reagent Selection Phase ü Check assay performance ü Characterize reagent ü Compare with

current/original reagent

TIER 2 – NEW REAGENT BATCH

TIER 1 – NEW REAGENT OR NEW LOT

DECISION TREE [

Qualify Min. 3 – Runs (independent for reagent in question)

(A) Assay performance same as current/original assay e.g.

-‐PK all QCs and Max. Signal meet A/C

-‐ADA all QCs and Min. Signal meet A/C, cutpoint within range

-‐Biomarker LOD, all QCs and Max. Signal meet A/C

Revalidate

(B) Assay performance altered but acceptable (Re-‐test to confirm)

(C) Assay performance outside of acceptable parameters (Re-‐test to confirm)

Partial Validation

Complete Documentation: Assay ready

Assess new reagents ID suitable alternate

Re-‐engineer assay

Assess new reagents No suitable alternate

L4: Reagents and their stability Draft Decision Trees For Comment

22 June 2012

Reagent Selection Phase ü Check assay performance ü Characterize reagent ü Compare with

current/original reagent

To TIER 1(B)

TIER 2 – NEW REAGENT BATCH

DECISION TREE [

Qualify Min. 1 – Run

(A) Assay performance same as current/original assay e.g.

-‐PK all QCs and Max. Signal meet A/C

-‐ADA all QCs and Min. Signal meet A/C, cutpoint within range

-‐Biomarker LOD, all QCs and Max. Signal meet A/C

(B) Assay performance altered but acceptable (Re-‐test to confirm)

(C) Assay performance outside of acceptable parameters (Re-‐test to confirm)

Min. 3 – Runs to complete qualification

REAGENT BATCH

Complete Documentation: Assay ready

Re-‐test acceptable

Re-‐test not acceptable No suitable alternate

Guidance and Literature review high level

summary slides

L4: Reagents and their stability Background and Rational

22 June 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 AddiQonal consideraQons: rare cri,cal reagents, difficult to replace, single source cri,cal reagent life

cycle management

“If there is only a single supplier of a criQcal reagent then it is advisable to consider some contractual relaQonship that warns the user about product changes or cessa,on of producQon.” Mire-‐Sluis 2004

“Successful lifecycle management of LBA criQcal reagents minimizes assay performance problems caused by declining reagent acQvity and can mi,gate the risk of delays during preclinical and clinical studies. “O’Hara 2012

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

26 March 2012

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)

26 March 2012

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 specifica,ons set, at least for those, which are most important. (EMA Guideline on Biotechnology-‐derived TherapeuQc Proteins, 2010)

•  should demonstrate that the labeling of the detec,on an,gen does not significantly obscure cri,cal an,genic determinants. (SecQon 3.B.2: FDA (Dran) Guidance Assay Development for Immunogenicity TesQng of TherapeuQc Proteins)

•  ….should assess which condiQons are likely to vary and design appropriate tests to examine the

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

26 March 2012

L4: White-Papers and Draft Guidance's Documentation

•  “Batch records documenQng the preparaQon of ligand binding reagents including the source of the raw material, the method of expression, purificaQon, final formulaQon buffer…” (Rup et. al. 2007)

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

•  “For ligand binding assays, the method of reagent qualificaQon 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 condiQons of use should be stated in documentaQon. .. further issues are now

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

26 March 2012

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

26 March 2012

L4: Agreement in Guidance

27 April 2012

Suggest a need to idenQfy and understand criQcal reagents Suggest a need to assess impact of lot changes

Acceptance criteria Suggest a need to assess reagent stability

Acceptance criteria Suggest a need to document Although focus is generally on PK and ADA we have included Biomarkers in our thinking

L4: GAPS in Guidance Guidance documents and whitepapers have broad expectations of critical

reagents “appropriate characterization” but how to achieve commonality in this effort needs to be delineated.

•  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 Stability testing of reagents is not well defined in the guidance and is the area

with the most gaps and range of practices •  The guidelines do not specify how reagent stability should be assigned and/or

tested. No standardized acceptance criteria.

•  Where to document reagent stability

26 March 2012

Documents

L4: Reagents and their stability Link with tiered approachbioanalysisforum.jp/images/2012_3rdJBFS/3rd_JBF_Sympo_L4.pdf · 6/22/2012 · L4: Reagents and their stability - Link with