mAb and ADC Analysis

64 Pages·2015·3.71 MB·English

by  Shanhua Lin

mAb and ADC Analysis
Shanhua Lin, Ph.D.
The world leader in serving science
Structure of IgG and Typical Forms of Heterogeneity
2
Protein and mAb Separation by HPLC
YES Size Exclusion
Size difference? MAbPac SEC-1
Chromatography (SEC)
MAbPac SCX-10
Ion Exchange
YES
ProPac WCX-10
Charge difference?
Chromatography (IEX)
CX-1 pH gradient Buffer
Reverse Phase
YES MAbPac RP
Chromatography (RPC)
Hydrophobicity difference?
MAbPac HIC-10
Hydrophobic Interaction
YES MAbPac HIC-20
Chromatography (HIC)
MAbPac HIC-Butyl
ProPac HIC-10
3
mAb Aggregate Analysis
4
mAb Aggregate Analysis
mAb monomer
50.0 (a) Column: MAbPac SEC-1, 5 µm,
Dimension: a. 7.8 × 300 mm
40.0
b. 4.0 × 300 mm
30.0 a. 2.1 × 300 mm
Aggregates
20.0 Mobile phase: 50mM Sodium Phosphate pH 6.8,
in 300 mM NaCl
10.0
Temp.: 30 ºC
0.0
Flow rate: a. 760 µL/min
0.0 2.5 5.0 7.5 10.0 12.5 15.0 18.0
b. 200 µL/min
150
(b) c. 50 µL/min
125 Inj. volume: a. 10 µL
100 b. 5 µL
75 a. 1 µL
50 Detection: 280 nm
25 Sample: mAb (1 mg/mL)
0
0.0 2.5 5.0 7.5 10.0 12.5 15.0 18.0
70.0
(c)
60.0
50.0
40.0
30.0
20.0
10.0
0.0
0.0 2.5 5.0 7.5 10.0 12.5 15.0 18.0
Retention Time (min)
5
Faster Aggregate Analysis
mAb monomer Column: MAbPac SEC-1, 5 µm,
140
Dimension: a. 4.0 × 300 mm
(a) 4.0 × 300 mm
b. 4.0 × 150 mm
100 Mobile phase: 50mM Sodium Phosphate pH 6.8,
in 300 mM NaCl
75 Temp.: 30 ºC
Flow rate: 200 µL/min
50
Aggregates Inj. volume: 2 µL
Detection: 280 nm
25
Sample: mAb (5 mg/mL)
-20 (b) 4.0 × 150 mm
400
300
Shorter 150 mm MAbPac SEC-1
200 column enables significant
reduction of run times
100
-50
0.0 2.0 4.0 6.0 8.0 10.0 12.0 14.0 16.0 18.0 20.0 22.0 25.0
Retention Time (min)
6
Non-denaturing SEC-MS
Column: MAbPac SEC-1, 5 µm,
(a) Dimension: 2.1 × 150 mm
monomer
Mobile phase: 20 mM ammonium formate
Temp.: 30 ºC
Flow rate: 50 µL/min
dimer
Inj. volume: 1 µL
Detection: Exactive Plus EMR
Samples: mAb (1 mg/mL)
+38
+39
(b)
+40
+37
+26
+25
(c)
+27
+24
+28
7
mAb Charge Variant Analysis by pH Gradient Method
8
Charge Variant Analysis
ProPac WCX-10 Column
Untreated mAb
YY

Lysine Variants
YY
K YY
10 µm Nonporous
K K
Polymeric Beads
Acidic Variants

Basic Variants
Polymeric grafts
WCX, SCX, WAX,

SAX

Boundary
YY
Carboxypeptidase B
(Cross-linked
hydrophilic layer) digested mAb
Core
(Highly cross-linked
EVB-DVB)
Acidic Variants
Basic Variants
9
Gold Standard for mAb Analysis
ProPac WCX-10 Column
10