multi-attribute monitoring of antibody modifications in a ... · multi-attribute monitoring of...

Multi-attribute Monitoring of Antibody Modifications in a Quality Control Laboratory: Points to consider

21st WCBP Conference, January 24 – 26, 2017Patrick Bulau, Roche Diagnostics GmbH, Germany

IntroductionWhat we do at Roche Pharma Penzberg?

Table of Contents

Mass Spectrometry for Quality Control

ConclusionThe take-home message…

Points to Consider

What we do at Roche Pharma Penzberg?Roche Penzberg - one of the largest Biotech centers of Europe

Research

• Discovery• Cell Science• Molecule Design

• Translational Medicine • Large Molecule Research

Development

• Manufacturing Process Development API*• Manufacturing Process Characterization & Validation• Clinical Supply of API*

Operations

API for …• Herceptin ®

• Mircera®

• NeoRecormon®

• Pegasys ®

• Gazyva®

• …

Roche Pharma

• Biomarker Assays• Cobas Elecsys Family• …

Diagnostics

Roche Diagnostics

* API = active pharmaceutical ingredient (drug substance)

IntroductionApplication of MS Methods in Pharma Development

• Purpose of MS Methods – Verification of the molecular mass (MALDI-MS, Electrospray MS) – Verification of the primary structure of proteins– Assessment of the glycosylation (LCMS Peptide Mapping, Electrospray

Mass Spectrometry, MALDI Mass Spectrometry) – Assessment of the microheterogeneity

• Deamidation of asparagine, Isomerization of aspartate• Oxidation of methionines and tryptophan• Variability of N- and C-terminus of the polypeptide chain• Glycation

– Sequence variant and Host Cell Protein Analysis, HDX, native MS – Detection of unexpected modifications

IntroductionApplication of MS Methods in our Laboratory

• Extended Characterization– Comparability studies– Isoform characterization– Process Development– Trouble shooting– Analysis of In-Process Samples– Other applications

• Release testing of Drug Substance– MALDI TOF MS for development products (Phase I-II) since 2009

• Identity test based on peptide analysis (“Genentech Approach”) since 2013• Identity test based on glycan analysis since 2009

– ESMS of reduced samples (mainly for antibody release) for Herceptin® since 2004• Confirmation of molecular weight of light and heavy chains• Quantification of main heavy chain variants (galactosylation variants)

– LCMS Peptide Mapping (Trypsin) for Pegasys® since 2002• Identity test with “impurity aspects”


Table of Contents



Points to Consider

ESMS of Reduced Protein Samples Purpose - Application

• Used for release testing of antibodies (e.g. trastuzumab)

• Confirm molecular mass of the light chain and of the main heavy chain variants

• Quantitative determination of the galactosylation variants (G0F, G1F, G2F, complex type biantennary core fucosylated oligosaccharide structures with 0, 1 or 2 galactose moieties)

F F

FF

“Setup ESI-Q-TOF MS”

ESMS of Reduced SamplesPrinciple

• Denaturation and reduction of samples using guanidinium hydrochloride and TCEP

• Rebuffering into medium suitable for ESMS analysis by means of gel filtration (NAP5 columns, GE Bioscience)

• Offline ESMS measurement of samples (mass spectrometer used: e.g. Q-TOF or LCT / Waters (Micromass))

• Automated calculation of molecular masses and relative contents of main heavy chain variants

ESMS of Reduced SamplesSum Mass Spectrum of a Sample

ESMS of Reduced SamplesZoom into Mass Spectrum and a Derivative Spectrum

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Intensity [arbitrary units]

m/z [amu]

HC 36+ HC35+ LC16+ HC34+ HC33+ LC15+

G0 G1

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ESMS of Reduced SamplesAcceptance Criteria Defined in Test Method

1. System Suitability (Based on Measurement of RS):– Accuracy of the molecular mass determined for light chain and main

heavy chain variants – Intensity of the Light Chain signals

2. Acceptance Criteria for Release of Batches– Comparison of molecular masses of the light chain and heavy chain

variants to the reference standard– Relative amounts of the main heavy chain variants (G0F, G1F, G2F)

must be within limits defined by the production process (production history)

ESMS of Reduced SamplesMethod Validation

• ESMS method used in our department is used for identification and quantification

• Validation Items (according to ICH 2Q(R1))– Accuracy of mass determination and quantification of the light and

heavy chain variants– Precision (Repeatability, Intermediate Precision)– Robustness– Linearity of quantification of the heavy chain variants– Limit of Detection (LOD) of heavy chain variants– Limit of Quantification (LOQ) of heavy chain variants

ESMS of Reduced SamplesValidation - Accuracy of Glycosylation Analysis

Comparison to HPAEC-PAD

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tive

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]

G0G0 Line FitG1G1 Line FitG2ESMS = PepMap

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tive

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nt E

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[%]

G0G0 Line FitG1G1 Line FitG2ESMS = OSP

Conclusion: Accuracy of Glycosylation Analysis Demonstrated

Comparison to LCMS Peptide Mapping

ESMS Results of Reference MaterialHC Galactosylation

0 20 40 60 80 100 12005

101520253035404550556065707580859095

100R

elat

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amou

nt [%

]

Analysis

G0F G1F G2F

ESMS of Reduced SamplesConclusion and Outlook

Conclusion:

The method can be used for release testing of the respective drug substance with regard to the determination of the molecular mass as well as the quantification of the main heavy chain variants

Trastuzumab: Since 2004 monitoring of > 500 drug substance batches: 3 SST deviations and 5 OOT results

Recent Improvements and Outlook:

Use of modern equipment for sample preparation and sample application to the mass spectrometer


Table of Contents



Points to Consider

Assessment of Trastuzumab PTM/Degradation Sites

BINDING• LC-Asn-30• HC-Asn-55• HC-Asp-102• No accessible Met or Trp

residue

PHYSICOCHEMICAL CHARACTERISTICS (Control Strategy) BIOLOGICAL CHARACTERISTICS

EFFECTOR FUNCTION• HC-Met-255• HC-Asn-387/392/393• HC-Met-431• C-terminal Lys heterogeneity is

classified as non CQA (for i.v.)

Constant quality of the purified pharmaceutical mAb requires a robust production process and a suitable control strategy.

• Free SH / Di-sulfide bond shuffling (no direct assessment)

• Deamidation/Isomerization (cIEC)• Oxidation (not monitored for DS)• N-term Pyro-Glu (E, low levels)• Glycosylation (Monitoring: CE-LIF /

ESMS, 2 AB HPLC)• Glycation (not detected)• Acetylation (not detected)• C-term Lys (not monitored)

• Aggregation/Fragmentation (SEC)

Shen et al., In: Techniques in Protein Chemistry, 1996; Harris et al., J. Chromatogr B, 2001; Chelius et al., Anal Chem , 2005

Assessment of Trastuzumab degradation sites IEC (CEC) – native antibody

Harris et al., J. Chromatogr B, 2001; Diepold et al., PLoS One, 2012

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LC-Asp-30 / HC-isoAsp-102

LC-Asn-30/ HC-Asn-55/ HC-Asp-102ab

sorb

ance

[mA

UFS

]

retention time [min]

Reference material

LC-Asp-30

HC-isoAsp-102

HC-isoAsp-55

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LC Asn 30 LC Asn 30 HC Asp 102

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tive

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nt [%

]

pH 7pH 6

Advantages of ph6 procedure vs. conventional Peptide Mapping for the Analysis of Trastuzumab

Deamidation Isomerisation

reduced methodartifacts (pH6)

Stable at pH 6

no chromatographic separation (pH7)

Succinimide

Assessment of Trastuzumab degradation sites LC-Asp-30: IEC (CEC) versus LC-MS


5 10 15 20 25 30

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1000LC-Asn-30/ HC-Asn-55/ HC-Asp-102

Relative abundance [%]IEC: 15.6 (± 0.3)LC-MS: 7.9 (± 0.4)


abso

rban

ce [m

AU

FS]


Reference material

LC-Asp-30

Assessment of Trastuzumab degradation sites HC-isoAsp-102: IEC (CEC) versus LC-MS


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Relative abundance [%]IEC: 11.9 (± 0.3)LC-MS: 4.6 (± 0.3)


LC-Asn-30/ HC-Asp-55/ HC-Asp-102ab

sorb

ance

[mA

UFS

]


Reference material

HC-isoAsp-102

Accuracy of LC-MS Quantification

Bomans et al., American Pharmaceutical Review, 2016

Linearity of LC-MS Quantification

Bomans et al., American Pharmaceutical Review, 2016

Points to consider for multi-attribute testing

• Separation based analysis (e.g. LC-UV) of intact or reduced protein samples

• (LC-)MS analysis of intact or reduced protein samples

• Peptide mapping combined with LC-MS analysis

Maximum information is only achieved by the combination of top-down and bottom-up approaches

Robustness

Resolution / Sensitivity

“Native analysis”

SummaryMass Spectrometry in a Quality Control Laboratory

• MS based methods (MALDI and ESI) can be used for robust and specific identity release testing of biologics

• Robust determination of the molecular mass as well as the quantification of the main heavy chain variants by ESI-MS of reduced antibody samples

• Multi-attribute monitoring of numerous pCQAs by LC-MS with quantitative aspects needs to be carefully evaluated and established with respect to robustness, method transfer, and acceptance criteria

Impact of multi-attribute monitoring on specification: Specification based on multiple and specific attributes versus total sum (e.g. HCP content, charge and size variants)

MS in QC should add value to the control strategy

Acknowledgement

• Katrin Bomans• Lea Bonnington• Manfred Wozny• Markus Haberger• Marco Thomann• Maria Maier• Susanne Eltner• Niklas Engler• Markus Dembowski• Dietmar Reusch

Doing now what patients need next

Points to Consider for Methods used as QC Test

• Laboratory fully embedded into our QA System and run under GMP– Instrument qualification with change control– Documentation requirements / Training of Personnel– Deviation management

• Methods are described in detail in SOP´s– How to perform method and how to evaluate data – Which reagents and which equipment must be used– System suitability criteria for equipment and test in general– Acceptance criteria for release of batches

• All methods used as QC tests have to be validated according to ICH Guideline 2Q(R1) “Q2(R1): Validation of Analytical Procedures: Text and Methodology“

ESMS of Reduced SamplesValidation –Precision of Mass DeterminationICH 2Q(R1):• “Repeatability” should be assessed using:

– a minimum of 9 determinations covering the specified range for the procedure (e.g., 3 concentrations/3 replicates each); or

– a minimum of 6 determinations at 100% of the test concentration”

• “Intermediate Precision”– establish the effects of random events on the precision of the

analytical procedure.– Typical variations to be studied include days, analysts, equipment”,

etc.

Experimental Setup Used:• Repeatability: six individual measurements at 100 % of the test

concentration

• Intermediate Precision: taken together six series at different days comprising 25 measurements, variation of operators, solutions and batches of gel filtration columns

ESMS of Reduced SamplesValidation – Precision of Glycosylation Analysis

Heavy Chain Variants Gx: glycosylated reduced heavy chain variant with x terminal galactose moieties NG: non-glycosylated reduced heavy chain variant

Statistical Analysis of the Data

(Relative Amounts in %) G0 G1 G2 NG

Number of Individual Determinations 10 10 10 10

Mean 44.5 44.0 9.2 2.3

Standard Deviation (SD) 0.7 0.7 0.5 0.9

Batch 1

SD • t(0.95) 1.5 1.5 1.2 2.1

Number of Individual Determinations 23 23 23 23

Mean 66.5 27.5 3.9 2.1

Standard Deviation (SD) 1.6 0.7 0.8 1.2

Batch 2

SD • t(0.95) 3.4 1.4 1.6 2.4

Two Batches (with different glycosylation pattern) were analyzed

Conclusion: High Precision of Glycosylation Analysis

ESMS of Reduced SamplesRobustness of the Method

• Variation from test method– Amount of protein used (50 % and 200 % of amount defined in SOP)– Concentration of guanidinium chloride (-20 % from SOP)– Concentration of TCEP (± 20 % from SOP)– Incubation time for reduction / denaturation (± 50 % from SOP)– Resolution of mass spectrometer (± 50 % from Range defined)– Sensitivity of the detector in the mass spectrometer (± 30 % from

range defined)

• Result– Decreased resolution of the mass spectrometer lead to slightly

different molecular mass of the light chain – Robustness could be demonstrated for all other parameters varied

ESMS of Reduced SamplesValidation –Precision and Accuracy of Mass Determination

Conclusion: High Precision and Accuracy of Mass Determination

Molecular Species

LC: reduced light chain Gx: glycosylated reduced heavy chain variant with x terminal galactose moieties NG: non-glycosylated reduced heavy chain variant

Statistical Analysis of the Data (all molecular masses are given in Da)

LC G0 G1 G2 NG Expected Molecular Mass Mth. 23443.1 50602.1 50764.2 50926.4 49157.5 Number of Individual Determinations 25 25 25 25 2 1)

Mean 23444.8 50599.7 50761.8 50923.5 - Mean - Mth. 1.7 -2.4 -2.4 -2.9 n.a.

Repeatability

Standard Deviation 0.6 0.1 0.2 0.5 n.a Mean 23443.2 50600.0 50762.3 50925.3 49154.4 Mean - Mth. 0.1 -2.1 -1.9 -1.1 -3.1 Minimum - Mth. -2.3 -2.7 -2.6 -3.6 -4.8 Maximum - Mth. 2.3 -1.4 -1.1 4.0 -1.4 Standard Deviation 1.3 0.4 0.4 2.0 n.a. Lower Confidence Limit - Mth.

-2.6 -2.9 -2.8 -5.4 n.a.

Intermediate Precision

Upper Confidence Limit - Mth. 2.8 -1.2 -1.0 3.1 n.a.

• Limit of Detection for G2: 0.8 %

• Limit of Quantification for G2: 2.5 %.

• Linearity: – Slope of correlation curve

1.0±0.02 (for G0)– Correlation Coefficient 1.00– Axis crossed at -0.5±1.3 %

ESMS of Reduced SamplesValidation – Linearity, Determination of LOD and LOQ

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• Experimental Design: Mixing of a batch with degalactosylated or deglycosylated material, Calculation according to ICH Guideline 2QB (based on slope and standard deviation of the response curve)

ESMS Results of Reference MaterialHC Non-Glycosylated (NG)

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Analysis

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Methods – sample preparation for UPLC-MS

Diepold et al., PLoS One, 2012

Assessment of Trastuzumab degradation sites HC-isoAsp-102: IEC versus LC-MS


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Relative abundance LC-MS [%] / 2M 40 °CLC-Asp-30: 33.3 (± 0.4)HC-isoAsp-55: 8.9 (± 0.6)HC-isoAsp-102: 49.9 (± 0.5)


LC-Asn-30/ HC-Asn-55/ HC-Asp-102ab

sorb

ance

[mA

UFS

]


Reference material 2 M 40°C

LC-Asp-30HC-isoAsp-102HC-isoAsp-55

Doing now what patients need next

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