A4: Reference standards and reagents

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A4: Reference standards and reagents

Team members:

Team lead

•  Joseph Bower – NA - [email protected] Other members

•  Andrew Warren – EU –

[email protected]

•  Carl Watson – EU – [email protected]

•  Jennifer McClung – NA – [email protected]

•  Kathy Wright – NA – [email protected]

•  Katia Pastre – LA- [email protected]

•  Mónica Cedrés Ercoli – LA - [email protected]

•  Takahiko Osumi – APAC - [email protected]

In scope

•  Recommendations for content in Certificate of

Analysis (COA) or equivalent documentation to be included with material if COA is not available for: •  Reference Standards

•  (small and large molecules)

•  Biomarkers

•  Metabolites

•  Internal Standards

•  Recommendations for preparation of:

•  Calibration standards and QCs.

•  Stock solutions

•  Internal standards

Out of scope

. Positive controls for Immunogenicity Assays . Bridging between lots of reference standards

Interdependencies with other teams – if any

–  L4 -‐ Reagents and their stability – Lindsay King

–  A11 – Biomarkers – Russ Weiner

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Team A4 Recommenda<ons

Reference Standards

All reference standards (including certified reference standards or stocks, commercially supplied reference standards, or other materials of documented purity) should be

provided with a COA, or equivalent documentation, that includes the following information:

•  Lot# / Batch#

•  Manufacturer

•  Purity “for use as is” or correction factor *

•  Expiration date (retest date)

•  Storage Conditions, noting any special handling requirements #

* Stated correction factor by purity or protein/peptide content

Chemical structure and formula (with salt form, water content, solvent content) # Light sensitivity etc..

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Team A4 Recommenda<ons

Reference Standards cont.

Large Molecules

Reference material used for preparation of calibrators and QCs should be of the highest purity available and from the same manufacturing process, and same batch when

possible, as that being dosed for non-clinical and clinical studies.

If reference material is provided in a buffer or solution, the information on the COA should be relative to the reference standard in solution. Any special handling

requirements (such as Freeze Thaw stability etc..) should also be noted.

Biomarkers

These reference standards are typically not well-characterized and prone to lot-to-lot

variability. It is desirable to use a reference standard from a single lot throughout a study. If it is not possible to use a single lot, appropriate bridging procedures are to be followed. The same process should be applied to kit assays.

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Team A4 Recommenda<ons

Metabolites

CoA is not required, but evidence of purity and idenDty should be documented.

AddiDonal cross-‐reacDvity should be evaluated as well as possibility of back conversion to parent and its impact on study

Based on suitability of use (e.g. metabolite is used as a primary endpoint), further documentaDon may be needed to include lot #, manufacturer (source), expiraDon (if known) and storage condiDons.

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Team A4 Recommenda<ons

Internal Standard

CoA is not required, but evidence of purity and idenDty should be documented. If purity is not available, suitability of use should be conﬁrmed.

Should demonstrate lack of lack of analyDcal interference and known impuriDes have been evaluated.

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Team A4 Recommenda<ons

Prepara<on of calibra<on standards & QCs

A calibration curve should be prepared for each analyte in the sample and prepared in the same biological matrix as sample for intended study. A single source of matrix may also be used for preparation of calibrators and QCs, provided selectivity has been verified.

Calibrators and QC samples should be prepared from a stock solution with proven solution stability and where accuracy have been verified.

During preparaDon of calibrators, soluDons should not be diluted by more than 5%; meaning that >95% matrix should be maintained. However, when working with low concentraDon (unique matrix) highly concentrated stock soluDons this may not be

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Team A4 Recommenda<ons

Prepara<on of calibra<on standards & QCs

In the case of rare matrix; altered/surrogate matrix may be used if equivalency is

demonstrated during assay validation. If endogenous analytes are present – assay buffer

may be used for preparation of calibrators. Altered or surrogates matrix should only be used if equivalency is demonstrated between surrogate matrix and authentic matrix.

(this may be achieved by spiking reference standard or immune depleting the endogenous analyte if the levels are too high)

If altered/surrogate matrix is used, QC samples should be prepared in authentic matrix. Stability should be assessed against freshly-spiked calibration curve and compared to nominal concentrations.

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Team A4 Recommenda<ons

Prepara<on of stock solu<ons

During Validation, two separate weightings are required to balance stock solutions. The

accuracy of stock soluDons should be demonstrated by balancing stock soluDons with deﬁned acceptance between separate preparaDons; such as within a percent diﬀerence

as appropriately jusDﬁed by scienDﬁc judgment, for example 10%. During sample

analysis, a single verified stock solution may be used.

If using working solutions, stability should be assessed by the lowest and highest concentrations of solutions.

If a stock solution is prepared from a reference standard within its expiration date, there is no need to prepare a new stock solution when reference expires.

Stability of the stock solution should be demonstrated with an appropriate solvent at known concentrations and at appropriate dilution. Stability at room temperature for at least 6 hours should be evaluated. If instability is observed at room temperature, then stability at alternate temperatures should be evaluated.

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Team A4 Recommenda<ons

Prepara<on of Internal Standards

When possible, stability such as room temperature for at least 6 hours and refrigerated or frozen stability should be evaluated . If using rare IS and formal stability is not possible, stability should be documented through suitability of use (eg lack of interference and appropriate assay controls).

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Team A4 RecommendaDons

QualiﬁcaDon of Reference Standards

Most approaches to bridging reference standard lots for large molecules involve quanDtaDng QCs prepared from the new lot versus calibraDon standards and QCs

prepared from the old lot. A single independent parDal validaDon run is used to support the change. The run includes duplicate curve (made from the old lot), 3 levels run

acceptance QC (old lot) in duplicate, and intra-‐assay QCs n=6 at low, medium and high levels prepared from the new lot.

Two-‐thirds of the run acceptance QCs (old lot) and at least 50% of the replicates from each QC level tested must quanDtate within accuracy acceptance limits in order for the run to be acceptable. The acceptance limits may be the assay acceptance or other deﬁned acceptance such as +/-‐20% of theoreDcal. The intra-‐assay QCs (new lot) must also meet these criteria for the new lot to be bridged with the old lot.

Other approaches involve making calibraDon standards and QCs from both lots and quanDtaDng both sets of QCs oﬀ each curve. Means should be with 20% of theoreDcal with a CV within 20%. ULOQ and LLOQ may be included as needed. It is recommended that reference standard lots be evaluated over Dme to ensure the performance is not trending outside the assay requirements.