NISTmAb Common Technical Document Case Study · 2018-04-02 · NISTmAb Common Technical Document...

53
NISTmAb Common Technical Document Case Study In order to provide attendees with an opportunity to evaluate “real world” data, we have assembled a mock IND filing for NISTmAb RM 8671, a humanized monoclonal antibody (IgG1κ) Reference Material (RM). NISTmAb RM 8671 embodies the quality and characteristics of a biopharmaceutical product, is widely available to the biopharmaceutical community, and is an open innovation tool for technology development and dissemination of results. The public nature of information pertaining to the NISTmAb product quality attributes presents a unique opportunity for cross-community discussion on best practices. The “mock” common technical document is a summation of NISTmAb data measured by numerous collaborators and formatted to model an elucidation of structure section of the ICH common technical document M4Q(R1). The case study is not intended to be a template for mAb filings, instead it should serve as a foundation upon which to build discussions on current best practices and potential innovative approaches to analytical and biophysical data submission. Disclaimers: RM 8671 is intended for research use only. RM 8671 is NOT intended for animal or human consumption, clinical testing, or therapeutic use. RM 8671 users should always refer to the official NIST Report of Investigation for their specific material lot to obtain NIST Reference Values and uncertainty ranges. Values and “specifications” reported herein are based on measurements performed on the NISTmAb Primary Sample 8670 and RM 8671 (other than potency), however they do not necessarily represent NIST Reference and/or Certified Values. This presentation/article reflects the views of the author(s) and should not be construed to represent FDA’s views or policies. Certain commercial equipment, instruments, or materials are identified to adequately specify the experimental procedure. Such identification does not imply recommendation or endorsement by the National Institute of Standards and Technology, nor does it imply that the materials or equipment identified are necessarily the best available for the purpose.

Transcript of NISTmAb Common Technical Document Case Study · 2018-04-02 · NISTmAb Common Technical Document...

Page 1: NISTmAb Common Technical Document Case Study · 2018-04-02 · NISTmAb Common Technical Document Case Study . In order to provide attendees with an opportunity to evaluate “real

NISTmAb Common Technical Document Case Study

In order to provide attendees with an opportunity to evaluate “real world” data, we have assembled a mock IND filing for NISTmAb RM 8671, a humanized monoclonal antibody (IgG1κ) Reference Material (RM). NISTmAb RM 8671 embodies the quality and characteristics of a biopharmaceutical product, is widely available to the biopharmaceutical community, and is an open innovation tool for technology development and dissemination of results. The public nature of information pertaining to the NISTmAb product quality attributes presents a unique opportunity for cross-community discussion on best practices. The “mock” common technical document is a summation of NISTmAb data measured by numerous collaborators and formatted to model an elucidation of structure section of the ICH common technical document M4Q(R1). The case study is not intended to be a template for mAb filings, instead it should serve as a foundation upon which to build discussions on current best practices and potential innovative approaches to analytical and biophysical data submission.

Disclaimers:

RM 8671 is intended for research use only. RM 8671 is NOT intended for animal or human consumption, clinical testing, or therapeutic use.

RM 8671 users should always refer to the official NIST Report of Investigation for their specific material lot to obtain NIST Reference Values and uncertainty ranges. Values and “specifications” reported herein are based on measurements performed on the NISTmAb Primary Sample 8670 and RM 8671 (other than potency), however they do not necessarily represent NIST Reference and/or Certified Values.

This presentation/article reflects the views of the author(s) and should not be construed to represent FDA’s views or policies.

Certain commercial equipment, instruments, or materials are identified to adequately specify the experimental procedure. Such identification does not imply recommendation or endorsement by the National Institute of Standards and Technology, nor does it imply that the materials or equipment identified are necessarily the best available for the purpose.

Page 2: NISTmAb Common Technical Document Case Study · 2018-04-02 · NISTmAb Common Technical Document Case Study . In order to provide attendees with an opportunity to evaluate “real

Document Organizers and Contact Information:

John Schiel, National Institute of Standards and Technology, [email protected]

Christina Vessely, Biologics Consulting, Inc., [email protected]

Contributing Authors

Contributors Organization John Schiel Marco Blanco Trina Formolo Abigail Turner Curtis Meuse Luke Arbogast Robert Brinson Katharina Yandrofski Srivalli Telikepalli

National Institute of Standards and Technology Institute for Bioscience and Biotechnology Research

Christina Vessely Biologics Consulting, Inc. David Hayes David Feder Boehringer Ingelheim

Iain Campuzano Amgen Brandon Ruotolo Yumie Tian University of Michigan

Doug Marshall Bernard Costello Applied Photophysics

TABLE OF CONTENTS

3.2.S.1 GENERAL INFORMATION

3.2.S.2 MANUFACTURE 3.2.S.3 CHARACTERIZATION

Page 3: NISTmAb Common Technical Document Case Study · 2018-04-02 · NISTmAb Common Technical Document Case Study . In order to provide attendees with an opportunity to evaluate “real

NIST 00001234 Humanized IgG1κ monoclonal antibody, RM 8671 0000

3.2.S.1 General Information [Humanized IgG1κ Monoclonal Antibody, NIST], Page 1

3.2.S.1 GENERAL INFORMATION [Humanized IgG1κ monoclonal antibody, NIST]

3.2.S.1.1 NOMENCLATURE

Chemical Name(s) NISTmAb humanized IgG1κ monoclonal antibody

Company or Laboratory Code RM 8671

Page 4: NISTmAb Common Technical Document Case Study · 2018-04-02 · NISTmAb Common Technical Document Case Study . In order to provide attendees with an opportunity to evaluate “real

NIST 00001234 Humanized IgG1κ monoclonal antibody, RM 8671 0000

3.2.S.1 General Information [Humanized IgG1κ Monoclonal Antibody, NIST], Page 2

3.2.S.1.2 STRUCTURE The NISTmAb is an ~150 homodimer of two light chain and two heavy chain subunits linked through both inter- and intra-chain disulfide bonds kDa (theoretical molecular mass of 148199.3 Da for the G0F/G1F glycoform). The amino acid sequence for the NISTmAb heavy and light chains, as deduced from the cDNA sequence and confirmed by ultrahigh performance liquid chromatography coupled with UV-Visible absorbance and tandem mass spectrometry detection (LC-UV-MS/MS peptide mapping) as well as intact mass spectrometry is shown in Section 01.

3.2.S.1.2.1 NISTmAb Sequence Summary Variable Fab–Constant Fab– Hinge –Constant Fc-secretory tail NISTmAb Heavy Chain AA QVTLRESGPALVKPTQTLTLTCTFSGFSLSTAGMSVGWIRQPPGKALEWLADIWWDDKKHYNPSLKDRLTISKDTSKNQVVLKVTNMDPADTATYYCARDMIFNFYFDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK NISTmAb Light Chain DIQMTQSPSTLSASVGDRVTITCSASSRVGYMHWYQQKPGKAPKLLIYDTSKLASGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCFQGSGYPFTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC The 3 complementarity-determining regions (CDRs) in each chain are bolded and underlined. The N-linked glycosylation site (Asn300) is marked in bold and italic. The Glu residue at position 1 of the heavy chain and the Asp residue at position 1 of the light chain constitute the N-termini of the mature chains.

1 Formolo, T.; Ly, M.; Levy, M.; Kilpatrick, L.; Lute, S.; Phinney, K.; Marzilli, L.; Brorson, K.; Boyne, M.; Davis, D.; Schiel, J., Determination of the Nistmab Primary Structure. In State-of-the-Art and Emerging Technologies for Therapeutic Monoclonal Antibody Characterization Volume 2. Biopharmaceutical Characterization: The Nistmab Case Study, American Chemical Society: 2015; Vol. 1201, pp 1-62.

Page 5: NISTmAb Common Technical Document Case Study · 2018-04-02 · NISTmAb Common Technical Document Case Study . In order to provide attendees with an opportunity to evaluate “real

NIST 00001234 Humanized IgG1κ monoclonal antibody, RM 8671 0000

3.2.S.1 General Information [Humanized IgG1κ Monoclonal Antibody, NIST], Page 3

3.2.S.1.3 GENERAL PROPERTIES The physical and chemical properties of RM 8671 are provided in Table 1.

Table 1: General Properties of RM 8671

Physical/Chemical Properties Description Physical form and appearance RM 8671 drug substance is an aqueous solution stored

frozen at ≤ -70 °C at a concentration of approximately 10 mg/mL in (12.5 mmol/L L-histidine, 12.5 mmol/L L-histidine HCl. The drug substance is a clear to opalescent solution, colorless to slightly yellow.

pH RM 8671 is formulated at a target pH of 6.0

pI The isoelectric point of RM 8671 was measured as 9.18 (± 0.01)

Page 6: NISTmAb Common Technical Document Case Study · 2018-04-02 · NISTmAb Common Technical Document Case Study . In order to provide attendees with an opportunity to evaluate “real

NIST 00001234 Humanized IgG1κ monoclonal antibody, RM 8671 0000

3.2.S.2 Manufacture [Humanized IgG1κ Monoclonal Antibody, {Manufacturer}], Page 1

3.2.S.2 MANUFACTURE [Humanized IgG1k monoclonal antibody, NIST]

3.2.S.2.1 MANUFACTURER(S) Table 1: Manufacturer Information

Facility Responsibility

National Institute of Standards and Technology Institute for Bioscience and Biotechnology Research National Institute of Standards and Technology 9600 Gudelsky Dr. Rockville, MD 20850 John Schiel

Manufacture of reference standard Release testing Extended Characterization Testing

3.2.S.2.2 DESCRIPTION OF MANUFACTURING PROCESS AND PROCESS CONTROLS

This NISTmAb was received as a bulk substance prepared using mammalian cell culture and downstream processing representative of industry state-of-the-art. The manufacturing process is proprietary and therefore will not be presented as part of this case study. This includes the description of in-process controls, control of materials (raw material and starting material specifications, and analytical methods for analysis of the materials, compendial grade of materials, etc), control of critical steps and intermediates, process validation and process development. Comparability assessments associated with process changes that occurred over the course of the development of the drug substance will also be excluded from of this document.

The drug substance that was filled as the primary reference standard (PS 8670) was manufactured in alignment with the scaled process. PS 8670 was prepared from a single lot of drug substance which was diluted 10 fold in USP grade formulation buffer (12.5 mmol/L L histidine, 12.5 mmol/L L-histidine HCl, pH 6.0) and 800 µL aliquots placed into internally threaded screw top. Vials were placed in racks of 96 units each for storage at –80 °C. Sample processing was completed in a sterile environment using pre sterilized single-use equipment and/or in a class 100 000 cleanroom environment. The drug substance that was filled as the working reference standard (RM 8671) was manufactured for NIST according to the commercial process. RM 8671 was prepared by first homogenizing multiple bulk substance containers to form the 14HB batch. Aliquots of 1 L each were made from the homogenized bulk and designated as individual lots. A single lot (14HB-002) was then diluted 10 fold in USP grade formulation buffer (12.5 mmol/L L histidine, 12.5 mmol/L L-histidine HCl, pH 6.0) and 800 µL aliquots placed into internally threaded screw top. Vials were placed in racks of 96 units each for storage at –80 °C. Sample processing was completed in a sterile environment using pre sterilized single-use equipment and/or in a class 100000 cleanroom environment.

Page 7: NISTmAb Common Technical Document Case Study · 2018-04-02 · NISTmAb Common Technical Document Case Study . In order to provide attendees with an opportunity to evaluate “real

National Institute of Standards and Technology 00001234 Humanized IgG1κ monoclonal antibody, RM 8671 0000

3.2.S.3 Characterization [Humanized IgG1κ Monoclonal Antibody, NIST], Page 1

3.2.S.3 CHARACTERIZATION [NISTmAb, National Institute of Standards and Technology]

TABLE OF CONTENTS 3.2.S .3.1 ELUCIDATION OF S TRUCTURE AND OTHER

CHARACTERIS TICS ...............................................................................................4

3.2.S.3.1.1 Introduction ..................................................................................................4 3.2.S.3.1.2 Primary Sequence and Structure ..................................................................5

3.2.S.3.1.2.1 MS/MS Peptide Mapping .....................................................................5 3.2.S.3.1.2.2 Intact Molecular Mass Analysis ........................................................12 3.2.S.3.1.2.3 Glycan Map with Mass Spectrometry ................................................14 3.2.S.3.1.2.4 Charge Variant Analysis.....................................................................17 3.2.S.3.1.2.5 Disulfide Structure..............................................................................19

3.2.S.3.1.3 Size Heterogeneity ....................................................................................19 3.2.S.3.1.3.1 Size Exclusion UltraHigh Performance Liquid

Chromatography (SE-UPLC) .............................................................19 3.2.S.3.1.3.2 Capillary Sodium Dodecyl Sulfate (CESDS) .....................................22

3.2.S.3.1.4 Biophysical Characterization....................................................................25 3.2.S.3.1.4.1 Fourier transform infrared (FTIR) Spectroscopy ...............................25 3.2.S.3.1.4.2 Circular Dichroism Spectroscopy.......................................................27 3.2.S.3.1.4.3 Differential Scanning Calorimetry .....................................................29

3.2.S.3.1.5 High Resolution Measurement of Higher Order Structure.......................31 3.2.S.3.1.5.1 Two dimensional Nuclear Magnetic Resonance Spectroscopy..........31

3.2.S.3.1.6 Additional Higher Order Structure Sensitive Methods..............................33 3.2.S.3.1.6.1 Sedimentation Velocity Analytical Ultra Centrifugation (SV-

AUC) ..................................................................................................33 3.2.S.3.1.6.2 Ion Mobility ........................................................................................35 3.2.S.3.1.6.3 Dynamic Light Scattering...................................................................38 3.2.S.3.1.6.4 Static Light Scattering ........................................................................41 3.2.S.3.1.6.5 Sub Visible Particle Analysis .............................................................43

3.2.S.3.1.7 Biological Function.................................................................................44 3.2.S.3.1.7.1 Biological Activity .............................................................................44 3.2.S.3.1.7.2 Protein Concentration .........................................................................44

3.2.S .3.2 IMPURITIES ............................................................................................................46

3.2.S.3.2.1 PROCESS-RELATED IMPURITIES .......................................................46 3.2.S.3.2.2 PRODUCT-RELATED IMPURITIES ......................................................46

LIST OF TABLES Table 1: Summary of NISTmAb Structure and Function Elucidation Strategy .........................4 Table 2: Identification of Peptides contributing to TIC and UV Chromatogram Peaks.............7 Table 3: Observed masses for RM 8671 intact mass analysis and corresponding peak

annotations ..................................................................................................................14

Page 8: NISTmAb Common Technical Document Case Study · 2018-04-02 · NISTmAb Common Technical Document Case Study . In order to provide attendees with an opportunity to evaluate “real

National Institute of Standards and Technology 00001234 Humanized IgG1κ monoclonal antibody, RM 8671 0000

3.2.S.3 Characterization [Humanized IgG1κ Monoclonal Antibody, NIST], Page 2

Table 4: Glycan compositions and associated abundance as determined via LC-F-MS/MS........................................................................................................................15

Table 5: CZE results for PS 8670 and RM 8671 ......................................................................18 Table 6: SE-UHPLC results for PS 8670 and RM 8671...........................................................22 Table 7: CE-SDS results for PS 8670 and RM 8671 ................................................................25 Table 8: Melting temperatures (Tm) of NISTmAb in 25mM His/His-HCl at pH 6.0 ..............30 Table 9: NMR Acquisition and Processing Parameters............................................................32 Table 10: Sedimentation Velocity Analytical Ultracentrifugation Results ................................35 Table 11: Best-fit parameters estimated from linear fit to Equation 6 of the collective

diffusivities of NISTmAb in 25 mM His/His-HCl at pH 6.0. Uncertainties on fitted parameters corresponds to 95% confidence intervals .......................................41

Table 12: Best-fit apparent molecular weight and second virial coefficient for NISTmAb in 25 mM His/His-HCl at pH 6.0. Error bounds correspond to 95% confidence intervals of best-fit values ................................................................43

Table 13: Observed subvisible particle concentration (n=4 vials for PS 8760 and n = 6 vials for D-002)...........................................................................................................44

Table 14: Reference Mass Concentration Value for PS 8670 and RM 8671 Lot 14HB-D-002 by UV Vis spectrophotometry (n = 10 vials). .................................................45

LIST OF FIGURES Figure 1: Primary Reference Standard Peptide Map....................................................................6 Figure 2: Sequence encoded by DNA construct for RM 8671 with variable fragment

antigen-binding (Fab) in normal font, constant Fab segment underlined, hinge in italics, and constant Fc in bold .....................................................................10

Figure 3: RM 8671 Lot 14HB-D-002 sequence coverage .........................................................11 Figure 4: Representative chromatograms of the reference peptide map generated from

the PS digest (top trace in panels A and B) and RM 8671 Lot 14HB-D-002 (bottom trace in panels A and B) ................................................................................12

Figure 5: Representative Intact Mass Spectrum for RM 8671 ...................................................13 Figure 6: LC-F-MS/MS-2-AB labeled glycan analysis .............................................................15 Figure 7: Representative CZE electropherogram for RM 8671 Lot 14HB-D-002 where

“*” denotes the C terminal lysine present on one HC and “**” denotes the C-terminal lysine present on both HC subunits ..............................................................18

Figure 8: Representative SE-UPLC Chromatogram for RM 8671 Lot 14HB-D-002................21 Figure 9: Representative electropherogram of RM 8671 Lot 14HB-D-002 by non-

reducing CE SDS. .......................................................................................................23 Figure 10: Representative electropherogram of RM 8671 lot 14HB-D-002 by rCE-SDS,

full scale (top figure) and expanded baseline (bottom figure)....................................24 Figure 11: FTIR spectra (upper panel) and second derivative plot (lower panel)

recorded for (1) Red PS 8670, (2) Grey 8671, (3), Blue RM 8671 minus PS 8670 ............................................................................................................................27

Page 9: NISTmAb Common Technical Document Case Study · 2018-04-02 · NISTmAb Common Technical Document Case Study . In order to provide attendees with an opportunity to evaluate “real

National Institute of Standards and Technology 00001234 Humanized IgG1κ monoclonal antibody, RM 8671 0000

3.2.S.3 Characterization [Humanized IgG1κ Monoclonal Antibody, NIST], Page 3

Figure 12: CD spectra of PS 8670 (red) and RM 8671 (blue) in far UV (plot A) and near UV (plot B) .........................................................................................................28

Figure 13: Quality range statistical treatment ..............................................................................29 Figure 14: Differential scanning calorimetry (DSC) profiles of NISTmAb in 25mM

His/His-HCl at pH 6.0 ................................................................................................30 Figure 15: A) Overlay 1H-13C methyl spectrum of intact PS 8670 (blue) and RM 8671

(red). B) Linear regression plot of the spectra shown in panel A. C) Overlay 1H-15N amide spectrum of the Fc domain from PS 8670 (blue) and RM 8671 (red). D) Overlay 1H-15N amide spectrum of the Fab domain from PS 8670 (blue) and RM 8671 (red).............................................................................33

Figure 16: Overlay of 3 replicate apparent sedimentation coefficient distribution functions for Primary Sample 8670. Inset is with Y axis magnified to show aggregate and fragment peaks ....................................................................................34

Figure 17: Overlay of 3 replicate apparent sedimentation coefficient distribution functions for Reference Material 8671 LOT 14HB-D-002. Inset is with Y axis magnified to minor peaks ....................................................................................35

Figure 18: Native MS analysis of NIST mAb PS8760 and RM 8671. Nitrogen-based collision cross sections were derived from the RF-confining drift tube instrument. Charge states and nitrogen collision cross sections are also annotated .....................................................................................................................37

Figure 19: Collision Induced Unfolding (CIU) profile of the NISTmAb ....................................38 Figure 20: Analysis of autocorrelation function for NISTmAb in 25 mM His/His-HCl at

pH 6.0. (a) Collective diffusion coefficient (DC) as a function of protein concentration. (b) Particle size distributions based on the average hydrodynamic radius (RH) ..........................................................................................40

Figure 21: Excess Rayleigh scattering (R90ex/K) of NISTmAb in 25 mM His/His-HCl at

pH 6.0. Symbols correspond to average R90ex/K over four independent

replicates for: (blue circles) Primary Sample 8670; and (red squares) Reference Material 8671. Error bars indicate 95% confidence intervals. Dashed lines represent best-fitted curves of the corresponding SLS data to Equation 6. ..................................................................................................................43

Figure 22: Representative images obtained for various particles in RM 8671 lot 14HB-D-002 ..........................................................................................................................44

Page 10: NISTmAb Common Technical Document Case Study · 2018-04-02 · NISTmAb Common Technical Document Case Study . In order to provide attendees with an opportunity to evaluate “real

National Institute of Standards and Technology 00001234 Humanized IgG1κ monoclonal antibody, RM 8671 0000

3.2.S.3 Characterization [Humanized IgG1κ Monoclonal Antibody, NIST], Page 4

3.2.S.3.1 ELUCIDATION OF STRUCTURE AND OTHER CHARACTERISTICS

3.2.S.3.1.1 Introduction The structural characterization of Humanized IgG1κ monoclonal antibody, RM 8671was performed as part of qualification studies for primary reference material (PS 8670) and working standard (RM 8671) lot 14HB-D-002. In addition to typical release specification methods, as described in Section S.4., the protein structure and biophysical properties were further characterized using a combinatorial toolbox approach consistent of numerous complimentary and orthogonal measurements of product quality as summarized in Table 1.

Table 1: Summary of NISTmAb Structure and Function Elucidation Strategy

S tructure Quality Attribute Assaya

Primary Structure Primary structure confirmation MS/MS Peptide Mapping

Post translational modifications Intact Mass Analysis

N-linked glycosylation profile Glycan Map with mass spectrometry

Charge Variant Analysis Capillary Zone Electrophoresis (CZE)

Capillary Isoelectric Focusing (cIEF)

Disulfide Structure Confirmation

Non-reduced MS/MS Peptide Map

Secondary / Tertiary Structure

General structural elements Fourier Transform Infrared Spectroscopy (FTIR)

Near-UV Circular Dichroism Spectroscopy (near-UV CD)

2D NMR

Quaternary Structure

Size variant analysis, aggregation, colloidal stability Sub-visible particle content Dynamics of Higher Order Structure Collisional cross section

Size Exclusion Chromatography

Reduced capillary sodium dodecyl sulfate electrophoresis (rCE-SDS)

Non-Reduced capillary sodium dodecyl sulfate electrophoresis (nrCE-SDS)

2D NMR

Analytical Ultracentrifugation (AUC)

Static and/or Dynamic Light Scattering

Microflow Imaging

Ion Mobility

Page 11: NISTmAb Common Technical Document Case Study · 2018-04-02 · NISTmAb Common Technical Document Case Study . In order to provide attendees with an opportunity to evaluate “real

National Institute of Standards and Technology 00001234 Humanized IgG1κ monoclonal antibody, RM 8671 0000

3.2.S.3 Characterization [Humanized IgG1κ Monoclonal Antibody, NIST], Page 5

S tructure Quality Attribute Assaya

Thermal Properties Thermal Stability Differential Scanning Calorimetry

Charge Variant Analysis

Charge Heterogeneity Isoelectic point

Capillary Zone Electrophoresis Capillary Isoelectric Focusing

a Additional characterization of the antibody by reduced/non-reduced denatured SEC (rdSEC and nrdSEC), X-Ray Chrystallography, and Hydrogen-Deuterium Exchange MS (HDX-MS) will be provided as a supplement to the submission when available.

A brief description of the extended characterization test methods and results are presented below. A complete description of the analytical procedures utilized in the release of Reference Material is provided in 3.2.S.4.2.

3.2.S .3.1.2 Primary S equence and S tructure

3.2.S .3.1.2.1 MS /MS Peptide Mapping

The peptide mapping method examines RM 8671 primary structure by monitoring its trypsin digested peptides which are resolved using reverse-phase ultrahigh pressure liquid chromatography instrumentation coupled to an ultraviolet wavelength detector and a high-resolution mass spectrometer with electrospray ionization source (together “LC-UV-MS”). A chromatographic trace, or peptide map, results from the signal generated as peptides eluting from the LC column pass through the UV and MS detectors producing “peaks”. Differing amino acid sequences give each peptide unique chromatographic properties and the presence of online MS/MS detection provides confident assignment of primary structure based on mass and fragmentation commensurate with the predicted amino acid sequence.

RM 8671 is prepared by first diluting the sample to approximately 1 mg/mL in denaturing buffer (6 mol/L guanidine HCl, 1 mmol/L EDTA in 0.1 mol/L Tris, pH 7.8). The samples are then reduced through the addition of Dithiothreitol (5 mmol/L), followed by alkylation of the resulting free sulfhydryl groups using iodoacetamide (IAM, 10 mmol/L). The samples are then digested using recombinant porcine trypsin at a 1:35 enzyme:sample mass ratio for a period of 4 hours at ambient temperature.

Reverse-phase ultrahigh pressure liquid chromatography is performed using the Dionex UltiMate™ Rapid Separation Binary Pump (Thermo Scientific, Waltham, MA; P/N HPG-3200RS). For digested samples, 5 µg of peptide digest is loaded via autosampler onto a C18 column (XSelect Peptide CSH C18 XP, 2.1 mm ID x 150 mm, 2.5 µm particle, 130 Å pore) (Waters Corp., Milford, MA; P/N 186006727) enclosed in a thermostatted column oven set to 40 °C. Samples are held at 7 °C while queued for injection. The chromatographic method is initiated with 98 % Mobile Phase A (0.1 % v/v formic acid in H2O) and 2 % Mobile Phase B (0.1 % v/v formic acid in acetonitrile) with the flow rate set at a constant 0.200 mL/min. After a 10 min wash, peptides were eluted over a 110 min gradient in which Mobile Phase B content rose at a rate of 0.39 % per min to reach a final composition comprising 45 % Mobile Phase B.

Page 12: NISTmAb Common Technical Document Case Study · 2018-04-02 · NISTmAb Common Technical Document Case Study . In order to provide attendees with an opportunity to evaluate “real

National Institute of Standards and Technology 00001234 Humanized IgG1κ monoclonal antibody, RM 8671 0000

3.2.S.3 Characterization [Humanized IgG1κ Monoclonal Antibody, NIST], Page 6

Prior to the next sample injection, the column was washed for 15 min with 97 % Mobile Phase B, then equilibrated to 98 % Mobile Phase A for 25 min.

Peptides or proteins eluting from the chromatography column are first passed through a variable wavelength detector (Dionex UltiMate™ 3000 Variable Wavelength Detector (Thermo Scientific, Waltham, MA; P/N VWD-3400RS) set to measure UV absorption at 214 nm. Ions are then introduced into an LTQ Orbitrap Discovery XL mass spectrometer (Thermo Scientific, Waltham, MA) fitted with a heated electrospray ionization source probe (HESI-II) (Thermo Scientific, Waltham, MA).

Representative chromatograms for the peptide sequence for the primary reference standard (PS 8670) are provided in Figure 1.

Figure 1: Primary Reference Standard Peptide Map

Primary Standard tryptic digest was analyzed by LC-UV-MS in quadruplicate. Mean retention times for TIC and UV chromatographic peaks were calculated as shown in Table 2. These values are used here to label their corresponding peaks (Panel A = TIC; Panel B = UV 214 nm) and together constitute the reference peptide map. In cases where post translational modifications are observed, lower case letters in the peptide sequence reflect the location of the observed modification.

Page 13: NISTmAb Common Technical Document Case Study · 2018-04-02 · NISTmAb Common Technical Document Case Study . In order to provide attendees with an opportunity to evaluate “real

National Institute of Standards and Technology 00001234 Humanized IgG1κ monoclonal antibody, RM 8671 0000

3.2.S.3 Characterization [Humanized IgG1κ Monoclonal Antibody, NIST], Page 7

Table 2: Identification of Peptides contributing to TIC and UV Chromatogram Peaks

Mean TIC RT (min)

Mean UV RT (min)

Peptide Modification

1.55 n/a (Buffer component) -

1.87 1.78

TISK -

APK -

QPPGK -

VEPK -

GQPR -

VDKR -

EAK -

EYK -

SCDK -

GEC -

RVEPK -

TKPR -

nd 1.89 (Buffer component) -

nd 1.93 (Buffer component) -

2.32 n/a EEMTK -

2.73 nd ADYEK -

2.79 2.71

SFNR -

VEIKR -

KHYNPSLK -

NKPGVYTK (Trypsin) -

nd 2.98 (Buffer component) -

3.34 3.26 (Buffer component) -

4.19 nd VEIK -

nd 5.12 Not determined -

5.61 5.55 LTVDK -

5.94 5.86 HYNPSLK -

WYQQKPGK -

6.63 6.55 (Buffer component) -

Page 14: NISTmAb Common Technical Document Case Study · 2018-04-02 · NISTmAb Common Technical Document Case Study . In order to provide attendees with an opportunity to evaluate “real

National Institute of Standards and Technology 00001234 Humanized IgG1κ monoclonal antibody, RM 8671 0000

3.2.S.3 Characterization [Humanized IgG1κ Monoclonal Antibody, NIST], Page 8

Mean TIC RT (min)

Mean UV RT (min)

Peptide Modification

7.98 7.91 VQWK -

10.14 10.07 NQVVLK -

10.58 10.51 LTISK -

12.92 nd DRLTISK -

nd 14.74 (Buffer component) -

21.01 20.94 LASGVPSR -

23.81 23.75

VTITCSASSR -

EEQYnSTYR G0F (N300)

EEQYnSTYR G1F (N300)

EEQYnSTYR G2F (N300)

27.17 27.12 SCSVMHEALHNHYTQK -

EVTHQGLSSPVTK -

28.75 28.67 SLSLSPGK -

VGYmHWYQQKPGK Oxidation (M34)

30.07 30.00 HKVYACEVTHQGLSSPVTK -

LSSPATLNSR (Trypsin) -

30.53 30.42 VGYMHWYQQKPGKAPK -

32.71 32.64 VDNALQSGNSQESVTEQDSK -

32.84 32.78 ALPAPIEK -

33.4 33.33 VGYMHWYQQKPGK -

35.2 35.13 qVTLR Gln -> pyroGlu (Q1)

VATVSLPR (Trypsin) -

35.77 35.69 DTLMISR -

nd 37.03 Not determined -

37.45 37.37 VYACEVTHQGLSSPVTK -

VGYMHWYQQK -

nd 37.67 Not determined -

39.52 39.43

SLSLSPGk Lys-loss (K450)

EPQVYTLPPSR -

TVLHQDWLNGK -

Page 15: NISTmAb Common Technical Document Case Study · 2018-04-02 · NISTmAb Common Technical Document Case Study . In order to provide attendees with an opportunity to evaluate “real

National Institute of Standards and Technology 00001234 Humanized IgG1κ monoclonal antibody, RM 8671 0000

3.2.S.3 Characterization [Humanized IgG1κ Monoclonal Antibody, NIST], Page 9

Mean TIC RT (min)

Mean UV RT (min)

Peptide Modification

40.26 (a) 40.18 (a) LLIYDTSK -

40.26 (a) nd WQQGNVFSCSVmHEALHNHYTQK Oxidation (M431)

40.26 (b) nd EPQVYTLPPSR -

42.38 42.31 STSGGTAALGCLVK -

DIQmTQSPSTLSASVGDR Oxidation (M4)

44.77 44.7 VTNMDPADTATYYCAR -

46.1 46.02 NQVSLTCLVK -

46.98 46.92 WQQGNVFSCSVMHEALHNHYTQK -

47.3 47.24 GPSVFPLAPSSK -

48.72 48.66 DIQMTQSPSTLSASVGDR -

WQQGNVFSC -

49.47 49.4 FNWYVDGVEVHNAK -

52.4 52.33 TPEVTCVVVDVSHEDPEVK -

54.55 54.48

DSTYSLSSTLTLSK -

nQVSLTCLVK Deamidation (N364)

LGEHNIDVLEGNEQFINAAK (Trypsin) -

56.18 56.12 SLSTAGMSVGWIR -

58.66 nd

RTVAAPSVFIFPPSDEQLK -

GPSVFPLAPSSKSTSGGTAALGCLVK -

IITHPNFNGNTLDNDImLIK (Trypsin) Oxidation (M94)

59.96 nd SCDKTHTCPPCPAPELLGGPSVFLFPPKPK -

FNWYVDGVEVH -

60.96 60.87 IITHPNFNGNTLDNDIMLIK (Trypsin) -

VVSVLTVLHQDwLNGK Dioxidation (W316)

62.58 62.48

THTCPPCPAPELLGGPSVFLFPPKPK -

ESGPALVKPTQTLTLTCTF -

VVSVLTVLHQDWLNGKEYK -

63.54 63.46 GFYPSDIAVEWESNGQPENNYK -

65.42 65.35 TTPPVLDSDGSFFLYSK -

Page 16: NISTmAb Common Technical Document Case Study · 2018-04-02 · NISTmAb Common Technical Document Case Study . In order to provide attendees with an opportunity to evaluate “real

National Institute of Standards and Technology 00001234 Humanized IgG1κ monoclonal antibody, RM 8671 0000

3.2.S.3 Characterization [Humanized IgG1κ Monoclonal Antibody, NIST], Page 10

Mean TIC RT (min)

Mean UV RT (min)

Peptide Modification

66.52 66.43 TVAAPSVFIFPPSDEQLK -

67.91 67.84 VVSVLTVLHQDWLNGK -

SGFSLSTAGMSVGWIR -

nd 69.29 Not determined -

70.32 70.25 VVSVLTVLHQDWLnGK Succinimide (N318)

70.61 70.52 ESGPALVKPTQTLTLTCTFSGF -

72.1 72.03 SGTASVVCLLNNFYPR -

74.82 74.74

ALEWLADIWWDDKK -

SLQPDDFATYYCFQGSGYPFTFGGGTK -

ESGPALVKPTQTLTLTCTFSGFSLSTAGMSVGWIRQPPGK -

80.14 80.06 ESGPALVKPTQTLTLTCTFSGFSLSTAGMSVGWIR -

81.87 81.78 DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTK -

83.54 83.45 ALEWLADIWWDDK -

86.51 86.42 FSGSGSGTEFTLTISSLQPDDFATYYCFQGSGYPFTFGGGTK -

nd 86.93 Trypsin digest component -

88.26 88.18 DMIFNFYFDVWGQGTTVTVSSASTK -

The theoretical amino acid sequence for RM 8671 is provided in Figure 2.

Figure 2: Sequence encoded by DNA construct for RM 8671 with variable fragment antigen-binding (Fab) in normal font, constant Fab segment underlined, hinge in italics, and constant Fc in bold

RM 8671 Heavy Chain AA QVTLRESGPALVKPTQTLTLTCTFSGFSLSTAGMSVGWIRQPPGKALEWLADIWWDDKKHYNPSLKDRLTISKDTSKNQVVLKVTNMDPADTATYYCARDMIFNFYFDVWGQGTTVTVSS ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRV EPKSCDKTHTCPPCP APELLGGPS VFLFPPKPKDTLMIS RTPEVTCVVVDVS HEDPEVKFNWYVDGVEVHNAKTKPREEQYNS TYRVVS VLTVLHQDWLNGKEYKCKVS NKALPAPIEKTIS KAKGQPREPQVYTLPPS REEMTKNQVS LTCLVKGFYPS DIAVEWES NGQPENNYKTTPPVLDS DGS FFLYS KLTVDKS RWQQGNVFSCS VMHEALHNHYTQKS LS LS PGK

Page 17: NISTmAb Common Technical Document Case Study · 2018-04-02 · NISTmAb Common Technical Document Case Study . In order to provide attendees with an opportunity to evaluate “real

National Institute of Standards and Technology 00001234 Humanized IgG1κ monoclonal antibody, RM 8671 0000

3.2.S.3 Characterization [Humanized IgG1κ Monoclonal Antibody, NIST], Page 11

RM 8671 Light Chain

DIQMTQSPSTLSASVGDRVTITCSASSRVGYMHWYQQKPGKAPKLLIYDTSKLASGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCFQGSGYPFTFGGGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

The mass spectrometry (MS/MS) data were used to assign the observed mass to the respective UV peaks for confirmation of sequence in each peptide. The results of the analysis confirm > 96% coverage of peptides in the UV chromatogram based on the expected RM 8671 amino acid sequence (96.89 % for heavy chain and 100 % for light chain), as illustrated in Figure 3.

Figure 3: RM 8671 Lot 14HB-D-002 sequence coverage

All post translational modifications identified in RM 8671 were consistent with those previously reported for the PS. Aglycosylation at Asn300 of the heavy chain was <1.0%, ≈90% of the heavy chain was missing the C-terminal lysine, and ≈99% of the light chain N-terminal Gln was converted to cyclized, pyroglutamic acid. The mass spectrometry results indicated low levels of glycation, oxidation, and deamidation as previously reported. No gross changes in the levels of post-translational modifications were reported compared to the PS material.

Page 18: NISTmAb Common Technical Document Case Study · 2018-04-02 · NISTmAb Common Technical Document Case Study . In order to provide attendees with an opportunity to evaluate “real

National Institute of Standards and Technology 00001234 Humanized IgG1κ monoclonal antibody, RM 8671 0000

3.2.S.3 Characterization [Humanized IgG1κ Monoclonal Antibody, NIST], Page 12

During certification of the working reference standard, RM 8671, a comparison was made of the peptide sequence to that of the primary reference standard. Representative chromatograms for the peptide sequence for the primary reference standard (PS 8670) and RM 8671 are provided in Figure 4.

Figure 4: Representative chromatograms of the reference peptide map generated from the PS digest (top trace in panels A and B) and RM 8671 Lot 14HB-D-002 (bottom trace in panels A and B)

A comparison of the peak traces shows a high degree of similarity upon visual inspection with no trace having a unique or missing peak as compared to the PS reference map for peptide-containing peaks. The retention times for RM 8671 peaks are also highly comparable to the reference chromatogram, displaying no significant differences (< 2 %) between the means of the reference peak retention times and those of each corresponding RM 8671 peak for either TIC (A) or UV (B) chromatograms.

3.2.S .3.1.2.2 Intact Molecular Mass Analysis Analysis of intact proteins using UHPLC-MS is a rapid method for empirically determining the molecular mass of a target protein along with information on post-translational modifications. The measured molecular mass is compared to a theoretically calculated molecular mass based on the expected amino acid composition as an orthogonal confirmation of primary structure.

Page 19: NISTmAb Common Technical Document Case Study · 2018-04-02 · NISTmAb Common Technical Document Case Study . In order to provide attendees with an opportunity to evaluate “real

National Institute of Standards and Technology 00001234 Humanized IgG1κ monoclonal antibody, RM 8671 0000

3.2.S.3 Characterization [Humanized IgG1κ Monoclonal Antibody, NIST], Page 13

The mass heterogeneity of intact RM 8671 lot 14HB-D-001 was analyzed by electrospray ionization mass spectrometry. LC-MS analysis was performed on an Agilent Infinity II UHPLC coupled with an Agilent 6545 QTOF mass spectrometer. Intact antibody was injected (3 µg) onto a PLRP-S Column (2.1 mm x 50 mm, 1000 Angstrom pore size, 5 µm particle size) held at a constant temperature of 60oC and eluted at 0.4 mL/min. Solvent A consisted of 0.1 % Formic acid in Water and Solvent B consisted of 0.1% Formic acid in Acetonitrile. The chromatographic method is initiated with 20 % Mobile Phase B. After a 2 min wash, peptides were eluted over an 18 min gradient in which Mobile Phase B content rose at a rate of 0.33 % per min to reach a final composition comprising 80 % Mobile Phase B. The QTOF was operated in 2 GHz Extended Mass Range (500 to 5000 m/z) mode at an acquisition rate of 1 spectra / sec. The 1221.990637 ion was used as a reference mass throughout the run. Deconvolution of the resulting spectrum was performed using BioConfrim 7 using the maximum entropy algorithm.

A representative deconvoluted mass spectrum for RM 8671 lot 14HB-D-002 is shown in Figure 5. The observed masses and corresponding peak identifications are listed in Table 3.

Figure 5: Representative Intact Mass Spectrum for RM 8671

Page 20: NISTmAb Common Technical Document Case Study · 2018-04-02 · NISTmAb Common Technical Document Case Study . In order to provide attendees with an opportunity to evaluate “real

National Institute of Standards and Technology 00001234 Humanized IgG1κ monoclonal antibody, RM 8671 0000

3.2.S.3 Characterization [Humanized IgG1κ Monoclonal Antibody, NIST], Page 14

Table 3: Observed masses for RM 8671 intact mass analysis and corresponding peak annotations

Proteoform Theoretical Mass (Da) Observed Mass (Da) ∆m ppm error

G0F/G0F –2GlcNAc 147630.8 147632.71 1.91 12.93768

G0F/G0F –GlcNAc 147834.0 147839.05 5.05 34.15994

G0F/G1F –GlcNAc 147996.1 147999.41 3.31 22.36545

G0F/G0F 148037.2 148041.74 4.54 30.66797

G0F/G0F + K 148165.3 148162.28 3.02 20.38264

G0F/G1F 148199.3 148203.32 4.02 27.12563

G0F/G1F + K 148327.5 148325.24 2.26 15.23655

G1F/G1F 148361.4 148365.00 3.6 24.26507

G1F/G1F +K 148489.6 148488.31 1.29 8.687477

G1F/G2F 148523.6 148525.83 2.23 15.01445

G1F/G2F + K 148651.8 148654.00 2.2 14.79969

G2F/G2F 148685.7 148685.10 0.6 4.035358

G2F/G2F + Hex 148847.7 148848.81 1.11 7.457287

G2F/G2F + 2Hex 149010.0 149007.79 2.21 14.83122

The results were consistent with the predicted NISTmAb structure containing N-linked, high mannose and core-fucosylated, biantennary glycans as well as minor species considered to be modified by non-enzymatic glycation. Minor variants consisting of des-lysine truncation at the C-terminus were also observed. An identical analysis of PS 8670 resulted in traces showing a high degree of similarity upon visual inspection with no trace having a unique or missing peak as compared to the RM 8671. The observed masses for PS 8670 are also highly comparable to RM 8671, displaying no significant differences (< 30 ppm) from the predicted molecular mass.

3.2.S .3.1.2.3 Glycan Map with Mass S pectrometry The distribution of N-linked oligosaccharides on PS 8670 was determined by hydrophilic liquid interaction UHPLC-Fluorescence-MS analysis. In this method glycans are enzymatically released from the NISTmAb and labeled at their reducing terminus with the fluorescent dye 2-aminobenzamide. Labeled glycans are resolved using hydrophilic liquid interaction UHPLC and quantified based on their relative fluorescence signal. Online MS/MS detection provides confident assignment of primary structure based on mass and fragmentation commensurate with a given glycan composition.

Glycan release was performed by mixing 500 µg of PS with 14 µL of 5X reaction buffer and 6 µL of PNGase F (Prozyme) and incubating for 120 minutes at 37 °C. Glykoprep H cartridges

Page 21: NISTmAb Common Technical Document Case Study · 2018-04-02 · NISTmAb Common Technical Document Case Study . In order to provide attendees with an opportunity to evaluate “real

National Institute of Standards and Technology 00001234 Humanized IgG1κ monoclonal antibody, RM 8671 0000

3.2.S.3 Characterization [Humanized IgG1κ Monoclonal Antibody, NIST], Page 15

(Prozyme) were then used to purify released glycan from remaining apoprotein. Samples were dried to completion in a centrifugal evaporator and stored at -20oC until labeling. Labeling of each glycan sample with 2-AB was performed by reconstituting in 10 µL of acetic acid/DMSO solution (3/7 v/v) containing 5 mg 2-AB / 100 µL and 6 mg NaCNBH3 / 100 µL. This sample was allowed to incubate at 65 oC for 2 hours. Each sample was then diluted with 190 µL of Acetonitrile and purified using GlykoPrep CU cartridges. Labeled glycans were dried to completion in a centrifugal evaporator, and stored at -20 oC until analysis.

A Dionex UltiMate 3000 UHPLC system with fluorescence detection was coupled to an OrbiTrap Elite mass spectrometer with heated electrospray ionization source (HESI-II) (Thermo Scientific, Waltham, MA). Dried 2-AB samples were reconstituted in 250 µL of 80% Acetonitrile. The samples were injected in volumes corresponding to glycans released from 80 µg of NISTmAb. Separation was conducted on a Waters Glycan column (150 mm x 2.1 mm i.d., particle diameter 1.7 µm) at 60oC. A binary gradient was used for the analytical separation consisting of solvent A = 50 mmol/L ammonium formate (pH 4.4) and solvent B = 0.1% formic acid (v/v) in acetonitrile (ACN). The gradient was delivered with a curve factor of 6 and compositions of 70% B at 0 min, 70% (v/v) B at 1.47 min, 55% (v/v) B at 15 min, 30% (v/v) B at 15.5 min, 30% (v/v) B at 16.25 min, 70% (v/v) B at 16.55 min, and 70% (v/v) B at 18.5 min. Flow rate was set at 0.561 mL/min at all timepoints with the exception of 16.25 min and 16.55 min, during which the flow rate was set to 0.300 mL/min. Eluent was monitored at λexcitation = 330 nm, λemission = 420 nm and then directed to the Orbitrap Elite. The mass spectrometer was set to collect in a data dependent MS1/MS2 collision induced dissociation mode (CID = 35) with dynamic exclusion enabled. The resulting 2-AB glycan fluorescence trace is illustrated in Figure 6.

Figure 6: LC-F-MS/MS-2-AB labeled glycan analysis

The major peaks corresponded to high mannose and core-fucosylated, biantennary structures with 0-2 terminal galactose residues, as described in Table 4. The level of charged glycans containing sialic acid was <2.0%.

Table 4: Glycan compositions and associated abundance as determined via LC-F-MS/MS

Oxford Nomenclature (proposed structure)

%RA St. Dev (n=3)

M3 0.3 0.01

Page 22: NISTmAb Common Technical Document Case Study · 2018-04-02 · NISTmAb Common Technical Document Case Study . In order to provide attendees with an opportunity to evaluate “real

National Institute of Standards and Technology 00001234 Humanized IgG1κ monoclonal antibody, RM 8671 0000

3.2.S.3 Characterization [Humanized IgG1κ Monoclonal Antibody, NIST], Page 16

Oxford Nomenclature (proposed structure)

%RA St. Dev (n=3)

FA1-Hex

FA1-Hex* 0.9 0.01

FM3 0.11 0.01

A1 0.35 0.01

FA1 2.25 0.03

A2 0.15 0.04

FA2 41.70 0.16

M5

3.05 0.02 FA1G1

FA3

FA2G1 28.84 0.06

FA2G1* 10.33 0.09

M5FA1

0.44 0.02 FA3G1

M5

FA3G1* 1.07 0.04

FA1G1Ga1

FA2G2 7.51 0.06

FA2G1G1a 0.21 0.01

FA1G1Gc1 0.76 0.02

FA3G2 0.30 0.00

FA3G2* 0.18 0.01

FA2G1Gc1 0.09 0.01

FA2G1Gc1* 0.14 0.01

FA2G2Ga1 1.21 0.02

M4A1G1Gc1 0.05 0.01

FA3G3 0.17 0.01

FA2G2Gc1 0.28 0.03

FA2G2Ga2 0.47 0.04

FA3G3Ga1 0.04 0.01

FA2G2Ga1Gc1 0.19 0.01

Page 23: NISTmAb Common Technical Document Case Study · 2018-04-02 · NISTmAb Common Technical Document Case Study . In order to provide attendees with an opportunity to evaluate “real

National Institute of Standards and Technology 00001234 Humanized IgG1κ monoclonal antibody, RM 8671 0000

3.2.S.3 Characterization [Humanized IgG1κ Monoclonal Antibody, NIST], Page 17

Oxford Nomenclature (proposed structure)

%RA St. Dev (n=3)

FA3G3Ga2 <0.03 NA

F2A3G3Ga2 <0.03 NA

FA3G3Ga3 <0.03 NA

3.2.S .3.1.2.4 Charge Variant Analysis

3.2.S .3.1.2.4.1 Capillary Zone Electrophoresis

Monoclonal antibody (mAb) charge purity and heterogeneity is quantified by capillary zone electrophoresis (CZE), wherein mAb charge variants are separated according to differential electrophoretic mobility in free solution within a uniform electric field applied across a buffer filled fused silica capillary. Samples of RM 8671 and of PS (as a system suitability control) were prepared by diluting 15 µL of sample (150 µg) with 85 µL distilled deionized (DDI) water. The instrument qualification (IQ) control was prepared by diluting 10 µL of pI 10.0 marker peptide with 90 µL DDI water.

All analyses were performed on a SCIEX PA800 plus capillary electrophoresis instrument fitted with a single-wavelength UV detector. All separations were performed in a previously-tested bare fused silica capillary (50 µm internal diameter, 40 cm effective length, 50.2 cm total length). The capillary was pre-conditioned with background electrolyte (BGE: 0.4 mol/L 6-aminocaproic acid, 2 mmol/L triethylenetetramine, 0.03 % mass concentration TweenTM 20, pH 5.7) and tested for adequate performance prior to analysis of RM 8671. Samples were hydrodynamically injected by application of 3.4 kPa pressure for 10 s (injection volume of 14 nL). The separation voltage was 30 kV (normal polarity, cathode at outlet) or +600 V/cm. Analyte bands were detected by absorbance at 214 nm by the fiber-coupled detector positioned 40 cm from the capillary inlet. All samples were analyzed in one sequence over the course of one day. The results of the CZE analysis demonstrate three charge groups: the main group, which comprises the majority of the sample; the basic variants, which migrate toward the cathode more rapidly than the main group; and the acidic variants, which migrate toward the cathode less rapidly than the main charge group as shown in Figure 7. The basic variants were identified as C-terminal lysine variants, with the C-terminal lysine present on either one (*) or both (**) heavy chain subunits through comparison to intact mass and peptide mapping analysis. The acidic variants co-migrate as a smear and comprise mAb presenting a variety of post-translational modifications (PTMs), including asparagine deamidation(s), lysine glycation(s), N-terminal glutamine, and sialic acid glycovariants. The charge purity of the NISTmAb is given as the relative abundance of the main charge group with respect to all detected charge species. Charge variant relative abundance (RA) was calculated according to Equation 1 where corrected area (CA) for each peak “x” and the sum of corrected areas for all peaks (CAtotal) were utilized:

Page 24: NISTmAb Common Technical Document Case Study · 2018-04-02 · NISTmAb Common Technical Document Case Study . In order to provide attendees with an opportunity to evaluate “real

National Institute of Standards and Technology 00001234 Humanized IgG1κ monoclonal antibody, RM 8671 0000

3.2.S.3 Characterization [Humanized IgG1κ Monoclonal Antibody, NIST], Page 18

𝑅𝑅𝑅𝑅𝑥𝑥�%� = 𝐶𝐶𝐶𝐶𝑥𝑥𝐶𝐶𝐶𝐶total

× 100 % (1) The charge purity is defined according to the Charge Purity (Equation 2): Charge Purity �%� =

𝐶𝐶𝐶𝐶main𝐶𝐶𝐶𝐶total

× 100 % (2)

Figure 7: Representative CZE electropherogram for RM 8671 Lot 14HB-D-002 where “*” denotes the C terminal lysine present on one HC and “**” denotes the C-terminal lysine present on both HC subunits

The results for RM 8671 compared to primary reference standard PS 8670 are presented in Table 5. The data indicate comparability of the two standards.

Table 5: CZE results for PS 8670 and RM 8671

Parameter PS 8670a RM 8671 lot 14 HB-D-002

b

Charge Purity (%) 74.69 (± 0.34) 73.82 (± 0.17)

Acidic Variants Relative Abundance (%) 16.76 (± 0.40) 16.55 (± 0.39)

Basic Variants Relative Abundance (%) 8.54 (± 0.28) 9.62 (± 0.22) a Values listed in parenthesis for 8670 represent the combined standard uncertainty based on ANOVA analysis for

8670 during qualification runs b Values listed in parenthesis for 8671 represent the combined standard uncertainty based on ANOVA analysis

incorporating intermediate precision data from qualification runs

Page 25: NISTmAb Common Technical Document Case Study · 2018-04-02 · NISTmAb Common Technical Document Case Study . In order to provide attendees with an opportunity to evaluate “real

National Institute of Standards and Technology 00001234 Humanized IgG1κ monoclonal antibody, RM 8671 0000

3.2.S.3 Characterization [Humanized IgG1κ Monoclonal Antibody, NIST], Page 19

3.2.S .3.1.2.4.2 Capillary Isoelectric Focusing CIEF separates charge variants based on differences in isoelectric point. A mAb sample is mixed with an ampholyte solution which forms a pH gradient when subjected to an applied electric field. The mAb variants migrate in the applied field until they reach the pH where their net charge and electrophoretic mobility go to zero; at this point, the pH is equal to the variant’s pI.

CIEF analyses were performed on a Sciex Separations PA800 plus Pharmaceutical Analysis system fitted with a neutral coated CIEF capillary (Sciex PN 477441), 50 µm i.d., cut to 30.5 cm (20 cm to detector). The system was configured with the UV detector module for absorbance detection at 280 nm. CIEF solutions were as follows: anolyte, 200 mmol/L phosphoric acid; catholyte, 300 mmol/L sodium hydroxide; chemical mobilizer, 350 mmol/L acetic acid; anodic stabilizer, 200 mmol/L iminodiacetic acid; cathodic stabilizer, 500 mmol/L arginine; capillary cleaning solution, 4.3 mol/L urea; separation gel, 3 mol/L (or as indicated) urea in CIEF gel buffer. The standard instrumental method for CIEF designed by the instrument manufacturer was used. Samples were prepared by diluting 10 µL of PS 8670 (in 12.5 mmol/L histidine/12.5 mmol/L histidine HCl, pH 6.0) with 240 µL of master mix.

The CIEF separation resulted in two basic peaks (corresponding to mAb with penultimate lysine present on one or both C-termini of the heavy chain), the main peak, and an acid variant peak group similar to the CZE separation. The apparent pI of the main peak was determind to be 9.18 (± 0.01) based on a best fit linear response of migration time versus pI for a series of pI markers.

3.2.S .3.1.2.5 Disulfide Structure

The disulfide bonds for PS 8760 were analyzed by peptide mapping under nonreducing conditions using RP-HPLC coupled with UV and mass spectrometry detection. The methods generally followed those described in section Section 3.2.S.3.1.1.1.1, with the exclusion of DTT as a reducing agent and the use of Lys C instead of trypsin.1 This protocol results in disulfide bonds that remain intact such that canonical disulfide linkages can be confirmed by mass in the high resolution mass spectrometer. The theoretical molecular mass of canonical disulfide-containing peptides based on the expected amino acid composition is compared to measured molecular masses observed for PS 8670. The expected disulfide bond structure for a human IgG1

was confirmed.1

3.2.S .3.1.3 S ize Heterogeneity

3.2.S .3.1.3.1 S ize Exclusion UltraHigh Performance Chromatography (S E-UPLC)

Size exclusion chromatography is a separation technique capable of resolving species based on size and/or conformation. Molecules in a solution are passed through a column packed with porous particles of a certain size. As the sample passes through the column, larger molecules

Page 26: NISTmAb Common Technical Document Case Study · 2018-04-02 · NISTmAb Common Technical Document Case Study . In order to provide attendees with an opportunity to evaluate “real

National Institute of Standards and Technology 00001234 Humanized IgG1κ monoclonal antibody, RM 8671 0000

3.2.S.3 Characterization [Humanized IgG1κ Monoclonal Antibody, NIST], Page 20

(e.g. aggregates) bypass the pores and are eluted first. The smaller molecules (e.g. fragments) sample an increased proportion of the pore volume, slowing down the rate at which they travel through the column, and elute last towards the end of the injection. The size heterogeneity and monomeric purity of NISTmAb was analyzed under non-denaturing conditions by SE-UHPLC with UV detection at 280 nm. All samples were analyzed on a Thermo Scientific/Dionex U3000 high-pressure liquid chromatography system. The SEC column was pre-conditioned with mobile phase for 30 min before sample analysis. Samples were injected (6 µL, except as noted for linearity and LOD/LOQ determination) into a pre-conditioned column using a flow rate of 0.300 mL/min, resulting in a pressure of ~180 bar. Isocratic elution was monitored for each injection using a total run time of 10 minutes. Peaks were detected by ultraviolet absorbance using the variable wavelength detector at a wavelength of 280 nm.

Figure 8 describes the elution pattern observed with SEC of the NISTmAb; there are three main species observed here defined as high molecular weight (HMW), monomer, and low molecular weight (LMW). In the high molecular weight series there is evidence of unresolved tetramer, trimer, and dimer peaks. In the low molecular weight species there is evidence of one or two resolved fragment peaks that elute immediately after the monomer peak. Note the unlabeled peak at approximately 6.25 minutes was verified to be the void volume of the column and due to elution of the L-histidine sample background buffer.

Page 27: NISTmAb Common Technical Document Case Study · 2018-04-02 · NISTmAb Common Technical Document Case Study . In order to provide attendees with an opportunity to evaluate “real

National Institute of Standards and Technology 00001234 Humanized IgG1κ monoclonal antibody, RM 8671 0000

3.2.S.3 Characterization [Humanized IgG1κ Monoclonal Antibody, NIST], Page 21

Figure 8: Representative SE-UPLC Chromatogram for RM 8671 Lot 14HB-D-002

The results for RM 8671 compared to primary reference standard PS 8670 are presented in Table 6. The data indicate comparability of the two standards.

The results for RM 8671 compared to primary reference standard PS 8670 are also presented in Table 6. The relative area of the monomer has decreased from ~ 98.7% to ~96.7% and the relative area of the high molecular weight species has increased from ~1% to ~3% as compared to PS 8670. Alterations in the homogenization and vial filling process versus the PS 8670 material are thought to have resulted in the increased HMW species. Despite a small increase in the %HMW versus 8670, two additional lots of 8671 have been shown to be consistent with one another in terms of monomeric purity, high molecular weight relative area and low molecular weight relative area demonstrating consistency in the production process.

Commented [CV1]: Please evaluate this paragraph, does this information cause any concern if y ou think of this in terms of process to process comparability ? How would y ou explain/justify the increase in aggregate? Would this material be acceptable if this did result from a process change?

Page 28: NISTmAb Common Technical Document Case Study · 2018-04-02 · NISTmAb Common Technical Document Case Study . In order to provide attendees with an opportunity to evaluate “real

National Institute of Standards and Technology 00001234 Humanized IgG1κ monoclonal antibody, RM 8671 0000

3.2.S.3 Characterization [Humanized IgG1κ Monoclonal Antibody, NIST], Page 22

Table 6: SE-UHPLC results for PS 8670 and RM 8671

Species PS 8670a RM 8671

b

Monomer (%) 98.783 (± 0.124)

96.627 (± 0.153)

High Molecular Weight RA (%) 1.020 (± 0.119)

3.175 (± 0.149)

Low Molecular Weight RA (%) 0.197 (± 0.008)

0.198 (± 0.008)

a Values listed in parenthesis for 8670 represent the combined standard uncertainty based on ANOVA analysis for 8670 during qualification runs.

b Values listed in parenthesis for 8671 represent the combined standard uncertainty based on ANOVA analysis incorporating intermediate precision from qualification runs.

3.2.S .3.1.3.2 Capillary S odium Dodecyl S ulfate (CES DS )

CE-SDS is the microelectrophoretic analogue of traditional slab-gel size based separations (e.g. SDS PAGE). Analytes are complexed with SDS and injected into a narrow bore glass capillary filled with an uncrosslinked polymer sieving matrix. A high voltage applied across the capillary drives analytes toward the outlet; their migration is retarded in a size-dependent manner by differential interaction with the sieving matrix.

NISTmAb monomeric purity was measured by capillary electrophoresis sodium dodecyl sulfate (CE-SDS) under non-reducing conditions (nrCE-SDS). Glycan occupancies of the heavy chain and relative abundance of non-reducible species were measured by CE-SDS under reducing conditions (rCE-SDS). For nrCE-SDS, 100 µg of NISTmAb in 10 µL of formulation buffer (10 mg/mL) was diluted to 1 mg/mL in fresh alkylating sample buffer (70 mmol/L citrate-phosphate buffer, pH 6.7, 1 % mass concentration SDS, 46 mmol/L iodoacetamide, and 1/50X 10 kDa internal standard protein). Samples were incubated for 5 min in a 70 °C water bath and cooled to room temperature prior to analysis. For rCE-SDS, 100 µg NISTmAb in 10 µL of formulation buffer was diluted to 1 mg/mL in fresh reducing sample buffer (70 mmol/L citrate-phosphate buffer, pH 6.7, 1 % mass concentration SDS, 5 % volume fraction 2-mercaptoethanol, 1/50X 10 kDa internal standard protein). Samples were incubated 10 min at 70 °C in a water bath and cooled to room temperature prior to analysis. Samples were analyzed within 24 h of preparation.

All analyses were performed on a SCIEX PA800 plus capillary electrophoresis instrument fitted with a photodiode array (PDA) multi-wavelength UV detector. All separations were performed in a previously-tested bare fused silica capillary (50 µm internal diameter, 20 cm effective length, 30.5 cm total length). Samples were electrokinetically injected into the pre-conditioned capillary by applying −5 kV across the capillary for 20 s. The separation voltage (15 kV, reverse polarity) was applied for 35 min. Sample bands were detected by the fiber optic-coupled PDA detector 20 cm from the capillary inlet using absorbance at 220 nm. Figure 9 depicts a representative nrCE-SDS electropherogram collected for Lot 14HB-D-002. The dominant peak in the electropherogram can be assigned to the monomeric NISTmAb based on

Page 29: NISTmAb Common Technical Document Case Study · 2018-04-02 · NISTmAb Common Technical Document Case Study . In order to provide attendees with an opportunity to evaluate “real

National Institute of Standards and Technology 00001234 Humanized IgG1κ monoclonal antibody, RM 8671 0000

3.2.S.3 Characterization [Humanized IgG1κ Monoclonal Antibody, NIST], Page 23

electrophoretic migration relative to bracketing injections of a molecular weight standard. Low abundance species corresponding to antibody fragments are also observed as indicated in the figure. Monomeric purity was calculated from the resultant corrected peak areas (CA) according to Equation 3.

Monomeric Purity �%� = 𝐶𝐶𝐶𝐶monomer𝐶𝐶𝐶𝐶monomer+ ∑𝐶𝐶𝐶𝐶fragments

× 100% (3) Figure 9: Representative electropherogram of RM 8671 Lot 14HB-D-002 by non-reducing CE SDS.

Figure 10 depicts a representative rCE-SDS electropherogram collected for Lot 14HB-D-002. The dominant peaks in the electropherogram can be assigned to the NISTmAb heavy chain and light chain based on electrophoretic migration relative to bracketing injections of a molecular weight standard. Low abundance species corresponding to aglycosylated/non-glycosylated heavy chain (NGH) and a non-reducible thioether-linked species (HC:LC thioether at heavy chain Cys223-light chain Cys213 were verified via mass spectrometry peptide mapping of a tryptic digest). For reduced samples, the relative abundance (RA) of each peak (LC, NGH, HC, thioether) was calculated according to Equation 4:

𝑅𝑅𝑅𝑅𝑋𝑋 �%� = 𝐶𝐶𝐶𝐶𝑋𝑋

𝐶𝐶𝐶𝐶total× 100 % (4)

Where (CA) is the corrected area for each peak “x” and CAtotal is the sum of all corrected areas for the NISTmAb peaks.

Page 30: NISTmAb Common Technical Document Case Study · 2018-04-02 · NISTmAb Common Technical Document Case Study . In order to provide attendees with an opportunity to evaluate “real

National Institute of Standards and Technology 00001234 Humanized IgG1κ monoclonal antibody, RM 8671 0000

3.2.S.3 Characterization [Humanized IgG1κ Monoclonal Antibody, NIST], Page 24

The glycan occupancy of the heavy chain was calculated for reduced samples and system suitability controls according to Equation 5:

Glycan Occupancy �%� = 𝐶𝐶𝐶𝐶𝐻𝐻𝐻𝐻

𝐶𝐶𝐶𝐶𝐻𝐻𝐻𝐻+𝐶𝐶𝐶𝐶𝑁𝑁𝑁𝑁𝐻𝐻× 100 % (5)

Figure 10: Representative electropherogram of RM 8671 lot 14HB-D-002 by rCE-SDS, full scale (top figure) and expanded baseline (bottom figure)

A summary of results obtained for PS 8670 and RM 8671 is presented in Table 7.

Page 31: NISTmAb Common Technical Document Case Study · 2018-04-02 · NISTmAb Common Technical Document Case Study . In order to provide attendees with an opportunity to evaluate “real

National Institute of Standards and Technology 00001234 Humanized IgG1κ monoclonal antibody, RM 8671 0000

3.2.S.3 Characterization [Humanized IgG1κ Monoclonal Antibody, NIST], Page 25

Table 7: CE-SDS results for PS 8670 and RM 8671

Species PS 8670a RM 8671 lot 14 HB-D-002

b

Monomer (%) (nrCE-SDS)

98.79 (± 0.38)

98.47 (± 0.79)

Light Chain RA (%) (rCE-SDS)

32.02 (± 0.20)

32.11 (± 0.21)

Heavy Chain RA (%) (rCE-SDS)

67.26 (± 0.20)

67.18 (± 0.20)

Thioether RA (%) (rCE-SDS)

0.31 (± 0.02)

0.30 (± 0.02)

Glycan Occupancy (%) (rCE-SDS)

99.40 (± 0.01)

99.39 (± 0.003)

a Values listed in parenthesis for 8670 represent the combined standard uncertainty based on ANOVA analysis for 8670 during qualification runs.b Values listed in parenthesis for 8671 represent the combined standard uncertainty based on ANOVA analysis incorporating intermediate precision from qualification runs.

3.2.S .3.1.4 Biophysical Characterization

3.2.S .3.1.4.1 Fourier transform infrared (FTIR) S pectroscopy The secondary structure of a protein can be investigated by Fourier transform infrared spectroscopy (protein measurements using FTIR are mostly performed in the mid-spectral infrared region where the amide I band is the most distinctive band for proteins. Since the amide I band is primarily dependent on the backbone structure, this band contains information about the secondary protein structure.

Data was acquired using a Bruker Optics Vertex 80 FTIR with a globar source, a KBr beam splitter, 100 mm focal length and a room temperature DLaTGS detector. The instrument has been frequency/wavelength limited using an optical filter with a cutoff of 1866 cm-1 purchased from Bruker Optics. The sample cell is made of calcium fluoride which restricts wavelengths longer than about 900 cm-1 from reaching the detector. By narrowing the band pass of the instrument, we are able to open the instruments optical aperture completely to 8 mm and slow the scanner velocity down to 1.6 KHz mirror crossings to more completely fill the instruments A to D converter. The calcium fluoride sample cell was purchased from BioTools LLC and its path is nominally 7.2 microns. BioTools reports the path length is 7.2 +/- 1.0 microns.

The data was collected using both a high and low pass filter (5KHz) and is the average of 40 forward and backward single sided mirror scans to collect 1316 data points to describe 4 cm-1 data between 2250 cm-1 and 0 cm-1. The signal was collected with an amplification of 1 to produce an interferogram amplitude on the order of 23000 positive and negative for background air scans and about 11000 positive and negative for scans with either sample or buffer. Then 2 levels of zero filling before Blackman-Harris 3-term apodization and Mertz 32 cm-1 phase

Page 32: NISTmAb Common Technical Document Case Study · 2018-04-02 · NISTmAb Common Technical Document Case Study . In order to provide attendees with an opportunity to evaluate “real

National Institute of Standards and Technology 00001234 Humanized IgG1κ monoclonal antibody, RM 8671 0000

3.2.S.3 Characterization [Humanized IgG1κ Monoclonal Antibody, NIST], Page 26

resolution calculations create our infrared spectra. Second derivative spectra with 25 point smoothing and spectral subtractions were performed in Bruker Optic’s OPUS 7.5 software.

To compare PS 8670 and RM 8671 samples using FTIR, three samples were acquired, in triplicate, one for each mAb sample and one from a sample of the buffer solution. The three samples must be temperature controlled so that sample differences related to temperature are minimized. For these experiments we utilized a Neslab RTE 111 temperature controlled water bath to control the temperature of an aluminum block to within +/- 0.02 degrees C next to the calcium fluoride cell that holds the samples.

Representative spectra for NISTmAb PS 8670 and RM 8671 are shown in Figure 11. The spectrum of the native NISTmAb is dominated by the band at 1639 cm–1 in the amide I region, indicative of intramolecular native β-sheets. Second derivative plots show additional bands. The two bands at 1658 cm–1 and 1686 cm–1 can be assigned to turns and β-sheets, respectively. The mode that is seen at 1620 cm–1 can be assigned to side chain vibration or β-sheets. Main peaks are relatively consistent between the samples, with minor variations in peak maxima and/or intensity thought to be within repeatability tolerance of the measurement.

Page 33: NISTmAb Common Technical Document Case Study · 2018-04-02 · NISTmAb Common Technical Document Case Study . In order to provide attendees with an opportunity to evaluate “real

National Institute of Standards and Technology 00001234 Humanized IgG1κ monoclonal antibody, RM 8671 0000

3.2.S.3 Characterization [Humanized IgG1κ Monoclonal Antibody, NIST], Page 27

Figure 11: FTIR spectra (upper panel) and second derivative plot (lower panel) recorded for (1) Red PS 8670, (2) Grey 8671, (3), Blue RM 8671 minus PS 8670

3.2.S .3.1.4.2 Circular Dichroism S pectroscopy Circular dichroism (CD) is a technique used to study tertiary and secondary structure in near- and far-UV modes respectively. CD spectroscopy measures the difference between the left-handed and right-handed circularly polarized light absorption of chirally active samples as a function of wavelength. The difference in these absorbances is called “ellipticity” and is affected by peptide bond orientation in secondary structural elements and tertiary structural interactions of certain UV-active chromophore side chains.

Page 34: NISTmAb Common Technical Document Case Study · 2018-04-02 · NISTmAb Common Technical Document Case Study . In order to provide attendees with an opportunity to evaluate “real

National Institute of Standards and Technology 00001234 Humanized IgG1κ monoclonal antibody, RM 8671 0000

3.2.S.3 Characterization [Humanized IgG1κ Monoclonal Antibody, NIST], Page 28

The secondary and tertiary structure of NISTmAb samples PS 8670 and RM 8671 were evaluated by circular dichroism (CD) spectroscopy and a quality range statistical approach. PS 8670 and RM 8671 were dialyzed into a common preparation of PBS and the dialysate was used for baseline correction. Samples were diluted to 1.28 mg/ml for far UV measurements at 0.1 mm pathlength and to 0.8 mg/ml for near UV measurements at 10 mm pathlength. High signal:noise spectra of known precision were derived from multiple independent repeat measurements (see Figure 12) enabled by using an autosampler and flow cell.

Figure 12: CD spectra of PS 8670 (red) and RM 8671 (blue) in far UV (plot A) and near UV (plot B)

Spectra are the average of n independent, baseline corrected, absorbance normalized measurements; n = 6 for far UV, n = 5 for near UV. A 202/218 nm peak/trough, characteristic of a beta sheet rich protein structure dominates the far UV CD spectra (Figure 12 A). Lower amplitude signals near 230 nm (inset plot) are likely to be caused by disulfide bonds and are expected to be sensitive to the dihedral angles of these bonds. CD signals in the near UV originate from the aromatic side chains of phenylalanine, tyrosine and tryptophan which, in the folded protein, are in a chiral environment (see Figure 12B). The characteristic wavelengths at which these amino acids tend to contribute to the spectra are indicated on the figure. A minor contribution from the disulfide bonds, centered at 260nm, is also expected. The CD spectra for PS 8670 and RM 8671 are close to being superimposable in the far UV with small differences present in near UV. The Office of Biostatistics and Office of Biotechnology Products, CDER/FDA, recommends a quality range method of statistical analysis for quality attributes of intermediate criticality (tier 2). The weighted spectral difference method described in Dinh N.N., et al 20142 was applied to yield data compatible with quality range testing and enable assessment of the significance of differences between PS 8670 and RM 8671 (see Figure 13). A reference group of similarity scores for comparisons within the PS 8670 dataset are plotted in blue and a test group of scores for comparisons which bridge the PS 8670 and RM 8671 datasets are plotted in red. Equivalence between the test and reference groups of scores was assessed using acceptance criteria of 2-

Page 35: NISTmAb Common Technical Document Case Study · 2018-04-02 · NISTmAb Common Technical Document Case Study . In order to provide attendees with an opportunity to evaluate “real

National Institute of Standards and Technology 00001234 Humanized IgG1κ monoclonal antibody, RM 8671 0000

3.2.S.3 Characterization [Humanized IgG1κ Monoclonal Antibody, NIST], Page 29

sigma (calculated from the reference group scores). Results show that the differences between PS 8670 and RM 8671 detected in the far UV are not significant and that those detected in the near UV are significant.

Figure 13: Quality range statistical treatment

.

3.2.S .3.1.4.3 Differential S canning Calorimetry DSC probes the thermodynamic state (through the heat capacity Cp) of the sample as a function of temperature. Cp represents the change in the enthalpy of the system as temperature is increased during a scanning analysis. In the case of protein solutions, these enthalpy changes are due to the thermal energy required/released to compensate for any change in internal and protein-protein/solvent interactions, as well as any residual energy that the protein requires to undergo a thermodynamic transition such as unfolding, self-association/dissociation, or phase separation. That is, the Cp profile for a given protein depends on both the protein conformational state and the solution conditions, as these factors directly affect the strength of intra- and intermolecular protein interactions. DSC can be used to probe the thermal stability of the protein solution by measuring the melting temperature (Tm) for any effective transition. Tm corresponds to the midpoint temperature during a transition, and it is given by the position of the corresponding local maximum in the Cp profile. The thermal stability of NISTmAb (both PS 8670 and RM 8671) was evaluated via capillary Nano-DSC (TA Instruments, DE). Cp profiles were measured from 30 oC to 110 oC at a ramp of 1oC/min. NISTmAb samples were diluted to a protein concentration of about 0.5 mg/mL and 4x dialyzed in 25 mM His/His-HCl at pH 6.0. Prior to each protein solution measurement, 5 buffer scans were acquired in order to establish thermal history, where each scan consists of a heating and a cooling ramp. No cleaning step was performed between buffer and sample scans. During analysis of DSC measurements, last buffer scan was subtracted from its corresponding sample scan followed by baseline determination using second-order polynomials. Figure 14 depicts the heat capacity profiles for NISTmAb after subtraction of the low-temperature baseline. Heat-capacity (Cp) of samples is shown with respect to that of the low-temperature state. Color code corresponds to the different protein samples: (blue) Primary Sample 8670; and (red) Reference Material 8671.

Commented [CV2]: The analy sis performed on the spectral data suggests a difference in tertiary structure between the two samples. Does this concern y ou from a comparability perspective? Why or why not?

Page 36: NISTmAb Common Technical Document Case Study · 2018-04-02 · NISTmAb Common Technical Document Case Study . In order to provide attendees with an opportunity to evaluate “real

National Institute of Standards and Technology 00001234 Humanized IgG1κ monoclonal antibody, RM 8671 0000

3.2.S.3 Characterization [Humanized IgG1κ Monoclonal Antibody, NIST], Page 30

The thermograms for NISTmAb show three effective thermal transitions at about 69, 83, and 94 oC (see Table 8). For multidomain proteins like mAbs, multiple transitions can be observed as the small interfaces between domains result on little or no dependence of the native structure of each domain on the conformational stability of the other domains. For the working case, these transitions have been previously assigned to the loss of native structure of the CH2 (69 oC), CH3 (83 oC), and Fab (94 oC), where the latter transition may also involves early stages of aggregation.3

Figure 14: Differential scanning calorimetry (DSC) profiles of NISTmAb in 25mM His/His-HCl at pH 6.0

.

Table 8: Melting temperatures (Tm) of NISTmAb in 25mM His/His-HCl at pH 6.0

Sample Tm 1 [oC] Tm 2 [oC] Tm 3 [oC]

PS 8670 69.196 ± 0.166 83.633 ± 0.166 93.897 ± 0.166

RM 8671 lot 14HB-D-002

69.123 ± 0.166 83.457 ± 0.166 93.804 ± 0.166

As Table 8 shows, no significant difference was observed between PS 8670 and RM 8671 for all the melting temperatures. However, discrepancies between both thermograms are evident when comparing the height of Cp (with respect to that of the native state) for the unfolding of the CH3 domain and the high-temperature baseline, where RM 8671 exhibits a larger heat capacity in these regions. From the analysis of SE-UHPLC, it was identified a slightly higher amount of HMW species in RM 8671 (~3% as compared to ~1% of HMW in PS 8670). Given that Cp is related to the energy required to balance out any conformational change (including association/dissociation), these variations on the thermograms might then be related to

Commented [CV3]: After reviewing this data and figure, do y ou concur with the conclusions in the text, and is this how y ou would present the information to the agency in an assessment of structural elucidation/characterization?

Page 37: NISTmAb Common Technical Document Case Study · 2018-04-02 · NISTmAb Common Technical Document Case Study . In order to provide attendees with an opportunity to evaluate “real

National Institute of Standards and Technology 00001234 Humanized IgG1κ monoclonal antibody, RM 8671 0000

3.2.S.3 Characterization [Humanized IgG1κ Monoclonal Antibody, NIST], Page 31

differences in the concentration of HMW species. This implies that the CH3 domain is directly involved in the self-assembly of NISTmAb.

3.2.S .3.1.5 High Resolution Measurement of Higher Order S tructure

3.2.S .3.1.5.1 Two dimensional Nuclear Magnetic Resonance S pectroscopy Nuclear magnetic resonance spectroscopy (NMR) can be utilized to simultaneously evaluate the primary, secondary, tertiary, and quaternary structure of protein therapeutics. The unique local electronic environment of each NMR-active nuclei differentially shields them from an external magnetic field and thus afford a specific and highly precise frequency position. Assuming sample conditions are matched (salinity, pH, temperature), deviations from the frequency position of a resonance may indicate a deviation in the higher order structure of a biologic. Typically two different fingerprints are extracted from 1H,15N and 1H,13C spectra to evaluate HOS. The 1H,15N amide region is considered the ‘gold standard’ for a protein spectral map, since every non-proline residue will afford a cross peak in the spectrum. On the other hand, methyl resonances have better properties that facilitate measurement for large proteins (> 30 kDa) at 13C natural isotopic abundance. While only six amino acids have methyl groups, these tend to be distributed throughout a protein and are reliable reporters of folding within hydrophobic pockets. Samples of intact PS 8670 and RM 8671 were prepared at a concentration of 40 mg/mL in 25 mM Bis-Tris d19, pH of 6.0. Fab and Fc domains from the respective lots were prepared by papain digest at 37 °C for 4 h using the Pierce Fab preparation kit (Thermo Fisher, Inc.). Final domain concentrations were ca. 20 mg/mL for RM 8671 and 13 mg/mL for PS 8670 in 25 mM Bis-Tris d19, pH of 6.0. All NMR data were collected at 50 °C on a Bruker Avance III 900 MHz spectrometer with cryogenically cooled HCN probe. 1H-13C methyl datasets were recorded using a gradient-selected heteronuclear single quantum spectroscopy (gsHSQC) experiment with parameters summarized in Table 9. 1H-15N datasets were recorded using a SOFAST-HMQC experiment with selective excitation and refocusing of amide resonances accomplished using PC-9 and Reburp selective pulses applied at 8.5 ppm with bandwidths of 5 ppm. Data were collected with 4096, 6144 or 12288 scans/transient for the PS 8670 domains, the RM 8671 Fc and RM 8671 Fab respectively. All data were processed with apodization using a shifted sine-square bell and then zero-filled to 512x4096 complex points prior to Fourier Transform. Calculation of Pearson’s correlation coefficients between spectra was accomplished by point-to-point linear regression analysis of the full processed frequency domain data matrices over the spectral window given in Table 9. To account for the sensitivity of spectral correlation coefficients to experimental noise, the S/N of each dataset was calculated by taking the RMS amplitude of the spectral window and dividing it by the RMS amplitude of the noise window given in Table 9. Raw spectral correlation coefficients were then normalized against the expected correlation coefficient between a replicate datasets at the given experimental S/N using a previously generated S/N vs. R calibration curve for each specific experiment.

Commented [CV4]: See previous question regarding assessment of the results.

Commented [CV5]: How is this ty pe of data being used in y our company ? Would this be a high priority if y ou have limited resources?

Page 38: NISTmAb Common Technical Document Case Study · 2018-04-02 · NISTmAb Common Technical Document Case Study . In order to provide attendees with an opportunity to evaluate “real

National Institute of Standards and Technology 00001234 Humanized IgG1κ monoclonal antibody, RM 8671 0000

3.2.S.3 Characterization [Humanized IgG1κ Monoclonal Antibody, NIST], Page 32

Table 9: NMR Acquisition and Processing Parameters

Parameter 1H-13C gsHSQC 1H-15N SOFAST HMQC

# of scans 128 Variable

Steady state scans 64 512

Recycle delay 2 s 0.4 s

Complex Points (F1xF2) 128x2018 62x1260

Acquisition times (F1/F2) 10 ms/80 ms 10 ms/50 ms

Spectral widths (F1/F2) 28 ppm/14 ppm 34 ppm/14 ppm

Carrier positions (F1/F2) 22 ppm/ H2O 118 ppm/ H2O

Spectral Window (F1/F2) 8—28.5 ppm/-0.5—2.4 ppm 101—135 ppm/6.0—11ppm

Noise Window (F1/F2) 8.6—27.6 ppm/8.25—10.5ppm 101—135 ppm /-1.5—2.25ppm

The higher order structure of PS-8670 and RM 8671 NISTmAb was evaluated by two-dimensional 1H-13C-methyl nuclear magnetic resonance spectroscopy (NMR). Both species exhibited well dispersed resonances with sharp spectral lines indicative of a well folded protein in dynamic conformational equilibrium. Visual comparison of PS 8670 and RM 8671 by spectral overlay (Figure 15 A) revealed no significant differences, suggesting both species adopt the same major HOS conformation. Statistical analysis of the two spectra by direct point-to-point linear regression analysis (Figure 15 B) shows a high degree of correlation, with a signal-to-noise (S/N) adjusted R value of 0.999. This indicates that the spectra are nearly identical within the precision of measurement. Since spectral mapping encodes primary, secondary, tertiary, and quaternary structure of a protein, this high statistical correlation affirms that the HOS of PS 8670 and RM 8671 are highly similar.

To further evaluate the structural similarity of PS 8670 and RM 8671, both species were subjected to fragmentation by enzymatic digest with papain, followed by purification of the resulting Fab and Fc domains by protein-A affinity chromatography. The individual domains were than evaluated by two-dimensional 1H-15N-amide NMR. Visual comparison of the domain spectra from PS-8670 and RM 8671 reveal no significant differences (Figure 15 C and D), and linear regression analysis of the spectra indicate strong degree of spectral correlation with S/N adjusted R values of 0.985 and 0.996 for the Fc and Fab respectively. These statistical correlations confirm that two protein lots adopt the same major HOS conformation.

Page 39: NISTmAb Common Technical Document Case Study · 2018-04-02 · NISTmAb Common Technical Document Case Study . In order to provide attendees with an opportunity to evaluate “real

National Institute of Standards and Technology 00001234 Humanized IgG1κ monoclonal antibody, RM 8671 0000

3.2.S.3 Characterization [Humanized IgG1κ Monoclonal Antibody, NIST], Page 33

Figure 15: A) Overlay 1H-13C methyl spectrum of intact PS 8670 (blue) and RM 8671 (red). B) Linear regression plot of the spectra shown in panel A. C) Overlay 1H-15N amide spectrum of the Fc domain from PS 8670 (blue) and RM 8671 (red). D) Overlay 1H-15N amide spectrum of the Fab domain from PS 8670 (blue) and RM 8671 (red).

3.2.S .3.1.6 Additional Higher Order S tructure S ensitive Methods

3.2.S .3.1.6.1 S edimentation Velocity Analytical Ultra Centrifugation (S V-AUC) SV-AUC is an analytical method orthogonal to size exclusion chromatography and field flow fractionation used to determine the content of intact molecule (monomer), high molecular weight species (aggregates) and low molecular weight species (fragments). SV-AUC is a first principle method that separates and characterizes macromolecular species without the need for molecular standards in a wide variety of possible solution conditions without the limitations intrinsic to size exclusion chromatography (SEC) such as filtering or interaction with a fixed matrix or the need for a special mobile phase. Sedimenting boundaries of macromolecules have a characteristic speed (quantified by the sedimentation coefficient) that depends on the molecular weight and frictional coefficient of the species and the viscosity and

Page 40: NISTmAb Common Technical Document Case Study · 2018-04-02 · NISTmAb Common Technical Document Case Study . In order to provide attendees with an opportunity to evaluate “real

National Institute of Standards and Technology 00001234 Humanized IgG1κ monoclonal antibody, RM 8671 0000

3.2.S.3 Characterization [Humanized IgG1κ Monoclonal Antibody, NIST], Page 34

density of the buffer. Curve fitting the speed and shape of sedimenting boundaries enables the calculation of the molecular weight of each species with enough precision to reliably identify fragments, monomer, and aggregates. SV-AUC experiments were performed on Primary Sample 8670, and on Reference Material 8671 LOT 14HB-D-002. SV-AUC experiments were performed with a Beckman XL-I analytical ultracentrifuge (Beckman Coulter, Brea, California). Each sample was diluted to 0.4 mg/ml and loaded into analytical double sector cells with 1.2 cm charcoal-filled epon centerpieces and sapphire windows. Matching buffer was loaded into the reference sector of the cells. Experiments were carried out using a four-hole titanium An-60 Ti rotor or an 8 hole An-50 Ti titanium rotor with standard radial calibration through a counterweight. After two hours of temperature equilibrium at 20°C, centrifugation at 50,000 RPM was initiated and raw data consisting of 100 c(r) scans of absorbance at 280 nm as a function of radial position and time were collected over about 7 hours using default optical settings.

Samples were examined in triplicate, with raw data being fit by SEDFIT 14.1. For each replicate, curve fitting produces an apparent sedimentation coefficient distribution function, or c(s) graph. The area under any segment of the curve corresponds to the loading concentration of sample apparently having a sedimentation coefficient between the endpoints of the x-axis of the curve. Figure 16 and Figure 17 present an overlay of the three replicate c(s) curves for the Primary Sample 8670, and the Reference Material 8671 LOT 14HB-D-002. Note that for fitted distribution functions, variance in peak width and position is expected and depends on the noise level in the raw data. Averages of the results of the three replicates are reported, along with the fitting parameters indicative of the quality of the curve fit are reported in Table 10.

Figure 16: Overlay of 3 replicate apparent sedimentation coefficient distribution functions for Primary Sample 8670. Inset is with Y axis magnified to show aggregate and fragment peaks

Page 41: NISTmAb Common Technical Document Case Study · 2018-04-02 · NISTmAb Common Technical Document Case Study . In order to provide attendees with an opportunity to evaluate “real

National Institute of Standards and Technology 00001234 Humanized IgG1κ monoclonal antibody, RM 8671 0000

3.2.S.3 Characterization [Humanized IgG1κ Monoclonal Antibody, NIST], Page 35

Figure 17: Overlay of 3 replicate apparent sedimentation coefficient distribution functions for Reference Material 8671 LOT 14HB-D-002. Inset is with Y axis magnified to minor peaks

Table 10: Sedimentation Velocity Analytical Ultracentrifugation Results

Rep # lot

fragments %

monomer %

aggregates %

S main peak (Svedbergs)

frictional ratio

RMSD (A280)

1 PS 8670 0.0% 99.8% 0.2% 6.359 1.573 0.005878 2 PS 8670 0.0% 99.8% 0.2% 6.386 1.574 0.006283

3 PS 8670 0.0% 99.2% 0.8% 6.356 1.574 0.006114 averages 0.0% 99.6% 0.4% 6.367 1.574 0.006092

1 RM 8671 14HB-D-002

0.0% 98.1% 1.9% 6.370 1.556 0.00744

2 RM 8671 14HB-D-002

0.0% 99.1% 0.9% 6.363 1.575 0.007175

3 RM 8671 14HB-D-002

0.0% 98.6% 1.4% 6.363 1.595 0.005785

averages 0.0% 98.6% 1.4% 6.365 1.576 0.0068

3.2.S .3.1.6.2 Ion Mobility The higher order structure of NIST PS 8670 and RM 8671 was investigated using Drift Tune and Travelling Wave Ion Mobility (TWIM) Collision Induced unfolding. IM spectrometry is a gas-phase chromatographic technique that separates ionic species based on their mobility differences as a result of their short- and long-range interactions with the neutral buffer gas, typically He (or

Commented [CV6]: Compare the AUC results to the previously discussed SEC-HPLC results. Do y ou see similarities in the data or discrepancies? How would y ou explain any differences that are observed? Which data set do y ou believe and why ?

Page 42: NISTmAb Common Technical Document Case Study · 2018-04-02 · NISTmAb Common Technical Document Case Study . In order to provide attendees with an opportunity to evaluate “real

National Institute of Standards and Technology 00001234 Humanized IgG1κ monoclonal antibody, RM 8671 0000

3.2.S.3 Characterization [Humanized IgG1κ Monoclonal Antibody, NIST], Page 36

N2), under the influence of a weak electric field. In principle, packets of ions are driven through a background gas using an electric field gradient across a drift-cell device, and a temporal separation of differing mobility species (ions) ensues. If we consider a protein with two distinct conformations, one extended and one compact, both with equal amounts of distributed surface charge, the more extended conformation would traverse the drift cell more slowly than the compact conformation. The modified Synapt G1 HDMS instrument4 was operated in positive nanoflow ESI mode. All critical instrument voltages and pressures were as follows: capillary voltage 0.8 to 1.0 kV; sample cone 40 V, extraction cone 1 V; source block temperature 30 °C; trap collision energy 4.0 V; transfer collision energy 60 to 200 V, corresponding to 3.3 V to 11.1 V/cm applied across the RF-confining drift cell; trap entrance 3.0 V; trap bias 16 V ; IMS DC entrance 5.0 V; IMS DC exit 0.0 V; transfer DC entrance 0.0 V; transfer DC exit 2.0 V; transfer wave velocity 70 m/sec; transfer wave amplitude 4.0 V; mobility trapping release time 250 μsec; trap height 20.0 V; extract height 0.0 V; source RF amplitude (peak-to-peak) 450 V; triwave RF amplitudes (peak-to-peak), trap 380 V, IMS 150 V, transfer 380 V; source backing pressure 6.0 mbar; trap/transfer pressure SF6, 3.3e-2 mbar (Pirani gauge indicated; flow rate 3.0 mL/min); IMS pressure N2 2.05 mbar (1.54 Torr; flow rate 38 mL/min); IMS pressure He 2.70 mbar (2.03 Torr; flow rate 70 mL/min). The pressure within the RF-confining drift cell was accurately measured using an MKS Baratron capacitance manometer, type 626 (range 10 Torr; accurate to 0.25%) and an MKS PDR2000 power supply. Ambient temperature was measured using an Oakton Temp10T thermocouple. The temperature was measured at the point where the capacitance manometer is connected to the instrument ion optics lid. Instrument control and data acquisition was carried out through MassLynx 4.1 SCN 639, SCN744. Mobilities and, therefore, the Ω values were made by accurately measuring the pressure within the drift cell device and the ambient temperature of drift cell and making up to 10 mobility measurements at different drift cell voltages (60 V to 200 V). This procedure was repeated twice and an overall average Ω value (in N2) between the two experiments was derived. Typical temperature and pressure variations over the mobility experiments were ≤ 0.2 °C and ≤ 0.002 Torr. Ion ATDs are measured for the ion of interest at each drift cell voltage gradient and plotted as a function of ATD versus the reciprocal of the drift tube voltage gradient (1/V). Typically, the relationship between ATD and 1/V is linear, with R2 correlation coefficients of greater than R2 = 0.9992. The mobility (K) of the ion can be calculated from the slope of the linear drift time versus 1/V relationship; and therefore, the Ω value of the ion also can be calculated. The Synapt G2 HDMS5 instrument was operated in positive nanoflow ESI mode. All critical instrument voltages and pressures are as follows: capillary voltage 0.8 to 1.0 kV; sample cone 25 V; extraction cone 1 V; source block temperature 30 °C; trap collision energy 4.0 V to 175 V in 5 V increments for the CIU experiments; trap entrance 3.0 V; trap bias 45 V; trap DC −2.0 V; trap exit 0.0 V; ion mobility spectrometer (IMS) entrance 10 V; IMS helium cell DC 25 V; IMS helium exit −5.0 V; IMS Bias 3.0 V; IMS exit 0.0 V; transfer entrance 4.0 V; transfer exit 5.0 V; IMS wave velocity 250 m/sec; IMS wave amplitude 21.0 V; transfer velocity 47 m/sec; transfer wave amplitude 4.0 V; mobility trapping release time 200 μsec; trap height 20.0 V; extract height 0.0 V; source RF-amplitude (peak-to-peak) 450 V; triwave RF-amplitudes (peak-to-peak) trap 380 V, IMS 250 V, transfer 380 V; source backing pressure 6.0 mbar; trap/transfer pressure SF6, 3.25e-2 mbar (Pirani gauge indicated; flow rate 4.5 mL/min); IMS pressure N2 3.54 mbar (Pirani

Page 43: NISTmAb Common Technical Document Case Study · 2018-04-02 · NISTmAb Common Technical Document Case Study . In order to provide attendees with an opportunity to evaluate “real

National Institute of Standards and Technology 00001234 Humanized IgG1κ monoclonal antibody, RM 8671 0000

3.2.S.3 Characterization [Humanized IgG1κ Monoclonal Antibody, NIST], Page 37

gauge indicated; flow rate 90 mL/min); He cell flow rate was set to 180 mL/min. Instrument control and data acquisition was carried out through MassLynx 4.1 SCN 781.

20 µL of samples PS 8760 and RM 8761 stock solutions (67 µM) were buffer exchanged into 200 mM ammonium acetate using a BioRad P6 spin column. An MS-working solution of 10 µM was created for nESI infusion and subsequent native IM-MS and CIU analysis. Figure 18 depicts the observed charge state distribution and associated arrival times observed for the NISTmAb PS 8670 and RM 8671. Drift time measurements were also recorded by varying the collision voltage to obtain a Collision Induced Unfolding (CIU) profile of the NISTmAb as shown in Figure 19.

Figure 18: Native MS analysis of NIST mAb PS8760 and RM 8671. Nitrogen-based collision cross sections were derived from the RF-confining drift tube instrument. Charge states and nitrogen collision cross sections are also annotated

Page 44: NISTmAb Common Technical Document Case Study · 2018-04-02 · NISTmAb Common Technical Document Case Study . In order to provide attendees with an opportunity to evaluate “real

National Institute of Standards and Technology 00001234 Humanized IgG1κ monoclonal antibody, RM 8671 0000

3.2.S.3 Characterization [Humanized IgG1κ Monoclonal Antibody, NIST], Page 38

Figure 19: Collision Induced Unfolding (CIU) profile of the NISTmAb

The difference plot was generated through the subtraction of two normalized CIU fingerprints and plotting the results as a contour plot, in which the CIU fingerprint of NIST_PS 8670 is shown on a red intensity scale and NIST_RM 8671 is shown in blue. In order to quantify the difference in the two CIU data shown, a root-mean-square deviation (RMSD) value was also computed using CIUsuite compare module.

3.2.S .3.1.6.3 Dynamic Light S cattering

For dynamic light scattering (DLS) experiments of protein solutions, estimated values of the protein size and the strength of the protein-protein interactions are obtained via analysis of the intensity autocorrelation function. Due to intermolecular interactions and transient concentration gradients across the solution, molecules exhibit Brownian motion that causes the number of scatters (i.e., molecules within the path of the incident light) to be constantly fluctuating. As a result, the size of the fluctuations in scattering intensity is correlated over short periods of time with the displacement of molecules. This function provides information regarding the average time that a molecule is located within the path of the incident light. By definition, such time is related to, and can therefore inform on, the diffusivity of the molecule (D), the hydrodynamic radius (RH), and the strength of the interactions experienced by the molecule. To characterize NISTmAb (both PS 8670 and RM 8671) via DLS, experiments were performed on a Wyatt DynaPro Nanostar (Wyatt Technology Inc., CA) as described in the SLS section. Intensity autocorrelation functions were obtained at a scattering angle of 90 for NISTmAb in 25 mM His/His-HCl at pH 6.0 and at protein concentrations between 1 and 10g/L. Prior to DLS experiments, samples were filtered through 0.1μm PVDF syringe filter (CELLTREAT Scientific Products, MA), and transferred into a 1.25μL quartz cuvette. Autocorrelation functions were averaged from five 10-second measurements, where the correlation time ranges from 0.5μs to 1s. For each protein concentration, four independent replicates were probed in order to reduce statistical uncertainties in the resulting diffusion coefficients. For all DLS experiments, measured autocorrelation functions correspond to a single exponential decay, and thus they were analyzed via the method of cumulants. In all the cases, the method of cumulants provided an excellent description of the autocorrelation function. Figure 20 depicts the resulting best-fit collective

Page 45: NISTmAb Common Technical Document Case Study · 2018-04-02 · NISTmAb Common Technical Document Case Study . In order to provide attendees with an opportunity to evaluate “real

National Institute of Standards and Technology 00001234 Humanized IgG1κ monoclonal antibody, RM 8671 0000

3.2.S.3 Characterization [Humanized IgG1κ Monoclonal Antibody, NIST], Page 39

diffusion coefficient (Dc) as a function of protein concentrations for NISTmAb (top panel) and an illustrative example for the estimated size distribution for those samples at 10g/L (bottom panel).

The results in Figure 20a show that D𝐶𝐶 values for both proteins are statistically undistinguishable from each other (within 95% confidence) at all evaluated protein concentrations. Nonetheless, best-fit values of the diffusivity for RM 8671 are systematically lower than those for PS 8670. Following the Stokes−Einstein equation, these differences suggest that RM 8671 is slightly larger than PS 8670, although such differences are not sufficient to be fully discerned by DLS. These results are qualitatively consistent with those obtained by SE-UHPLC, where the amount of HMW species (mainly, protein dimer) increases from ~1% to ~3% for RM 8671 as compared to PS 8670. A similar conclusion is also obtained when comparing the estimated size distributions (cf. Figure 20b for NISTmAb at 10g/L). Both proteins yield equivalent distributions for the hydrodynamic radius (with a maximum around 4.5nm), but the distribution for RM8670 is slightly shifted towards larger values of R𝐻𝐻 . Interestingly, fitted polydispersity indexes indicate monodisperse protein solutions (i.e. < 0.05 for all samples), which lead to narrow size distributions with resulting standard deviations of less than 2.5μm2/s (or equivalently, the variability on the apparent R𝐻𝐻 is less than 0.5nm as shown in Figure 20b).

Page 46: NISTmAb Common Technical Document Case Study · 2018-04-02 · NISTmAb Common Technical Document Case Study . In order to provide attendees with an opportunity to evaluate “real

National Institute of Standards and Technology 00001234 Humanized IgG1κ monoclonal antibody, RM 8671 0000

3.2.S.3 Characterization [Humanized IgG1κ Monoclonal Antibody, NIST], Page 40

Figure 20: Analysis of autocorrelation function for NISTmAb in 25 mM His/His-HCl at pH 6.0. (a) Collective diffusion coefficient (DC) as a function of protein concentration. (b) Particle size distributions based on the average hydrodynamic radius (RH)

Additionally, D𝐶𝐶 vs. concentration data for NISTmAb was linearly fitted (dashed lines in Fig. 20a) in order to characterize the self-diffusion coefficient, the effective hydrodynamic radius, and the interaction parameter 𝑘𝑘𝐷𝐷. Table 11 summarizes the best-fit values for these three parameters. Overall, no significant difference was observed when comparing the values of these parameters for both proteins. The calculated self-diffusion coefficient D0 is of ~45.5μm2/s, which yields an effective hydrodynamic radius of ~5.35nm. In the case of the parameter for protein interactions, the measured diffusivities for NISTmAb exhibit a positive slope with respect to protein concentration, resulting in a 𝑘𝑘𝐷𝐷 value of about 2900L/mol that indicates net repulsive protein interactions. Additionally, the best-fit values for these parameters are in excellent agreement with a previous DLS study on NISTmAb-PS 8670 under equivalent conditions.3

Page 47: NISTmAb Common Technical Document Case Study · 2018-04-02 · NISTmAb Common Technical Document Case Study . In order to provide attendees with an opportunity to evaluate “real

National Institute of Standards and Technology 00001234 Humanized IgG1κ monoclonal antibody, RM 8671 0000

3.2.S.3 Characterization [Humanized IgG1κ Monoclonal Antibody, NIST], Page 41

Table 11: Best-fit parameters estimated from linear fit to Equation 6 of the collective diffusivities of NISTmAb in 25 mM His/His-HCl at pH 6.0. Uncertainties on fitted parameters corresponds to 95% confidence intervals

Sample D0 [μm2/s] RH [nm] kD [L/mol]

PS 8670 46.00 ± 0.58 5.31 ± 0.06 2802.78 ± 205.86

RM 8671 45.23 ± 0.68 5.40 ± 0.08 3.2 200.61

3.2.S .3.1.6.4 S tatic Light S cattering

In static light scattering (SLS) experiments of protein solutions, time-averaged scattered intensities are collected as a function of protein concentration at a given angle θ. Because of the small length-scale of most proteins with respect to the wavelength of the incident light, proteins effectively behave as isotropic particles (from the standpoint of SLS) and the resulting scattering intensity is nearly independent of the detection angle (typically, θ is set to 90° for convenience). In order to obtain estimations of the protein size and the strength of protein-protein interactions, Rayleigh scattering data at low protein concentration (generally, < 10 g/L) is analyzed via Equation 6.

𝑅𝑅90𝑒𝑒𝑥𝑥

𝐾𝐾= 𝑀𝑀𝑤𝑤

𝑎𝑎𝑎𝑎𝑎𝑎𝑐𝑐1+2𝐵𝐵22𝑐𝑐

(6)

where 𝑅𝑅90𝑒𝑒𝑥𝑥 corresponds to the Rayleigh ratio at 90° of the protein solution at a total protein mass

concentration 𝑐𝑐 minus that of an equivalent solution at zero protein concentration. 𝐾𝐾 is an optical constant and is given by 𝐾𝐾 = 4𝜋𝜋2𝑛𝑛2�𝑑𝑑𝑛𝑛/𝑑𝑑𝑐𝑐�2𝑁𝑁𝐶𝐶

−1𝜆𝜆−4. 𝑁𝑁𝐶𝐶 is Avogadro’s number; 𝑛𝑛 and 𝑑𝑑𝑛𝑛/𝑑𝑑𝑐𝑐 denote the refractive index of the solution and its derivative with respect to protein concentration, respectively; and 𝜆𝜆 is the wavelength of the incident light. 𝑀𝑀𝑤𝑤

𝑎𝑎𝑎𝑎𝑎𝑎 is the apparent molecular weight of the protein, and it is a function of the strength of protein-solvent/excipients interactions. Similarly, 𝐵𝐵22 corresponds the protein osmotic second virial coefficient, and it is formally related to the solvent-average protein-protein interactions in the limit of infinite-dilution of protein. To characterize the NISTmAb, SLS experiments were carried out using a Wyatt DynaPro Nanostar (Wyatt Technology Inc., CA) equipped with a solid-state laser (λ=658nm) and a 512-channel, multi-tau correlator with a sampling time of 100ns. Scattering measurements were performed on 1.25 μL quartz cuvettes; scattering measurements were performed at 25 ± 0.3°C. The scattered light intensity and its autocorrelation function were obtained at 90° for NISTmAb (both PS 8670 and RM 8671) in 25 mM His/His-HCl at pH 6.0 and protein concentration ranging from 0.5 to 5 g/L. Prior to light scattering experiments, samples were filtered through 0.1 μm PVDF syringe filter (CELLTREAT Scientific Products, MA). Intensities in SLS were obtained by time-averaging the collected intensities over a time window of 1 min for a given sample. Four independent replicates of each protein concentration were measured to reduce statistical uncertainties in the resulting Rayleigh ratio at 90o (i.e. 𝑅𝑅90

𝑒𝑒𝑥𝑥 values). SLS data were fitted against Equation 6 for evaluation of the second virial coefficient (B22) for NISTmAb, with 𝑛𝑛 = 1.333 and 𝑑𝑑𝑛𝑛/𝑑𝑑𝑐𝑐 = 0.185. Figure 21 illustrates 𝑅𝑅90

𝑒𝑒𝑥𝑥 as a function of protein concentration

Page 48: NISTmAb Common Technical Document Case Study · 2018-04-02 · NISTmAb Common Technical Document Case Study . In order to provide attendees with an opportunity to evaluate “real

National Institute of Standards and Technology 00001234 Humanized IgG1κ monoclonal antibody, RM 8671 0000

3.2.S.3 Characterization [Humanized IgG1κ Monoclonal Antibody, NIST], Page 42

for PS 8670 and RM 8671 together with their best-fit curve to Equation 6. The resulting fitted values for apparent molecular weight ( 𝑀𝑀𝑤𝑤

𝑎𝑎𝑎𝑎𝑎𝑎 ) and 𝐵𝐵22 are summarized in Table 12. The results in Figure 21 clearly show that both PS 8670 and RM 8671 are equivalent from the standpoint of SLS, as no significant difference, within statistical uncertainty, is observed for 𝑅𝑅90

𝑒𝑒𝑥𝑥 vs. protein concentration. Similarly, best-fit curves of Equation 6 to SLS data for both proteins (dashed lines in Figure 21 ) lead to statistically equivalent results for 𝑀𝑀𝑤𝑤

𝑎𝑎𝑎𝑎𝑎𝑎 and 𝐵𝐵22 (Table 12), with an apparent molecular weight of ~149 kDa and a virial coefficient of ~2360 L/mol. Notably, the estimated molecular weight is in excellent agreement with the theoretical value (148.2 kDa). Although 𝑀𝑀𝑤𝑤

𝑎𝑎𝑎𝑎𝑎𝑎 is not anticipated to match the true molecular weight since it depends on the presence of other macromolecules in solution (e.g., protein oligomers and impurities), its equivalence with the theoretical 𝑀𝑀𝑤𝑤 suggests that the contribution of any HMW species is negligible due to their relatively small size and/or low concentration. In the case of the second virial coefficient, the estimated value indicates net repulsive protein-protein interactions (i.e., 𝐵𝐵22 > 0). This best-fit 𝐵𝐵22 value agrees with a previous SLS analysis NISTmAb under equivalent solution conditions.3 In that work, Rayleigh scattering were evaluated using an alternative methodology that linearized Equation 6 to yield an apparent virial coefficient𝑅𝑅2 = 𝐵𝐵22/𝑀𝑀𝑤𝑤

𝑎𝑎𝑎𝑎𝑎𝑎. For NISTmAb (PS 8670) in 25 mM His/His-HCl at pH 6.0, 𝑅𝑅2 was reported as 1.64x10-7 mol L g2.3

Page 49: NISTmAb Common Technical Document Case Study · 2018-04-02 · NISTmAb Common Technical Document Case Study . In order to provide attendees with an opportunity to evaluate “real

National Institute of Standards and Technology 00001234 Humanized IgG1κ monoclonal antibody, RM 8671 0000

3.2.S.3 Characterization [Humanized IgG1κ Monoclonal Antibody, NIST], Page 43

Figure 21: Excess Rayleigh scattering (R90ex/K) of NISTmAb in 25 mM His/His-HCl at pH 6.0.

Symbols correspond to average R90ex/K over four independent replicates for: (blue

circles) Primary Sample 8670; and (red squares) Reference Material 8671. Error bars indicate 95% confidence intervals. Dashed lines represent best-fitted curves of the corresponding SLS data to Equation 6.

Table 12: Best-fit apparent molecular weight and second virial coefficient for NISTmAb in 25

mM His/His-HCl at pH 6.0. Error bounds correspond to 95% confidence intervals of best-fit values

Sample Mwapp [kDa] B22 [L/mol] A2 [1x108 L.mol/g2]

PS 8670 150.01 ± 2.50 2662.67 ± 543.58 11.99 ± 2.45

RM 8671 148.18 ± 2.32 2063.21 ± 405.58 3.2 1.83

3.2.S .3.1.6.5 S ub Visible Particle Analysis

3.2.S .3.1.6.5.1 Flow Imaging

Flow imaging can be used for the analysis of subvisible particles (2 µm to 100 µm in size) in suspension. As a sample stream passes through a flow cell positioned in the field of view of a microscopic system, bright-field images are captured in successive frames. The digital images of the particles present in the images are stored in a database that can be retrieved and analyzed for counts, size, transparency, and various other morphological parameters. Figure 22 shows representative images obtained for various particles in RM 8671 lot 14HB-D-002 Commented [CV7]: With respect to data content and

presentation, how does the current dataset compare to what one might expect to include in a regulatory filing?

Page 50: NISTmAb Common Technical Document Case Study · 2018-04-02 · NISTmAb Common Technical Document Case Study . In order to provide attendees with an opportunity to evaluate “real

National Institute of Standards and Technology 00001234 Humanized IgG1κ monoclonal antibody, RM 8671 0000

3.2.S.3 Characterization [Humanized IgG1κ Monoclonal Antibody, NIST], Page 44

Figure 22: Representative images obtained for various particles in RM 8671 lot 14HB-D-002

Table 13 shows the average subvisible particle concentrations in the PS 8670 and RM 8671 lot 14HB-D-002. Each of the vials of 8670 was obtained from the same rack of material, such that vial to vial variability due to the fill process can be assumed to be negligible. The 8671 vials, on the other hand, were obtained from across the fill rack. The % RSD of each of the 8671 samples are on the same order as observed for 8670, indicating little to no vial to vial heterogeneity with regard to protein particle content was introduced due to the filling process. It should be noted that although the concentrations of particles were slightly dissimilar from PS 8670 and RM 8671, a student’s t-test at the 95% confidence level indicate that the lots are statistically identical to one another. In addition, the amount of protein present in the particles is very minute, on the order of tens to several hundreds of ng/mL, corresponding to < 0.005 % protein concentration in solution.

Table 13: Observed subvisible particle concentration (n=4 vials for PS 8760 and n = 6 vials for D-002)

Lot Conc. (≥ 2 µm), mL-1 One Standard Deviationc

PS 8670 4271 1413 RM 8670 14HB-D-002 6068 1696

c (n=4 vials for PS 8760 and n = 6 vials for D-002)

3.2.S .3.1.7 BIOLOGICAL FUNCTION

3.2.S .3.1.7.1 Biological Activity

NISTmAb biological activity was not measured as part of this exercise, although this would be a critical component of a typical CTD.

3.2.S .3.1.7.2 Protein Concentration Concentration of protein material is often performed using UV-Visible spectrophotometry wherein the measured absorbance is assumed to be equivalent to the decadic attenuance. The decadic attenuance, D, is computed as the negative logarithm (base 10) of the transmittance, and is analogous to absorbance except for the inclusion of scattering and luminescence effects upon the radiant power exiting the sample. 6 Concentrations reported are based on decadic attenuance at 280 nm (D280). Concentration of the NISTmAb RM 8671 lot 14HB-D-002 was determined by using a theoretical extinction coefficient (ε) of 1.42 mL · mg−1 · cm−1 and a glycan mass

Page 51: NISTmAb Common Technical Document Case Study · 2018-04-02 · NISTmAb Common Technical Document Case Study . In order to provide attendees with an opportunity to evaluate “real

National Institute of Standards and Technology 00001234 Humanized IgG1κ monoclonal antibody, RM 8671 0000

3.2.S.3 Characterization [Humanized IgG1κ Monoclonal Antibody, NIST], Page 45

fraction correction factor of 0.977. 3, 7 Reference value assignment was performed using a 0.5 mm path length quartz cuvette. Primary decadic attenuance measurements of the NISTmAb were measured using the MML Transfer Spectrophotometer, which is traceable to the national reference instrument for absorbance (HAS II).

Reference standard concentration values for PS 8670 and RM 8671 Lot 14HB-D-002 is summarized in Table 14.

Table 14: Reference Mass Concentration Value for PS 8670 and RM 8671 Lot 14HB-D-002 by UV Vis spectrophotometry (n = 10 vials).

Material Average Concentration (mg/mL)

Combined Standard Uncertainty, uc (mg/mL)

PS 8670 10.015 0.0403

RM 8671 Lot 14HB-D-002

10.003 0.0441

Page 52: NISTmAb Common Technical Document Case Study · 2018-04-02 · NISTmAb Common Technical Document Case Study . In order to provide attendees with an opportunity to evaluate “real

National Institute of Standards and Technology 00001234 Humanized IgG1κ monoclonal antibody, RM 8671 0000

3.2.S.3 Characterization [Humanized IgG1κ Monoclonal Antibody, NIST], Page 46

3.2.S.3.2 IMPURITIES

3.2.S.3.2.1 PROCESS-RELATED IMPURITIES Potential process-related impurities include residual host cell proteins (HCP), residual host cell DNA, residual Protein A, and retrovirus-like particles. The residual levels of HCP, DNA and Protein A are controlled by appropriate specifications. Based on the applicant’s experience in the manufacture of monoclonal antibodies, it is expected that the purification process will have excess capacity to remove HCP, DNA, retroviruses, and Protein A to acceptable levels. Evaluation of process related impurities in PS 8670 has been previously reported. 8, 9 RM 8671 was produced using the same upstream and downstream processing and is expected to contain similar levels of process-related impurities, analysis is ongoing.

3.2.S.3.2.2 PRODUCT-RELATED IMPURITIES Potential product-related impurities include multimers, fragments, charge variants, and post-translationally modified forms of the product. Covalent multimers and fragments are monitored by capillary sodium dodecyl sulfate electrophoresis (CE-SDS); covalent and non-covalent multimers are monitored by SE-UHPLC; charge-related variants are monitored with CZE. Other post-translational modifications will be monitored by peptide and oligosaccharide mapping. All product-related impurities are within method performance criteria established for PS 8670 except the presence of increased levels of multimers observed in SE-UHPLC and AUC. Despite a small increase in the %HMW versus 8670, two additional lots of 8671 have been shown to be consistent with one another in terms of monomeric purity, high molecular weight relative area and low molecular weight relative area demonstrating consistency in the production process.

A full characterization of each impurity species in each method is currently underway, and the ability to detect each relevant impurity will be determined as part of analytical method validation.

1. Formolo T, Ly M, Levy M, Kilpatrick L, Lute S, Phinney K, et al. Determination of the NISTmAb Primary Structure. State-of-the-Art and Emerging Technologies for Therapeutic Monoclonal Antibody Characterization Volume 2 Biopharmaceutical Characterization: The NISTmAb Case Study. ACS Symposium Series. 1201: American Chemical Society; 2015. p. 1-62. 2. Dinh NN, Winn BC, Arthur KK, Gabrielson JP. Quantitative spectral comparison by weighted spectral difference for protein higher order structure confirmation. Analytical biochemistry. 2014;464:60-2. Epub 2014/07/23. doi: 10.1016/j.ab.2014.07.011. PubMed PMID: 25051254. 3. Gokarn Y, Agarwal S, Arthur K, Bepperling A, Day ES, Filoti D, et al. Biophysical Techniques for Characterizing the Higher Order Structure and Interactions of Monoclonal Antibodies. State-of-the-Art and Emerging Technologies for Therapeutic Monoclonal Antibody Characterization Volume 2 Biopharmaceutical Characterization: The NISTmAb Case Study. ACS Symposium Series. 1201: American Chemical Society; 2015. p. 285-327. 4. Bush MF, Hall Z, Giles K, Hoyes J, Robinson CV, Ruotolo BT. Collision cross sections of proteins and their complexes: a calibration framework and database for gas-phase structural

Page 53: NISTmAb Common Technical Document Case Study · 2018-04-02 · NISTmAb Common Technical Document Case Study . In order to provide attendees with an opportunity to evaluate “real

National Institute of Standards and Technology 00001234 Humanized IgG1κ monoclonal antibody, RM 8671 0000

3.2.S.3 Characterization [Humanized IgG1κ Monoclonal Antibody, NIST], Page 47

biology. Anal Chem. 2010;82(22):9557-65. Epub 2010/10/29. doi: 10.1021/ac1022953. PubMed PMID: 20979392. 5. Giles K, Williams JP, Campuzano I. Enhancements in travelling wave ion mobility resolution. Rapid communications in mass spectrometry : RCM. 2011;25(11):1559-66. Epub 2011/05/20. doi: 10.1002/rcm.5013. PubMed PMID: 21594930. 6. May W, Parris R, Beck II C, Fassett J, Greenberg R, Guenther F, et al. Definitions of terms and modes used at NIST for value-assignment of reference materials. Special Publication 260-136. Washington, DC: Government Printing Office; 2000. 7. Pace CN, Vajdos F, Fee L, Grimsley G, Gray T. How to measure and predict the molar absorption coefficient of a protein. Protein Sci. 1995;4(11):2411-23. Epub 1995/11/01. doi: 10.1002/pro.5560041120. PubMed PMID: 8563639; PubMed Central PMCID: PMCPMC2143013. 8. Chen W, Doneanu CE, Lauber M, Koza S, Prakash K, Stapels M, et al. Improved Identification and Quantification of Host Cell Proteins (HCPs) in Biotherapeutics Using Liquid Chromatography-Mass Spectrometry. State-of-the-Art and Emerging Technologies for Therapeutic Monoclonal Antibody Characterization Volume 3 Defining the Next Generation of Analytical and Biophysical Techniques. ACS Symposium Series. 1202: American Chemical Society; 2015. p. 357-93. 9. Prakash K, Chen W. Analytical Methods for the Measurement of Host Cell Proteins and Other Process-Related Impurities. State-of-the-Art and Emerging Technologies for Therapeutic Monoclonal Antibody Characterization Volume 2 Biopharmaceutical Characterization: The NISTmAb Case Study. ACS Symposium Series. 1201: American Chemical Society; 2015. p. 387-404.