NISTmAb RM 8671 Humanized Monoclonal IgG1κ · PDF file5 NISTmAb Attributes by CE Methods...

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Transcript of NISTmAb RM 8671 Humanized Monoclonal IgG1κ · PDF file5 NISTmAb Attributes by CE Methods...

  • 1

    NISTmAb RM 8671 Humanized Monoclonal IgG1

    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

  • 2

    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

  • 3

    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 (IgG1) 10 mg/mL, 800 L 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

  • 4

    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

  • 5

    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.

  • 6

    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

  • 7

    CE-SDS for Size Variants

  • 8

    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

  • 9

    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 m 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 (3) 16 ( 3) pg 0.17 ( 0.03) %

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

    R = 0.9993

    R = 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

  • 15

    Charge Sensitive Methods

  • 16

    CIEF Method Development

    R = 0.9996

    R = 0.9997

    R = 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

    R = 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 m 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

  • 17

    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 m 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 m i.d., 40 cm LTD 30 kV; UV 214 nm

  • 20

    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 T