A Reserch Tool to Advance Biopharmaceutical Characterization

NISTmAb RM 8671

Regulatory

NIST Industry

John E. Schiel, Trina Mouchahoir , Abby Turner, Katharina Yandrofski, Srivalli Telikepalli, Jason King, Paul DeRose, Dean Ripple, Karen Phinney

Monoclonal Antibody = biological drug that is 150X larger than Lipitor

BIOLOGICAL PRODUCTION STRUCTURAL COMPLEXITY

Atorvastatin (Lipitor)

Small chemical molecule 800-1000 Da

Well established chemical synthesis

Key challenges: • Complexity of characterizing biological molecules (larger, complex and dynamic structures and consist

of diverse populations of molecules) • Expanding catalogue analytical/biophysical methods – which methods to choose? • Understanding critical quality attributes of a biologic, identifying relevant product attributes and defining

acceptable manufacturing process ranges to ensure safety and efficacy

Monoclonal Antibody (IgG) Complex Biologic

150,000 Da, ~1300 Amino acids (with host cell modifications) Produced in mammalian cells

NISTmAb model courtesy of Joseph Curtis, Travis Gallagher, Monica Castellanos,

Monoclonal Antibody as a Platform

N-terminal pyro-Glu ~99% Pyro-Glu

PTMs intrinsic to mAbs Complex Molecule

Yet as a class they share common attributes

Industry-wide Reference Material must embody quality characteristics of product class

o Product-related substances : Intrinsic heterogeneity expected from manufacture process

o Product-related impurities : Undesirable degradation products

30+ Glycoforms

~90% % -K

N-glycosylation

C-terminal truncation

Glycation

Deamidation

Oxidation

Dioxidation

“Everyone should be using a common standard. …we know they (regulators) are getting crushed with applications….” – Small Biotech

“It would be great to have a huge production of this standard antibody, and everyone has the same system suitability criteria….” – Small Biotech

Low Abundance:

“The more robust they [standards] are, particularly when we start doing the multi-attribute approach (when using mass spec to give 4 or 5 different results) – using these standards and being able to show system suitability and some assay acceptance qualities would be very critical and very helpful.” - Large Biopharma

NH

CH C

CH2

O

CH2

S

CH3

NH

CH C

CH2

O

CH2

S

CH3

OMet Oxidation

NH

CH C

CH2

O

C

NH2

O

NH

CH C

CH2

O

C

OH

OAsp Deamidation

POTENTIAL ROLES OF RM FOR BIOTHERAPEUTIC CLASSES • Assess method variability

• Support method qualification & system suitability

• Assess new analytical technologies

• Support comparative studies of analytical methods

• Common framework for pre-competitive analytical methods development

QUALITY CONSIDERATIONS TO ENSURE SUITABILITY FOR USE • Must be voluntary, product neutral standard

• Exhaustively characterized for physicochemical and biophysical attributes

• Reference Values, stability, homogeneity with respect to • Physicochemical and biophysical attributes • Concentration

• Long term availability without alteration in product attributes

Emphasis on Material Properties

Attribute-Specific Control

Unique Lifecycle Approach

Unique Approach for IgG RM: • Completed rigorous interlaboratory characterization

• Results used for book compilation • Reference Material 8671

• Product Lifecycle --> Quality and Availability • Attribute-specific methods rigorously qualified • Value assignment incorporating method experience • Homogeneity, purity, stability based physicochemical

method control ranges

NIST mAb Attributes: • Open Innovation Humanized mAb (IgG1κ) RM 8671

• 10 mg/mL, 800 µL per unit • 12.5 mM L-His, 12.5 mM L-His HCl (pH 6.0)

•Peptide mapping by LC-MS/MS

• Primary Sequence • S-S Bridge Analysis • PTM analysis

•Intact, middle down MS •Glycosylation Analysis •LC: SEC, RP, IEX, HIC •CE: cIEF, cSDS, CZE •SDS-PAGE •MS/MS library compilation •HOS: NMR, HDX, XRD •Neutron scattering •Biophysical: CD, FTIR, DSC, DLS, AUC, SLS, DSF •Protein particulates •Many emerging technologies

NISTmAb Reference Material

Representative of IgG1ĸ Therapeutic Class

http://pubs.acs.org/isbn/9780841230262, http://pubs.acs.org/isbn/9780841230293 , http://pubs.acs.org/isbn/9780841230316

Reference Value Combined Standard

Uncertainty (uc) Coverage Factor (k)

Expanded Uncertainty (U)

Charge Purity (%) 73.81 0.17 3 0.51

Acidic Variants (%)

16.52 0.35 3 1.05

Basic Variants (%)

9.67 0.22 3 0.66

Method Attribute Recommended Storage

−80 °C Max F/T (cycles)

−20 °C Max F/T (cycles)

Max storage at 4 °C (days)

Control Range

UV Concentration −80 °C 5 5 28 ±2uc SEC -80 °C −80 °C 5 5 7 ±3uc

nrCE-SDS Monomeric Purity −80 °C 5 5 28 ±3uc

rCE-SDS

Glycan Occupancy, Thioether Content

−80 °C 5 5 28 ±3uc

CZE Charge Purity −80 °C 5 5 28 ±3uc

Table 1. Alternate storage conditions under which RM 8671 is expected to yield results within the indicated control rang

• Reference Values • UV, CZE, CE-SDS, SEC

• Informational Values • MFI, DLS

• LC-MS/MS MAM peptide map

• Representative Dataset • Chromatogram, etc.

• Handling storage independently reported for each intended use

Physicochemical Property Values, Homogeneity, and Stability

Five paper series accepted to Anal. Bioanal. Chem. describing in detail lifecycle, method details, qualification, value assignment

Interlaboratory Measurement Comparisons Using NISTmAb • Assess variability between labs and analytical technologies

• De-risking and/or lifecycle appropriate implementation • Fosters collaboration across global biopharma community

• 22 participants, ~ 8 industrial • Manuscript in preparation • Jeff Hudgens, Ioannis Karageorgos

HDX-MS of NISTmAb Fab

• 108 participants, ~ 50 industrial • Manuscript in preparation • Lorna DeLeoz

Glycoanalysis of NISTmAb

• 30 participants, ~ 11 industrial • Manuscript in preparation • Robert Brinson, Frank Delaglio, Luke Arbogast, John Marino

2D-NMR of NISTmAb Fab • 31 industrial participants • Data Received • Trina Mouchahoir, John Schiel

Multi-Attribute Method Consortium

• Primary sequence confirmation

• PTMs • Site-specific

glycan identification

Digestion

• C18 Reversed phase most common • Acetonitrile/water with formic acid gradient

LC-MS/MS

MS1

m/z

MS2 Collision Cell Data Analysis

m/z

m/z

LC-MS/MS Multi-Attribute Peptide Mapping

High Resolution ESI-MS

DTLMISROxidation

SLSLSPG(K) C-term Lys-

loss

DIQMTQSPSTLSASVGDR Oxidation WQQGNVFSCSVMHEALHNHYTQK;

FNWYVDGVEVHNAK Deamidation/Isomerization

5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 Time (min)

20

40

60

80

100 68.59 50.96

49.98 41.57 75.37 65.73

46.42 34.03 83.36

75.90 28.53 82.46

63.74 21.57 61.58 12.01

1.56 9.66 83.80 23.88 56.76 71.13 6.08 81.06 87.97 37.51 18.36 92.37

EEQYNSTYR G0F, G1F, G2F

Glycan

QVTLR N-term pyro-Glu

• Leverage LC-MS peptide mapping method to monitor multiple product quality attributes

• Potential for attribute-specific (rather than “peak”) acceptance criteria

• Automated new peak detection for impurity profiling

• New m/z detection combined with historical library allows simultaneous impurity identification

• State-of-the-art technology/software will drive lifecycle appropriate implementation

Multi-Attribute Method and New Peak Detection Round Robin Strength in Numbers

• Time-to-market fueled by high resolution, information rich analytics • NISTmAb Inter-laboratory LC-MS peptide mapping method to evaluate

• Ability for LC-MS to perform industry-relevant purity evaluation • Detection of spiked peptides and PTMs on the NISTmAb when stressed

• Evaluate LC-MS peptide mapping lifecycle appropriate implementation

DTLMISROxidation

SLSLSPG(K) C-term Lys-

loss

DIQMTQSPSTLSASVGDR Oxidation WQQGNVFSCSVMHEALHNHYTQK;

FNWYVDGVEVHNAK Deamidation/Isomerization

5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 Time (min)

20

40

60

80

100 68.59 50.96

49.98 41.57 75.37 65.73

46.42 34.03 83.36

75.90 28.53 82.46

63.74 21.57 61.58 12.01

1.56 9.66 83.80 23.88 56.76 71.13 6.08 81.06 87.97 37.51 18.36 92.37

EEQYNSTYR G0F, G1F, G2F

Glycan

QVTLR N-term pyro-Glu

Richard Rogers, JUST Trina Mouchahoir John Schiel

Digest performed at NIST -NISTmAb -pH stressed -peptide spike -unknown

• CASSS HOS Conference Workshop • NISTmAb data formatted to model ICH M4Q(R1) • Prepared as Phase III BLA section 3.2.S.3 • The NISTmAb was utilized as a test case

• Actual data on non-IP molecule • Facilitate discussion on experience/hurdles • Identify potential analysis gaps

Common Technical Document Case Study

NOTE: Not intended to be the “perfect” CTD, meant to spur discussion

TRAINIGN HERE!!!

• Industry Relevant Workforce Training • Fundamental LC and LC-MS with hands on training

• Agilent 6545 QTOF • Agilent Infinity II UHPLC • Exposure to NMR, HDX, CE, XRD, etc.

• Execute NISTmAb Standard Operating Protocols • Size-exclusion chromatography • Glycoanalysis • Intact and middle down MS

BREP Offers Cell Culture, Upstream, and Downstream Modules as well

http://www.brep.umd.edu/workforce-training.html

June 2018

Advanced Analytical

Technologies

Reference Materials

Process Analytics & Manufacturing

Structure- Function Studies

Computational Tools, Models

& Analytics

Maturation over time

Comprehensive models for product

attributes

Predicting biological outcomes

Reference materials & qualified methods

Lifecycle appropriate analytical

technologies

Predicting clinical performance and

feedback to manufacturing

VISION A national resource that provides measurement science and reference materials to advance the development, manufacture and regulatory approval of biopharmaceuticals.

Partnership Limitless Potential

Novel technology development Regulatory readiness evaluation Collaboration

NISTmAb Reference Material •Focus on Material Properties

• Large structural knowledgebase •IP free, thus dynamic utility for open innovation

• Novel technology development and collaboration • De-risk technology for lifecycle appropriate implementation

•Common framework for harmonization • Lifecycle directed toward long-term availability

Additional Class-Specific Materials •Future: Candidate IgG2 and IgG4 Materials

Expected Impact •Fuel technology innovation •Underpin regulatory decisions •Higher-order characterization •Method accuracy, precision, comparability •Translate to product safety and efficacy

Intended Use Drives Measurement System

Summary

http://www.nist.gov/mml/bmd/nist-mab.cfm

Acknowledgements

ACS Book Darryl Davis Oleg Borisov

NISTmAb Team Trina Mouchahoir Abigail Turner Katharina Yandrofski Srivalli Telikepalli Paul DeRose Dean Ripple Karen Phinney

NPD Round Robin Rich Rogers Trina Mouchahoir

NISTmAb CTD Workshop

Mike Tarlov Karen Phinney John Marino Dean Ripple

The hundreds of industry, academic, and government

collaborators who have fueled the NISTmAb through innovative

measurements Mark Schenerman MedImmune

http://www.nist.gov/mml/bmd/nist-mab.cfm