2
Automated MAb Workflow: from Harvest Cell Culture to Intact Mass Analysis of Variants
Overview
Purpose:
Demonstrate an automated monoclonal antibody (MAb) analysis
two-dimensional (2D) workflow and intact mass detection.
Methods:
Automated analysis is achieved with the Thermo Scientific Dionex UltiMate
3000 x2 Dual Titanium Biocompatible Analytical LC System using Thermo Scientific
Dionex Chromeleon Chromatography Data System (CDS) software. The intact mass
information is acquired on the Thermo Scientific Q Exactive mass spectrometer.
Results:
This workflow enables the completion of affinity purification, size-exclusion
analysis, and charge variant analysis in less than one hour. The intact mass analysis
characterizes the structural difference of the MAb variants.
Introduction
During development of recombinant MAbs, a large of number of harvest cell culture
(HCC) samples must be screened for IgG titer, aggregations, and charge variants.
Affinity chromatography is often used first to purify MAbs, with typical yields of more
than 95%. Size-exclusion chromatography (SEC) is used to identify and quantify MAb
aggregations. Finally, ion-exchange chromatography (IEC) characterizes charge
variants. For the final biopharmaceutical product approval and subsequent
manufacturing processes, a comprehensive characterization of MAb purity, aggregate
forms, and charge variants is required by the regulatory agencies.
In the present study, we automate a 2D high-performance liquid chromatography
(HPLC) workflow using an integrated HPLC system. This system consists of a
dual-gradient pump, a UV/VIS detector, a column oven, and an autosampler capable of
both sample injection and fraction collection. First, the HCC is injected onto the
POROS
®
A Protein A Affinity column and IgG fractions are collected by the autosampler.
Subsequently, the IgG fractions are injected separately onto Thermo Scientific MAbPac
SEC-1 and MAbPac
™
SCX-10 columns for further analysis. The MAbPac SCX-10, 3 µm
column was recently introduced in 4 × 50 mm format for high-throughput MAb variant
analysis. This column delivers high resolution separation with a shorter run time using
either salt or pH gradients. Incorporating this column into the workflow, we completed
affinity purification, SEC and charge variant analyses in less than one hour.
Furthermore, the fractions collected off the MAbPac SCX-10 column were analyzed by
mass spectrometry (MS), and intact mass information of the MAbs demonstrated the
presence of lysine variants.
Methods
Harvest Cell Culture
MAb HCC was a gift from a local biotech company. The HCC was filtered through a
0.22 µm membrane prior to sample injection.
Columns
§
MAbPac SCX-10, 3 µm, 4 × 50 mm (P/N 077907)
§
MAbPac SCX-10, 10 µm, 4 × 250 mm (P/N 074625)
§
MAbPac SEC-1, 4 × 300 mm (P/N 074696)
§
POROS A Protein A Affinity 20 µm Column, PEEK
™
, 4.6 mm x 50 mm, 0.8 ml
(P/N 1-5022-24)
Liquid Chromatography System
HPLC experiments were carried out using an UltiMate™ 3000 x2 Dual Titanium System
equipped with SRD-3600 Integrated Solvent and Degasser Rack,
DGP-3600BM x 2 Dual-Gradient Micro Pump, TCC-3000SD Thermostated Column
Compartment with two biocompatible 10-port valves, WPS-3000T(B)FC Analytical Dual-
Valve Wellplate Sampler, VWD-3400RS Four Channel Variable Wavelength Detector
equipped with a Micro Flow Cell, and PCM-3000 pH and Conductivity Monitor.
pH-Based Ion-Exchange Chromatography
In a scale-up purification, 1 mL of IgG was purified from the 3.8 mL HCC using Thermo
Scientific Pierce Protein A Plus Agarose beads (P/N 22810). The protein concentration
was determined at ~ 0.5 mg/mL. Approximately 100 µL of the purified IgG was injected
onto a MAbPac SCX-10, 10 µm, 4 × 250 mm column and separated via pH gradient
from pH 7.8 to pH 10.8. Mobile phase buffers contained 9.6 mM Tris, 11 mM imidazole,
and 6 mM piperazine with pH values of either 6.8 (Buffer A) or 10.8 (Buffer B). The
column was equilibrated at 40% B. Three min after sample injection, a linear gradient
was run from 40% to 100% B in 30 min. Fractions were collected onto a 96-wellplate at
a rate of 0.2 min per fraction from 17 to 27 min.
FIGURE 1. Fluidic configuration of th
the wellplate bio-inert autosampler
2D-LC Workflow
The workflow and LC conditions for aut
§
Injection of 50 µL of an unpurified H
§
A first-dimension (
1
D ) affinity chrom
2.0 mL/min using the following step
–
A column wash/equilibration step
–
An elution step of 1 min
–
Automated time-based fraction c
–
Protein A column is regenerated
for the next analysis
Total analysis time is approximately
§
A second-dimension (
2
D) separatio
following:
–
SEC separation at a flow rate of
–
Strong cation-exchange separati
gradient
LC-MS
HPLC: Thermo Scientific ProSwift RP-
(1.0 mm i.d. × 5 cm) was used for desa
(Solvent A) and 0.1% formic acid in ac
ºC during analysis. Flow rate was 100
from 10% B to 95% B was used to elut
MS: Using Q Exactive
™
instruments, in
molecular mass. The spray voltage wa
Auxiliary gas flow rate was set at 5. Ca
was set at 55. In-source CID was set a
target was set at 3E6 for full scan. Max
Data Processing: Full MS spectra of int
Scientific Protein Deconvolution softwa
molecular mass determination. Mass s
averaging spectra across the most abu
The averaged spectra were subsequen
2000 to 4000
m/z
, an output mass ran
150000 Da, and minimum of at least 8
spectrum to produce a deconvoluted p
