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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