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