mAb and ADC Analysis

32 Pages·2017·2.11 MB·English

by  Shanhua Lin

mAb and ADC Analysis
Shanhua Lin, Ph.D.
R&D Manager
Bioseparation, Chromatography Consumables

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Structure of IgG and Typical Forms of Heterogeneity
2
Protein and mAb Separation by HPLC
YES Size Exclusion
Size difference? Thermo Scientific™
Chromatography (SEC)
MAbPac™ SEC-1
MAbPac SCX-10
Ion Exchange
YES
CX-1 pH Gradient Buffer
Charge difference?
Chromatography (IEX)
Thermo Scientific™
ProPac ™ WCX-10

Reverse Phase
MAbPac RP
YES
Chromatography (RPC)
Hydrophobicity difference?
MAbPac HIC-10
Hydrophobic Interaction
YES MAbPac HIC-20
Chromatography (HIC)
MAbPac HIC-Butyl
ProPac HIC-10
3
IEC Analysis for Charge Variant
• ProPac WCX-10 and MAbPac SCX-10 columns
• Separate acidic and basic variants
• CX-1 pH Gradient Buffer Kit provides a platform solution
4
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
5
Next-generation CEX Column
YY YY
MAbPac SCX-10
Lysine Variants
YY
Column
K YY
YY Acidic K K
K Variants
Lysine Basic Variants
Variants
YY
K K
ProPac WCX-10 column, 4×250 mm
22
MAbPac SCX-10 column,
Acidic
×
4 150 mm
Variants
15 min. total analysis time
Basic Variants U
A
m
0
MAbPac SCX-10 column, 4×250 mm
0 5 10 15
Minutes
60 min. total analysis time
Improve resolution or sample throughput through column chemistry
6
Thermo Scientific CX-1 pH Gradient Buffers
Buffer A Buffer B
pH 5.6 10.2
Form Liquid Liquid
Concentrate 10X 10X
Shipping Room Room
• Dilute buffers 10-fold with DI water
condition Temp Temp
• A linear pH gradient (pH 5.6 - 10.2)
is generated by running a linear
pump gradient from 100% Buffer A
Storage
4 ~ 8 °C 4 ~ 8 °C
to 100% Buffer B
condition
• It is platform, fast, and high-res!

US 8921113 B2: Buffer kit and method of generating a linear pH gradient
7
pH Gradient Buffers – How Do They Work?
Protein Elution Mechanisms on IEX
Isoelectric
Point (pI)
+
COO-
+
+ + R NH+
3
Buffer pH typically < pI + Catiboinnidc sp troo tein +
negatively charged
 Cation-Exchange cationexchanger
Chromatography + + +
COOH
R +
NH
3
Buffer/System pH
0
4 5 6 7 8 9 10 11 12
- -
-
pH range covered by CX-1 pH gradient buffers
Buffer pH typically > pI
Anionic protein -
- binds to
 Anion-Exchange positively charged
anion exchanger
Chromatography - - -
-
COO
R
NH
– 2
Protein net charge vs. pH
8
Linear pH Gradient by Zwitterionic Buffer Cocktail
10.5
y = 0.1577x + 4.9755
R² = 0.9996
9.5
e
u
l
a
v 8.5

H
p

d
e Measured Value
r
u 7.5
s Linear (Measured Value)
a
e
M
6.5
5.5
0 10 20 30 40
Retention Time [min]
9
Protein Standard – CX-1 pH Gradient Buffer
MAbPac SCX-10 Column, 4 × 250 mm
Peak label: protein name – elution pH
90 11
Lectin-1 - 6.11
10
pH trace
60
9
40 8
Cytochrome C - 10.15
Lectin-2 - 6.28
Ribonuclease A - 8.72 7
20 Lectin-3 - 6.45
Trypsinogen - 7.75
6
0
-10 5
0 5 10 15 20 25 30 35 40
10