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Assigning Potency to Reference Standards Matthew Borer, Ph.D. Corporate Reference Standards Advisor, Analytical Sciences Eli Lilly and Company

CMC Strategy Forum - Summer 2013: Reference Standards for Therapeutic Proteins - Current Regulatory and Scientific Best Practices and Remaining Needs July 15 – 16, 2013 Gaithersburg, Maryland USA

M. Borer, CASSS CMC Strategy Forum - July 2013

Copyright © 2013 Eli Lilly and Company 2

Terminology



Inconsistent Nomenclature

Certified Property Value (ISO)

Calculation Value (USP)

Assigned Content (PhEur)

Defined Potency (Lilly)

(JP)



Expression of Certified Properties

Attribute

• Main Peak (Free)

• Main Peak (Compound + Salt)

• Total Enantiomers

• Total Isomers

• Identity

• Bioactivity

• Monomer

• Total Protein Content

Basis

• Weight Percent

– As Is

– As Is (after defined equilibration)

– As Is (with exposure limits)

– Anhydrous (corrected for water at time of use)

– Dried (specified conditions)

– Volatile Free

• Solution Concentration

– mg/ml

• Per Vial Basis

– Milligrams

– Units



Reference Standard Intended Use



Regulations: Characterization

Reference standard characterization must be customized to support its specific intended use

ICH Q3a, IV • Reference standards used in the analytical procedures for control of impurities should

be evaluated and characterized according to their intended uses.

WHO, Annex 3, Technical Report Number 885,3. • It is necessary to consider all data obtained from testing the material by a wide variety

of analytical methods. When taken as a whole, this will ensure that the substance is suitable for its intended use. The extent of the analyses required depends on the purpose(s) for which the chemical reference substance is to be employed, and may involve a number of independent laboratories.

ISO 34, 4.1.1 • It should be recognized that a reference material needs to be characterized mainly to

the level of accuracy required for its intended purpose…



Certified Property Value: A Key Expression of Intended Use

68.4% LY123456 on an "as is" basis. 99.9% compound LY123456 Dimesylate on an "as is" basis.

84.9% LY456789 on an anhydrous basis when corrected for KF titration at time of use

1394 units of activity per mg when dried at 60 C for 3 hours under vacuum (20-25 mm Hg)

3.80 mg itsamab main peak per vial for HPLC assay; 3.95 International Units per vial for biopotency assay; 2.99 mg total protein per vial for non-specific analysis. DO NOT WEIGH; reconstitute entire vial contents.

2.0 mg total protein per mL. 100% potency for bioassay.



High-Level Approaches for Determination of Property

Values



Determining Certified Properties

Mass Balance • Weight based

• Total protein based

Comparative Assay • HPLC

• UV

• Bioassay

“Absolute” Measures • Titration

• Thermal Methods

• NMR

• UV Extinction Coefficient



Chaining by Comparison to Previous Reference Standard

Chaining can lead to a drift in assignment



Assign Chemical Content: Independent Means



Assign By Comparison to a Primary Reference Standard

The range of relative potencies depends on the overall uncertainty in the reference standard property value



Desired Situation

No measurable shift between RS batches

Example: Reporter Gene Assay for a mAb



Reference Standard Transition



Specific Property Value Assignment Strategies: Monoclonal Antibodies

Potency Assignment Strategy: Initial Batch

First Laboratory RS is assigned a value of 100% Relative Potency (RP)

• Subsequent Laboratory RS batches are assigned relative to the previous Laboratory RS

The Primary RS is assigned a value of 100% RP • Confirmation of a dose response relationship

• For-information-only Bioassay comparison to Laboratory RS

– more extensive for IgG1 mAbs due to greater potential for potency change

• Extensive physicochemical comparability testing to Laboratory RS

The initial Secondary RS is based on the same characterization tests as the Primary RS (same source material)

• A subsequent Secondary RS is assigned a value of 100% RP if the bioassay versus the Primary RS is within a pre-defined window



Laboratory RS

Primary RS

Secondary RS

Potency Assignment Strategy: Replacement Batch

Analyzed by the biological assay using 5 independent measurements relative to the previous RS

If the average relative potency is within 80-120% RP, the new reference standard will be assigned a value of 100% RP

If the average potency falls outside of the interval, an investigation is conducted.

• Question the suitability of the material

• Consider a larger number of replicate measurements in order to assign the RP directly to the previous standard



Laboratory RS

Potency Assignment Strategy: Replacement Batch

Analyzed by the biological assay using 10 independent measurements relative to the Primary RS

If the average relative potency is within 95-105% RP, the new reference standard will be assigned a value of 100% RP

If the average potency falls outside of the interval, an investigation is conducted

• Question the suitability of the material

• Consider a larger number of replicate measurements in order to assign the RP directly to the previous standard



Secondary RS

Sample Size Determination



Sample size determination

S an estimate of the intermediate precision of the method performed as written

a the desired level of confidence for the computed confidence interval, e.g. if 95% confidence is desired, then a=0.05 , calculated as ((100-95)/100)

w the desired half-width of the confidence interval for the reference standard defined potency (expressed as a percent of the defined potency)

g the probability that the confidence interval half-width is greater than w (expressed as the decimal equivalent of the percent probability)

For most situations, a=0.05, g=0.10 .

Then choose the smallest n that meets the criteria ag

1,1,1

2

1,1

2

1 nn Fw

snn

where 2

1,1 g n is the upper one-sided (1-g) percentile of a chi-square

distribution with (n-1) degrees of freedom

a 1,1,1 nF is the upper one-sided (1-a) percentile of an F-distribution

with 1 numerator degree of freedom and (n-1) denominator

degrees of freedom

Source: Kupper, Lawrence L., Hafner, Kerry B., “How Appropriate Are Popular Sample Size

Formulas?”, The American Statistician, 43(2), May 1989, pp. 101-105.

Sample Size Calculation

s = 10.0%, estimated intermediate precision of the

Bioassay method

a = 0.05, desired level of confidence for the confidence

interval (95% CI)

w = 20.0%, desired half-width of the confidence interval

for the Reference Standard Potency (80% - 120%)

g = 0.10, probability that the confidence interval half-

width is greater than w (10% chance of being out)



ag

1,1,1

2

1,1

2

1 nn Fw

snn …is true at n = 5

Independent Measurement Defined

Independent Measurement - In General • An average reported result based on a replication strategy that

encompasses appropriate variance components associated with the measurement

Independent



Independent Measurement for Bioassay

• Three plates

• Three replicates at each dose level for each:

• Sample

• Standard

• Control sample

Why Assign 100% Instead of the Comparative Test Result?

Avoids drift due to chaining

Useful when a material can be assumed to be stable – extensive measures have been taken to assure stability

• In this situation, departures from 100% potency are more likely to represent method variability.

Useful in situations where the reference standard is sourced from a validated process

Assumes that method variability is greater than material variability

Comparative assay is still done, so changes will be detected M. Borer, CASSS CMC Strategy Forum - July 2013


Conclusions

Terminology differences exist – propose use of “Certified Property Value”

The Certified Property Value must be specifically designated on the CoA and must tie directly to the intended use

A Primary Reference Standard must be established prior to pivotal clinical studies to serve as the line-of-sight to the original clinical experience

It is not necessary to directly assign property values using comparative results as long as the assigned value maintains the most consistent measurement of potency



Thanks To:

Kimberley B. Dancheck

Kristi L. Griffiths

Bryan J. Harmon

Jerry J. Lewis

David L. Lytle

Jerry Zhirui Lian

Bhavin S. Parekh

13-May-2011, M. Borer, et al. © 2011 Eli Lilly and Company 24



Thank you!

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