The Use of Mass Spectrometry in FDA Applications for ... · CDER/OTR/DPA The Use of Mass Spectrometry in FDA Applications for Biotherapeutics: A Retrospective Review Sarah Rogstad,

CDER/OTR/DPA CDER/OTR/DPA

The Use of Mass Spectrometry in FDA Applications for Biotherapeutics: A

Retrospective Review

Sarah Rogstad, Anneliese Faustino, David Keire, Michael Boyne, and Jun Park

CASSS Mass Spec September 25, 2015

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This presentation reflects the views of the author and should not be construed to represent FDA’s

views or policies

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Protein Therapeutics

• Protein based biotherapeutics comprise a large and promising market within the United States.

• Complex heterogeneity requires elaborate technical analysis.

• Modern analytical techniques are necessary for their characterization.

• MS is an indispensable tool for the analysis of these products.

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Monoclonal Antibody

Fusion Protein

Antibody Drug Conjugate

Protein

CDER/OTR/DPA

Protein Therapeutic Characterization Physicochemical • Structure (primary and higher

order) • Molecular weight • Degree of heterogeneity

(chemical, conformational, etc) • Functional, receptor binding and

immunochemical • Biological activity (e.g. potency) • Functional domain

characterization • Enzyme kinetics • Receptor binding studies • Protein‐target binding

Impurity profiles • Product‐related impurities

– Inactive protein variants generated during manufacture or storage

• Process‐related impurities – Host cell proteins & DNA – Media components – Column leachates

Stability • Degradation profiles under real

time, accelerated, stress (freeze‐thaw, light high temperature exposure, agitation) conditions

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Collectively, these quality attributes define identity, purity, potency, and stability of the product and if critical, they correlate with efficacy and safety

CDER/OTR/DPA

Glycosylation Data Mining

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Read et al., Biotech & Appl Biochem, 2011.

• Survey of glycosylation analysis in mAb drug applications.

• Found high incidence of MS-based assays.

Inspired all-inclusive analysis of MS in protein therapeutics.


MS Data Mining Overview Goal: Analyze usage of MS in protein therapeutic biologics license applications (BLAs). Purpose: To inform the public and industry of the trends in MS usage in BLAs. 67 protein therapeutic BLAs approved between 1989-2015 were analyzed. Data Mining Focus:

– What was being analyzed? (ex. AA sequence, PTM, etc) – How was it being analyzed? (ex. Peptide mapping, Intact mass) – What methodology was used? (ex. MS1, MS2, MS3) – What instrumentation was used? (ex. MALDI-TOF, LC-ESI) – How was data used? (ex. Characterization)

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7

CDER/OTR/DPA

MS Data Mining Analysis Quality Attribute Targets • What is being analyzed • ex) Molecular Mass, amino acid

sequence, PTMs

MS Approach • General analytical method • ex) Intact mass, Peptide mapping

MS Instrumentation • Type of MS/ionization used • ex) LC-ESI, ESI, MALDI

MS Methodology • Specific analytical method • ex) MS, MS/MS

8 Target Approach Instrumentation Methodology

CDER/OTR/DPA

Protein BLA Overview

Antibody 49%

Other Protein

36%

Fusion Protein

13%

Antibody Drug

Conjugate 2%

Product Type Distribution

0

2

4

6

8

10

12

1989 1994 1999 2004 2009 2014

# of

BLA

s

Product Type Over Time

Antibody Protein

Fusion protein Antibody Drug Conjugate

Total

9

CDER/OTR/DPA

MS Usage Over Time

10

0

2

4

6

8

10

12

# of

BLA

s

MS No MS

MS used in all but 5 of the BLAs surveyed. MS data was used predominantly for API and impurity characterization purposes.

CDER/OTR/DPA

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Quality Attribute Targets • What is being analyzed • ex) Molecular Mass, amino acid

sequence, PTMs




CDER/OTR/DPA

0

10

20

30

40

50

60

# of

BLA

s

Quality Attribute Targets Analyzed via MS


30 Distinct Quality Attribute Targets were analyzed via MS.

CDER/OTR/DPA

Top 8 Targets Over Time


MS usage for the analysis of the top 8 targets generally increased over time.

0

2

4

6

8

10

12

2004 2005 2006 2007 2008 2009 2010 2011 2012 2013 2014 2015

# of

BLA

s

Amino Acid Sequence Analysis

Molecular Mass

Glycosylation

Disulfide bonds

Deamidation

Oxidation

Sequence Variants (N-term pyroGlutamateFormation)Sequence Variants (C-term Lysine Clipping)

Total

0102030405060708090

100

Molecular Mass Amino AcidSequenceAnalysis

Glycosylation Deamidation Disulfide bonds Oxidation SequenceVariants (N-termpyroGlutamine

Formation)

SequenceVariants (C-termLysine Clipping)

% o

f BLA

s

1989-2000 2001-2005 2006-2010 2011-2015

CDER/OTR/DPA

Number of MS Targets per BLA

0

2

4

6

8

10

12

# of

Tar

gets

per

BLA

Average # of Targets

R² = 0.9396

0

2

4

6

8

10

12

2009 2010 2011 2012 2013 2014 2015 2016

# of

Tar

gets

per

BLA

Average # of Targets


# of Targets per BLA

The number of targets analyzed via MS increased over time.


Quality Attribute Target Summary

• MS usage for 30 quality attribute targets was monitored. • MS was widely used in the analysis of those targets. • Amino acid sequencing and molecular weight analysis

were the most widely used and accounted for the MS usage in 60 applications.

• Over time, MS usage has increased in all of the top 8 targets, with respect to the number of BLAs and without.

• Number of MS targets per application has increased steadily over time.


CDER/OTR/DPA

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sequence, PTMs





MS Approaches Used in BLAs

• Peptide Mapping (Bottom-up) – Proteins digested prior to MS analysis

• Intact Mass (Top-down) – Whole proteins analyzed via MS

• Glycan Profiling – MS analysis of cleaved glycans

• Other – GC-MS, ICP-MS, Middle-down

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General Analytical Methods

CDER/OTR/DPA

MS Approaches Used in BLAs

# of Approaches per BLA

4321

0

2

4

6

8

10

12

2004 2006 2008 2010 2012 2014 2016

# of

BLA

s

MS Approaches over time

Intact Mass Peptide Mapping Glycan Profiling

Other Total

0

10

20

30

40

50

60

70

Peptide Mapping Intact Mass Glycan Profiling Other

# of

BLA

s

MS Approaches


General Analytical Methods

5%

19%

53%

23%

CDER/OTR/DPA

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sequence, PTMs




CDER/OTR/DPA

MS Instrumentation Used in BLAs

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0

5

10

15

20

25

30

Intact Mass Peptide Mapping Glycan Profiling#

of B

LAs

MS Instrumentation

QTOF MALDI-TOF Orbitrap

Target Approach Instrumentation Methodology

0

10

20

30

40

50

60

Peptide Mapping Intact Mass Glycan Profiling

# of

BLA

s

Ionization Method

LC-ESI MALDI ESI (no LC)

0

2

4

6

8

10

12

2004 2006 2008 2010 2012 2014 2016

# of

BLA

s

Ionization Method Over Time

LC-ESI MALDI ESI (no LC) Total

Types of MS and Ionization used

CDER/OTR/DPA

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sequence, PTMs




CDER/OTR/DPA

MS Methodology Used in BLAs


0

5

10

15

20

25

30

35

40

Peptide Mapping Intact Mass Glycan Profiling

# of

BLA

s

Methodology Used

MS1 MS2 MS3

0

2

4

6

8

10

12

2004 2006 2008 2010 2012 2014 2016

# of

BLA

s

Methodology Used Over Time

MS1 MS2 MS3 Total

Specific Analytical Methods


Methods Summary

• Peptide mapping and intact mass analysis are the most widely used MS approaches.

• Most applications (77%) used more than one MS approach. Small number of approaches being applied to multiple targets.

• LC-ESI was used most often. • LC-ESI and ESI usage increased over time. • MS and MS/MS were both widely used in peptide

mapping analyses. • Top-down MS/MS was not used.


CDER/OTR/DPA

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sequence, PTMs




CDER/OTR/DPA

Amino Acid Sequence Analysis 89%

11% Tested

NotTested

0

2

4

6

8

10

12

1985 1990 1995 2000 2005 2010 2015 2020

# of

BLA

s

Total MS AA Analysis

AA Analysis Total

Peptide Mapping


0

2

4

6

8

10

2004 2006 2008 2010 2012 2014 2016

# of

BLA

s

Ionization Method

LC-ESI MALDI AA Analysis

0

2

4

6

8

10

2004 2006 2008 2010 2012 2014 2016

# of

BLA

s

MS Methodology

MS1 MS2 AA Analysis

0

2

4

6

8

10

2004 2006 2008 2010 2012 2014 2016

# of

BLA

s

MS Instrumentation


Unknown AA Analysis


Molecular Mass Analysis

71%

29%

Tested Not Tested

0

2

4

6

8

10

12

1985 1990 1995 2000 2005 2010 2015 2020

# of

BLA

s

Total Molecular Mass MS

Mol. Mass Total

Intact Mass


0

2

4

6

8

10

12

2004 2006 2008 2010 2012 2014 2016

# of

BLA

s

Ionization Method

LC-ESI ESI (No LC) MALDI Mol. Mass

0

2

4

6

8

10

12

2004 2006 2008 2010 2012 2014 2016

# of

BLA

s

MS Instrumentation


Unknown Mol. Mass


Glycosylation Analysis

Tested 60%

Not Tested

40%

Tested Not Tested

21

9 6 1

11

15

3

1 0

5

10

15

20

25

30

35

Antibody Protein Fusion protein Antibody DrugConjugate

# of

BLA

s

MS Glyco-analysis by Drug Type

No MS Glyco Test

MS Glyco Test

69%

42%

67%

50%

0

10

20

30

40

50

60

70

80

Peptide Mapping Intact Mass Glycan Profiling Other

% o

f MS

Gly

cosy

latio

n Te

sts

Types of MS-based Glycosylation Testing

Other = GC-MS and Middle Down


0

2

4

6

8

10

12

1985 1990 1995 2000 2005 2010 2015 2020

# of

BLA

s

Total MS Glycosylation Analysis

Glycosylation Tests Total


Glycosylation Analysis

0

2

4

6

8

10

2004 2006 2008 2010 2012 2014 2016

# of

BLA

s

Ionization Method

LC-ESI ESI (no LC)

MALDI Total Glycosylation Tests

0123456789

10

2004 2006 2008 2010 2012 2014 2016

# of

BLA

s

MS Methodology

MS1 MS2 MS3 Total Glycosylation Tests


0

2

4

6

8

10

2004 2006 2008 2010 2012 2014 2016

# of

BLA

s

MS Approach

Peptide Mapping Intact Mass

Glycan Profiling Other

Total Glycosylation Tests

CDER/OTR/DPA

Other Targets of Interest

Higher Order Structure • 3 BLAs (2014)

– 1 mAb, 1 protein, 1 fusion protein

• Hydrogen Deuterium Exchange MS (HDX) – Intact Mass and Peptide

Mapping

Host Cell Proteins • 2 BLAs (2005, 2014)

– Both proteins

• In gel digest with LC-MS(/MS) of major bands.

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6%

94%

Tested Not Tested

3%

97%

Tested Not Tested

Target Approach Instrumentation Methodology


Quality Attribute Target Methods Summary

• MS based amino acid sequence analysis and molecular weight analysis are fundamental in protein BLAs.

• Multiple attributes are being analyzed using the same versatile MS approaches (peptide mapping and intact mass analysis) within the same applications.

• Similar overall trends for target analysis as for analysis as a whole.

• Complex targets are being analyzed using emerging approaches.



Future Roles of MS

• Continued increase in usage. • Increased complexity of assays.

– Top-down MS/MS, MS3, HDX

• Adapted methodology for more complex products. – Bispecific antibodies, biosimilars

• Broader application. – QC, comparability and similarity, clinical assays

• Coupling with orthogonal techniques. – CE, NMR

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Conclusions

• MS is critical to protein therapeutic characterization, and its usage is increasing proportionally to new applications.

• The number of quality attribute targets MS is being used to assess is also growing.

• Room for growth in MS assays as analytical techniques improve and products become more complex.

• Although MS is currently being used primarily for characterization, we anticipate that it will be used increasingly for QC and comparability and similarity.

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CDER/OTR/DPA

Acknowledgements

CDER/OPQ/OTR/DPA • Anneliese Faustino • David Keire • Ashley Gucinski • Michaella Levy • Melissa Zarr

CDER/OPQ/OBP •Jun Park

Other •Michael Boyne •CASSS MS

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CDER/OTR/DPA

Questions

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Documents

The Use of Mass Spectrometry in FDA Applications for ... · CDER/OTR/DPA The Use of Mass Spectrometry in FDA Applications for Biotherapeutics: A Retrospective Review Sarah Rogstad,