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NISTmAb RM 8671 Humanized Monoclonal IgG1κ

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

The First Publicly Available, Well-Characterized Reference Material for:

System Suitability

Technology Development

Method Control

Open Innovation

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The NISTmAb: A Comprehensively Characterized Reference Material to

Support Biopharmaceutical Analytical Technology Development

Abigail Turner, John Schiel

CE Pharm 2016

September 28, 2016

San Diego, CA

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• Certify for concentration traceable to transmittance

• Lifecycle management and stability program Intended uses – Develop and implement innovative

technology – Assist method qualification – System suitability – Assess method variability

What is the NISTmAb? Reference Material 8671

– Open Innovation Humanized mAb (IgG1κ) – 10 mg/mL, 800 µL per unit

In-House Standard: Manufacturer-specific drug substance

Reference Material: Class-specific material issued under NIST trademark and established to be fit for intended use in measurement of nominal property values.

Completed rigorous interlaboratory characterization documented in ACS book compilation

Regulatory

NIST Industry

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Comprehensive RM Characterization

• Peptide mapping by LC-MS/MS and CE-MS/MS – Primary Sequence

– S-S Bridge & PTM analysis

• Intact, middle down MS

• MS/MS library compilation • Glycosylation Analysis

• LC: SEC, RP, IEX, HIC

• CE: CIEF, CE-SDS, CZE

• SDS-PAGE

• HOS: NMR, HDX, XRD • Neutron scattering • Biophysical: CD, FTIR, DSC,

DLS, AUC, SLS, DSF

• Protein particulates • Many emerging technologies! On-going characterization and quality monitoring program

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NISTmAb Attributes by CE Methods • CE-SDS

– Qualified method – Monomeric purity, HC glycan occupancy,

stability • CIEF

– Characterization method – Apparent pI

• CZE – Qualified method – Charge purity, stability

• CE-ESI-MSn

– Characterization method – Peptide mapping (PTMs) – Focus on glycopeptides

Formolo, Ly, Levy, Kilpatrick, Lute, Phinney, Marzilli, Brorson, Boyne, Daviss, Schiel. State-of-the-Art and Emerging Technologies for Therapeutic mAb Characterization Volume 2.; American Chemical Society: ACS Symposium Series 1201; American Chemical Society, Washington, DC, 2015.

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CE Methods: Qualification Approach Qualification designed to fit the needs of our Lifecycle Management Plan: • For quantitative assays • Using Primary Standard (PS) 8670 • ICH Q2(R1)

– “Fit For Purpose” – LOD/LOQ – Linearity – Specificity – Precision

• Repeatability • Limited intermediate precision

Production Lot 1

PS 8670

Homogenized Multiple Prod. Lots

8671/x

D-001 RM 8671 D-002 D-003

Split into lots

NISTmAb

Method Qualification

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CE-SDS for Size Variants

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CE-SDS Sample Prep Optimization

60

70

80

90

100

2 4 6 8 10 12 14 16 18 20 22

Mon

omer

ic P

urity

(%)

Incubation Time (min)

70 °C 80 °C 90 °C 100 °C

60

70

80

90

100

-5 0 5 10 15 20 25 30 35 40 45 50

Mon

omer

ic P

urity

(%)

[Iodoacetamide] (mmol/L)

pH 9.0, time 0 pH 9.0, time 12 h

pH 6.7, time 0 pH 6.7, time 12 h

Optimized conditions: Dilute in pH 6.7, 1% SDS sample buffer with 5% BME (reduced) or 46 mM IAM (non-reduced); incubate 10 min (reduced) or 5 min (non-reduced) at 70 °C

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Optimized CE-SDS Profiles

Minutes11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35

AU

-0.002

0.000

0.002

0.004

0.006

0.008

0.010

0.012

0.014

0.016

0.018

0.020

0.022

0.024

0.026

AU

-0.002

0.000

0.002

0.004

0.006

0.008

0.010

0.012

0.014

0.016

0.018

0.020

0.022

0.024

0.026

PDA - 220nmSS_02

Minutes11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35

AU

-0.0012

-0.0010

-0.0008

-0.0006

-0.0004

-0.0002

0.0000

0.0002

0.0004

0.0006

0.0008

0.0010

0.0012

0.0014

AU

-0.0012

-0.0010

-0.0008

-0.0006

-0.0004

-0.0002

0.0000

0.0002

0.0004

0.0006

0.0008

0.0010

0.0012

0.0014

PDA - 220nmSS_02

Minutes11 12 13 14 15 16 17 18 19 20 21 22 23 24

AU

-0.002

0.000

0.002

0.004

0.006

0.008

0.010

0.012

0.014

0.016

0.018

0.020

0.022

0.024

0.026

0.028

0.030

0.032

AU

-0.002

0.000

0.002

0.004

0.006

0.008

0.010

0.012

0.014

0.016

0.018

0.020

0.022

0.024

0.026

0.028

0.030

0.032

PDA - 220nmSS_04

Minutes11 12 13 14 15 16 17 18 19 20 21 22 23 24

AU

-0.0015

-0.0010

-0.0005

0.0000

0.0005

0.0010

0.0015

0.0020

0.0025

0.0030

0.0035

0.0040

0.0045

0.0050

AU

-0.0015

-0.0010

-0.0005

0.0000

0.0005

0.0010

0.0015

0.0020

0.0025

0.0030

0.0035

0.0040

0.0045

0.0050

PDA - 220nmSS_04

Non-Reduced Reduced

10 kDa marker

monomer L:H:H:L

10 kDa marker

light chain/L

heavy chain/H

aglyco- heavy chain/

NGH

non-reducible species

(thioether)1

non-reducible species

(thioether)

L NGH

H H:L H:H H:H:L

“clip”

50 µm i.d. x 30.5 cm (20 cm LTD) BFS 25 °C UV 220 nm Sciex SDS-MW sieving gel (1) Tous, et al. Anal. Chem. 2005, 77(9), 2675-2682

12 Minutes

12 14 16 18 20 22 24 26 28 30 32 34A

U

-0.0005

0.0000

0.0005

0.0010

0.0015

0.0020

0.0025

0.0030

0.0035

0.0040

AU

-0.0005

0.0000

0.0005

0.0010

0.0015

0.0020

0.0025

0.0030

0.0035

0.0040

PDA - 220nmAHT-UV_NR

PDA - 220nmAHT+UV_NR

10 kDa

L H H:L H:H

H:H:L clip

monomer

CE-SDS Qualification Intermediate Precision Average CV (%)

Monomeric Purity (%) 98.8 (±0.4) 0.4

Monomer Migration Time (min) 28.2 (±0.3) 1.0

Heavy Chain MT (min) 19.3 (±0.2) 1.2

Light Chain MT (min) 15.3 (±0.2) 1.2

Glycan Occupancy (%) 99.40 (±0.01) 0.01

Thioether (%) 0.31 (±0.02) 4.9

Figures of Merit Absolute Relative to Target (1 mg/mL)

Linear range 0.25 – 2.0 mg/mL 25 – 200 %

Limit of Detection (3σ) 16 (± 3) pg 0.17 (± 0.03) %

Limit of Quantification (10σ) 53 (± 8) pg 0.57 (± 0.09) %

R² = 0.9993

R² = 0.9996

0

5000

10000

15000

20000

25000

30000

0.00 1.00 2.00 3.00

Corr

ecte

d Ar

ea

Total Concentration (mg/mL)

LC

HC

+ UV, 21 h - UV, 21 h

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Charge Sensitive Methods

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CIEF Method Development

R² = 0.9996

R² = 0.9997

R² = 0.9994

0

5000

10000

15000

20000

25000

0 0.2 0.4 0.6 0.8

Corr

ecte

d Ar

ea (m

AU*c

m/s

2)

[NISTmAb] (mg/mL)

Basic Group Main Group Acidic Group

Minutes18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33

AU

0.00

0.01

0.02

0.03

0.04

0.05

0.06

0.07

0.08

0.09

0.10

0.11

0.12

0.13

pI 10 pI 7

75% PL 8-10.5 : 25% PL 3-10 1.5 mol/L Urea

100% PL 3-10 3 mol/L Urea

2K

1K

Main

Acidic Basic

R² = 0.9897

202224262830

8.5 9 9.5 10Mig

ratio

n Ti

me

(min

)

pI

Sciex CIEF Kit: CIEF separation gel Catholyte: 300 mM NaOH; Anolyte: 200 mM H3PO4; Cathodic Stabilizer: 40 mM Arginine; Anodic Stabilizer: 1.6 mM Iminodiacetic acid; 4.8% Pharmalytes (GE); Mobilizer: 350 mM HAc 30.5 cm neutral coated capillary, 50 µm i.d., 20 cm LTD; UV 280 nm

• LOD: 1.5% (3.6 ng) • LOQ: 5.1% (12 ng) • Range (Main Peak): 0.1-0.6

mg/mL • Does not consistently detect 2K

peak at loading ≤ 0.4 mg/mL

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CZE Method Development

Minutes7 8 9 10 11 12 13 14

Minutes6 7 8 9 10 11 12 13

Repl

icat

e in

ject

ions

Tween 20 (0.03%) HPMC (0.05%)

He, et al. J. Sep. Sci. 2011

EACA

TETA

HPMC

Tween 20

Minutes6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 10.0 10.5 11.0 11.5

AU

-0.01

0.00

0.01

0.02

0.03

0.04

0.05

0.06

0.07

0.08

0.09

2K

1K

Main

Acidic

Basic

400 mM EACA + 2 mM TETA, pH 5.7 50.5 cm BFS capillary, 50 µm i.d., 40 cm LTD 30 kV; UV 214 nm

400 mM EACA + 2 mM TETA, pH 5.7 0.03% Tween 20 50.5 cm BFS capillary, 50 µm i.d., 40 cm LTD 30 kV; UV 214 nm

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CIEF CZE Intra-Day Precision (1 day, 1 column)

Average (± SD) CV Intermediate Precision (6 days, 3 columns)

Average (± u) u = total uncertainty from ANOVA*

CV

Main Peak Migration Time (min) 25.54 (± 0.20) 0.78 % Main Peak Migration Time (min) 9.67 (± 0.17) 1.8%

Main Peak Apparent pI 9.2 (± 0.01) 0.11 % IQ Standard Migration Time (min) 5.79 (± 0.06) 1.0%

pH Gradient Linearity (RSQ) 0.990 (± 0.001) 0.07 %

Main Group RA (%) 72.5 (± 0.4) 0.6 % Main Group RA (%) 74.7 (± 0.3) 0.5 %

Acidic Group RA (%) 19.9 (± 0.5) 2.5 % Acidic Group RA (%) 16.8 (± 0.4) 2.4 %

Basic Group RA (%) 7.6 (± 0.2) 2.6 % Basic Group RA (%) 8.5 (± 0.3) 3.3 %

Figures of Merit

Limit of Detection (3σ) 3.6 (± 0.2) ng 1.5 (±0.1) % at Target Limit of Detection (3σ) 0.044 (± 0.012) ng 0.2 (± 0.1) % at Target

Limit of Quantification (10σ) 11.9 (± 0.7) ng 5.1 (± 0.3) % at Target Limit of Quantification (10σ) 0.150 (± 0.039) ng 0.7 (± 0.2) % at Target

Linear Range (Main Peak) 0.1─0.6 mg/mL 25-150% of Target Linear Range (Main Peak) 0.25─2.5 mg/mL 17─170 % of Target

Resolution (1K:Main) 1.6 (± 0.1) Resolution (1K:Main) 0.9 (± 0.001)

Theoretical Plates (Main Peak) 8 x 104 Theoretical Plates (Main Peak) 6 x 104

Sample Consumption 240 ng Sample Consumption 20 ng

Run Time per Sample 55 min Run Time per Sample 25 min

CZE Comparison to CIEF

*Total uncertainty u (for CZE only) was calculated using ANOVA to identify within-day and between-day variances in the intermediate precision dataset.

Minutes20 21 22 23 24 25 26 27 28 29 30 31 32 33 34

AU

0.00

0.01

0.02

0.03

0.04

0.05

0.06

0.07

0.08

0.09

0.10

0.11

0.12

pH 6.0, -80 °C

pH 6.0, 40 °C

pH 3.7, 40 °C

pH 8.9, 40 °C

Minutes7.5 8.0 8.5 9.0 9.5 10.0 10.5 11.0 11.5 12.0 12.5

AU

0.00

0.01

0.02

0.03

0.04

0.05

0.06

0.07

0.08

0.09

0.10

0.11

0.12

0.13

0.14

pH 6.0, -80 °C

pH 6.0, 40 °C

pH 3.7, 40 °C

pH 8.9, 40 °C

CIEF CZE

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NISTmAb RM 8671 Attributes by Qualified CE Methods

NISTmAb Homogeneity and Stability by CE-SDS

Code Condition

FZ-80001 1 Freeze/Thaw (F/T) from -80 C

FZ-80005 5 F/T from -80 C

FZ-20001 1 F/T from -20 C

FZ-20005 5 F/T from -20 C

ASXXX00d Control (no stress, -80 C storage)

AS04C07d 7 days at 4 C

AS04C28d 28 days at 4 C

AS0RT07d 7 days at room temperature/light

AS0RT28d 28 days at room temperature/light

AS40C07d 7 days at 40 C

AS40C28d 28 days at 40 C

Lots are homogeneous by ANOVA (95% significance level)

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NISTmAb Homogeneity and Stability by CZE

RM 8671 Lot D-001

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CE-MSn

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Tryptic Peptide Maps by CE- and LC-MS2

CE-MS2 Map of Heavy Chain

LC-MS2 Map of Heavy Chain

MS: Thermo Orbitrap Discovery • CE- and LC-specific MS

parameters Data Analysis: ByonicTM

LC raw data courtesy of Trina Formolo

• Excellent coverage by both methods.

• Different peptides and PTMs detected by LC and CE

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Glycopeptide Detection by CE/MS2 and LC/MS2

LC raw data courtesy of Trina Formolo

BFS sheathless cartridge, Sciex CESI 8000 + Thermo Orbitrap Discovery

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Conclusions • NISTmAb RM 8671 is well-characterized by CE methods

– Molecule-specific methods, qualified where possible – Publicly available protocols and data – On-going method development for new technologies (CE-MS)

• NISTmAb is a valuable tool to assist – Method assessment/comparability – Technology development and open innovation – Inter-laboratory comparisons/establishing best practices

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Acknowledgments NISTmAb Certification Team John Schiel (CASSS MS Poster P-118-W ) Catherine Formolo (CASSS MS Poster P-142-W ) Katharina Yandrofski Srivalli Telikepalli Paul DeRose

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Laurie Locascio (MML Lab Director) Michael Tarlov (Division Chief) Karen Phinney (Bionalytical Sciences Group Leader) John Marino (Biomolecular Structure GL) Dean Ripple (Process Meas. GL) Trina Formolo (Peptide Mapping) Abigail Turner (CE, CE-MS) Katharina Yandrofski (SEC, Glycoanalysis) Srivalli Telikepalli (Protein particles) Paul DeRose (UV) John Marino (NMR) Rob Brinson (NMR) Luke Arbogast (NMR) Frank Delaglio (NMR data processing & informatics) Marc Cicerone (Lyo Protein stability, optical methods) Miaochan Zhi (Lyo Protein stability, optical methods) Young Lee (Broadband Raman optical activity) Ian Ryu (Broadband Raman optical activity) Mark Lowenthal (Glycan Reference Materials) Joseph Curtis (SANS of IgGs) Monica Castellanos (SANS of IgGs)

Acknowledgements: NIST Biomanufacturing Team

Ronald Jones (nSoft) Steve Hudson (Protein rheology, viscometry) John Royer (Protein rheology, viscometry) Dick Cavicchi (Protein particle measurement technologies) Wyatt Vreeland (Particle and mAb AFFFF) Adrian Defante (Particle and mAb AFFFF) Travis Gallagher (NISTmAb x-ray crystallography) Alex Grishaev (Protein x-ray scattering) Jeff Hudgens (HDX-MS) Ioannis Karageorgos (HDX-MS) Steve Stein (MS Library) Lorna de Leoz (Glyco MS library) Kelly Telu (CHO & cell culture media MS library) Tytus Mak (Cell culture metabolite MS library) Vince Shen (Coarse-grain modeling of mAbs) Marco Blanco (Coarse-grain modeling of mAbs) Ken Cole (Cell-line ID) Sumona Sukar (Cell counting)