2
Intact Mass Analysis of Monoclonal Antibody (MAb) Charge Variants Separated Using Linear pH Gradient
Overview
Purpose:
Intact mass analysis of monoclonal antibody (MAb) charge variants
separated using linear pH gradient.
Methods:
The separation of MAb charge variants is achieved using linear pH gradient
method on a cation-exchange column. The intact mass information is acquired on the
Thermo Scientific™ Q Exactive™ hybrid quadrupole-Orbitrap mass spectrometer.
Results:
This linear pH gradient enables the high resolution separation of MAb
charge variants. The intact mass analysis characterizes the structural difference of the
MAb variants.
Introduction
Monoclonal antibodies can be highly heterogeneous due to modifications such as
sialylation, deamidation, and C-terminal lysine truncation. Salt gradient cation-
exchange chromatography has been used with some success in characterizing MAb
charge variants. However, additional effort is often required to tailor the salt gradient
method for an individual MAb. In the fast-paced drug development environment, a
platform method is desired to accommodate the majority of the MAb analyses.
In 2009, Dell and Moreno reported a method to separate MAb charge variants using
pH gradient ion-exchange chromatography. The buffer employed to generate the pH
gradient consisted of piperazine, imidazole, and tris, covering a pH range of 6 to 9.5.
While good separation was observed, the slope of the pH increase was shallow at the
beginning and steep towards the end.
1
In this study, we present a novel pH gradient
method for cation-exchange chromatography that is more linear. This method features
a multi-component buffer system in which the linear gradient was run from 100%
eluent A (low pH buffer) to 100% eluent B (high pH buffer). Using an online pH meter,
it was confirmed that a linear pH gradient was achieved. Furthermore, a plot of
measured pH values at the retention time of model proteins versus their pI values
exhibited a high correlation. Once the approximate pH elution range of the target MAb
has been established in the initial run, further optimization of separation can simply be
achieved by running a shallower pH gradient in a narrower pH range.
Methods
Sample Preparation:
All standard proteins were purchased from Sigma. Harvest cell culture and
monoclonal antibodies were a gift from a local biotech company. Proteins and MAb
were dissolved in deionized water.
Column and Buffer
Thermo Scientific™ MAbPac™ SCX-10, 10 µm, 4
×
250 mm (P/N 074625)
CX-1 pH Gradient Buffer Kit (P/N 083274)
Liquid Chromatography
HPLC experiments were carried out using a Thermo Scientific™ Dionex™
UltiMate™ 3000 BioRS System equipped with:
- SRD-3600 Membrane Degasser
- DGP-3600RS Biocompatible Dual Gradient Rapid Separation Pump
- TCC-3000SD Thermostatted Column Compartment with two biocompatible
10-port valves
- WPS-3000TBRS Biocompatible Rapid Separation Thermostatted Autosampler
- VWD-3400RS UV Detector equipped with a Micro Flow Cell
- PCM-3000 pH and Conductivity Monitor
Column and Buffer
The CX-1 pH buffer kit consists of one bottle of 10X buffer A (pH 5.6) and one bottle of
10X buffer B (pH 10.2). Eluent A and B each was prepared by simply diluting the
corresponding 10 X buffer 10 fold using deionized water.
Results
Linear pH gradient
The linear pH gradient was achieved
containing multiple zwitterionic buffer
Eluent A was titrated to pH 5.6 and el
each buffer species was either neutra
retained by cation-exchange column
mobile phase and the stationary phas
Using the gradient method shown in
6 to 10 were effectively separated on
These proteins were lectin (including t
trypsinogen, ribonuclease A, and cyto
Figure 1. The pH value measured in t
Figure 2. The pH gradient was essent
minute period. The correlation coeffici
An analysis was performed to show t
the peaks and the corresponding pI v
graph comparing the measured pH va
as a function of the corresponding pI
component peaks exhibited a strong li
Thus, after a calibration procedure, th
coupled with the gradient method des
protein component based on the peak
Table 1. 30 min linear gradient met
mm, cation exchange columns.
Tot
from pH 5.6 to pH 10.2. UV waveleng
Linear pH Gradient Chromatograph
The linear pH gradient was generated
(pH 5.6) to 100% eluent B (pH 10.2).
MAbPac SCX-10, 10 µm, 4
×
250 m
method in Table 1 was used unless fu
LC-MS
First dimension HPLC: in a scale up p
3.8 mL of HCC using Thermo Scientifi
concentration was determined at ~ 0.
injected onto a MAbPac SCX-10, 10
pH gradient from pH 6.52 to pH 9.28.
minutes after sample injection, a linea
minutes. Fractions were collected ont
from 10 to 26 min.
Second dimension LC-MS: Thermo S
(1
×
50 mm) was used for desalting.
A) and 0.1% formic acid in acetonitrile
analysis. Flow rate was 100 µL/min. A
to 95% B was used to elute the mAbs
MS: The Q Exactive Orbitrap mass sp
analyzed by ESI-MS for intact molecu
flow rate was set at 10. Auxiliary gas f
275 ºC. S-lens level was set at 55. In-
17,500. The AGC target was set at 3
Data Processing: Full MS spectra of i
Scientific™ Protein Deconvolution 1.0
molecular mass determination. Mass
averaging spectra across the most ab
The averaged spectra were subseque
to 4000 m/z, an output mass range of
and a minimum of at least eight cons
produce a deconvoluted peak.
Time
(minutes)
Flow r
(mL/mi
0-1
1
1-31
1
31-34
1
34-40
1
