Significance of the Topic
The detailed characterization of biotherapeutics is critical for ensuring product safety, efficacy, and stability.
Advances in liquid chromatography are essential to resolve complex protein structures, detect low‐level impurities, and support regulatory requirements in biopharma QC and R&D.
Objectives and Study Overview
This evaluation examines the performance and flexibility of the ACQUITY UPLC H-Class PLUS Bio System for high-resolution bioseparations.
The primary goals are to demonstrate:
- Compatibility with multiple chromatographic modes (IEX, SEC, HILIC, HIC, RP, MDLC)
- Enhanced separation resolution and sensitivity using UPLC technology
- Robust operation with bio-inert flow paths under demanding buffer conditions
Methodology and Instrumentation
The system integrates UPLC-optimized small-particle columns with precise quaternary or binary solvent delivery and a flow-through-needle injector to minimize carryover.
Key methodological features include:
- Auto•Blend Plus for on-demand mobile phase generation by pH, ionic strength, or organic content
- Gradient SmartStart for seamless method transfer from legacy LC platforms
- Low system dispersion (band spread <10 µL) to preserve chromatographic resolution
Instrumentation
- ACQUITY UPLC H-Class PLUS Bio System with bio-inert, iron-free flow path
- BioQuaternary and bioBinary Solvent Managers (up to 4 solvents, optional 6-solvent select valve)
- Flow-through-needle injector (0.1–1000 µL) with ultra-low carryover
- Sample Organizer for vials and ANSI plates; temperature-controlled fraction collector
- Detectors: PDA, UV/Vis, fluorescence, refractive index, ELSD, MS
- Support for analytical columns up to 300 mm with eCord Intelligent Chip Temperature Control
Key Results and Discussion
1. Peptide Mapping and Protein Separations
• UPLC BEH Peptide columns delivered five-run overlay of infliximab with highly reproducible retention times and peak shapes.
2. Size Exclusion Chromatography (SEC)
• ACQUITY UPLC BEH SEC (450 Å, 2.5 µm) resolved monomer and dimer species with Rs = 2.49 under 0.35 mL/min, improving on conventional HPLC (Rs = 1.42).
3. Hydrophobic Interaction Chromatography (HIC)
• Six-protein standard separated on Protein-Pak Hi Res HIC column using high ionic strength mobile phases, demonstrating robust retention and peak symmetry.
4. Hydrophilic Interaction Chromatography (HILIC)
• RapiFluor-MS-labeled glycans separated in under 30 min on UPLC BEH Amide 1.7 µm column vs. 120 min on HPLC, showing superior speed and resolution.
Benefits and Practical Applications
The system supports a broad spectrum of biopharma assays:
- Peptide mapping and intact protein analysis
- Aggregate and fragment detection by SEC
- Charge variant profiling by IEX
- Glycan and glycopeptide analysis by HILIC
- ADC and difficult hydrophobic separations by HIC
- Multidimensional LC workflows for enhanced characterization depth
Future Trends and Potential Applications
Emerging developments may include:
- Deeper integration with high-resolution mass spectrometry and multidimensional platforms
- Automated method scouting using AI-driven solvent and column selection
- Expanded bio-inert materials for challenging buffers and denaturants
- High-throughput QC workflows for biosimilar and gene therapy products
Conclusion
The ACQUITY UPLC H-Class PLUS Bio System offers unparalleled resolution, flexibility, and robustness for modern biotherapeutic analyses.
Its combination of low-dispersion flow paths, on-demand mobile phase blending, and versatile detection addresses critical QC and R&D needs while facilitating rapid method transfer and development.
Content was automatically generated from an orignal PDF document using AI and may contain inaccuracies.
ACQUITY UPLC
H-Class PLUS Bio
FOR YOUR MOST CHALLENGING BIOSEPARATIONS
www.waters.com/hclassbio
With its flow-through-needle injector and
robust, quaternary or binary solvent delivery
system, the ACQUITY UPLC H-Class PLUS
Bio System has been specifically engineered
with a bio-inert flow path and Auto•Blend™
Plus technology to address your most
challenging bioseparations. The complete
flow path is engineered with iron-free,
bio-inert materials, helping your laboratory
extend the lifetime of your instrument
and maintain optimal bioseparations
performance.
Analytical laboratories around the world are leveraging the advanced
resolution capabilities of the ACQUITY UPLC H-Class PLUS Bio System.
■
Reversed-phase for peptide mapping and protein separations
■
SEC for detecting clips and aggregates
■
IEX for charge variants
■
HILIC for released glycan analysis and
glycoprotein/glycopeptide separations
■
HIC for ADC analysis
■
Multidimensional liquid chromatography (MDLC)
■
Reversed-phase for amino acid analysis
Increasing complexity of modern biotherapeutic characterization, monitoring, and quality control assays
require even more demanding analytical techniques to accurately define and assess critical quality
control attributes (CQAs) related to product safety, stability, and efficacy.
The ACQUITY™ UPLC™ H-Class PLUS Bio System provides the same high resolution, high sensitivity
separations you have come to expect from UPLC Technology, with the flexibility and ruggedness
to run all the chromatography modes you require – ion exchange (IEX), size exclusion (SEC),
hydrophilic interaction (HILIC), hydrophobic interaction (HIC), or reversed-phase – all on one system.
PURPOSE BUILT TO CHARACTERIZE THE COMPLEX
Low dispersion
True UPLC performance with band
spread of less than 10 µL for highest
chromatographic resolution.
Wide range of
detection capabilities
UPLC-optimized detectors to match
your application needs, including
photodiode array, UV/Vis, fluorescence,
refractive index, evaporative light
scattering, and mass detection.
Fraction collection
Analytical scale, temperature controlled
fraction collection from your high efficiency
UPLC separations to enable additional
targeted studies.
Versatile column management
Support for analytical LC column
dimensions up to 300 mm with
capacity to automatically select
between as many as six analytical
columns. Equipped with independently
controlled temperature zones, active
pre-heating, and eCord™ Intelligent
Chip Technology for Waters™ columns.
Bio-inert flow-through-
needle injector
Volume range of 0.1 to 1000 µL
and ultra-low sample carryover
performance compatible with your most
sensitive LC and LC-MS applications.
Auto•Blend Plus technology
Program gradients directly in terms of pH,
molarity, or organic composition to minimize
manual mobile phase preparation, reduce
human error, and accelerate method robustness
testing for chromatographic methods.
bioQuaternary or bioBinary
solvent management
Precise and accurate blending of up to
four solvents with automated solvent
compressibility compensation available
with the bioQuaternary solvent manager.
Increase method development flexibility with
an optional, integrated solvent select valve,
providing access to six additional solvents.
Deliver precise and accurate binary solvent
blending with the bioBinary solvent manager
at pressures up to 15,000 psi.
Flexible sample support
Accepts either vials or ANSI well plate
formats. Extend capacity for high
throughput and open access environments
with the optional Sample Organizer.
Gradient SmartStart
Easily adjust for system volume
differences when transferring methods
from an alternative LC system without
alteration to the method table.
HIGHEST RESOLUTION OF ANY BIO-INERT QUATERNARY OR BINARY LC SYSTEM
A BETTER PERSPECTIVE FOR YOUR BIOSEPARATIONS
Leverage the benefits of UPLC Technology to improve your HPLC methods. By scaling your bioseparations methods
to UPLC, your assays will benefit from the combination of increased speed, resolution, and sensitivity that comes with
modern small particle chemistries.
Purpose built for biopharmaceutical
characterization, the ACQUITY UPLC
H-Class PLUS Bio System leverages high
resolution column technology, advanced
detection, and intelligent informatics to
increase the information you receive from
each and every sample.
With exceptional quaternary or binary gradient
precision and accuracy formation, reliable and
robust separations can be achieved for your
most challenging bioseparations, day-after-
day and year-after-year.
UPLC and ACQUITY UPLC BEH Peptide Columns deliver unparalleled resolution and
reproducibility in peptide mapping, here of infliximab (five overlayed chromatograms).
AU
0.00
0.02
0.04
0.06
0.08
0.10
0.12
Retention time (min)
10
12
14
16
18
20
22
24
26
28
30
32
34
36
38
40
42
0.00
0.03
0.06
0.09
0.12
0.0
5.0
10.0
15.0
Time (min)
UV absorbance (220 nm
)
Hydrophobic interaction chromatography separation of six proteins (HIC Protein Standard) by the Protein-Pak™ Hi Res
HIC 2.5 µm, 4.6 x 100 mm Column. Mobile phase A: 2M (NH4)2SO4 in 50 mM Na2HPO4 at pH 6.9; Mobile phase B:
50 mM Na2HPO4/Na2HPO4 at pH 6.9; Flow rate: 0.6 mL/min; Gradient: 0–100% B in 15 minutes.
RELIABLE OPERATION UNDER DEMANDING CONDITIONS
Assays utilizing IEX, SEC, and HIC modes of chromatography typically utilize high ionic strength mobile phases
containing denaturing agents. The ACQUITY UPLC H-Class PLUS Bio System’s bio-inert flow path is made of
non-stainless steel and iron-free materials to minimize undesirable protein interactions and maximize system
robustness. The result: a system that is ideally suited for routine, trouble-free operation with maximum recovery
of biomolecules and consistently high system performance.
ON-DEMAND MOBILE PHASE CREATION
The bioQuaternary Solvent Manager (bioQSM) automatically blends mobile phase combinations and
gradients on demand, enabling you to obtain the optimum retention and selectivity requirements for
your analysis. Designed with bio-inert materials, the bioQSM accommodates the aqueous,
high ionic strength mobile phases and organic solvents used in biopharmaceutical applications.
Auto•Blend Plus technology automatically blends your mobile phase to a specified pH and ionic
strength or organic modifier percentage, for an infinite set of analytical conditions. By blending
online to the desired pH and ionic strength conditions, you can significantly reduce the number
of buffers you need to prepare for method development and routine everyday use.
ACQUITY UPLC BEH SEC, 450Å (2.5 µm, 4.6 x 300 mm)
Flow rate = 0.35 mL/min
Injection volume = 5 µL
XBridge™ Protein BEH SEC, 450Å (3.5 µm, 7.8 x 300 mm)
Flow rate = 1.0 mL/min
Injection volume = 14 µL
Monomer
Dimer
Rs = 2.49
Monomer
Dimer
Rs = 1.42
0.0
0.0200
0.0050
0.0100
0.0150
0.0250
0.0200
0.0050
0.0100
0.0150
0.0250
By combining 450Å
sub-3-µm packing materials
with a low dispersion ACQUITY
UPLC H-Class PLUS Bio System,
separations with improved
resolution and high throughput
of SEC-UPLC can be realized
for highly aggregated proteins
with molecular weights of up to
approximately 2000 KDa.
ACCELERATE YOUR PRODUCTIVITY
Adopting a proven analytical technology that improves laboratory efficiency is advantageous for
supporting your new projects and products. Designed to support your HPLC, UHPLC, and UPLC
methods, the ACQUITY UPLC H-Class PLUS Bio System is the ideal solution to address legacy
methods, while enabling the transition to true UPLC performance when you are ready.
■
Reproduce established HPLC methods while enabling seamless transfer to UPLC
■
Faster methods development with expanded solvent and column capacity and fully scalable
column chemistries
■
Get results faster with high pressure mixing binary solvent management
Comparison of UPLC and HPLC HILIC separations of RapiFluor-MS™-labeled N-glycans from the Waters Glycan
Performance Test Standard diluted in water to a concentration of 20 pmole/µL. Injection volumes of 1.2 and 3.7 µL
for UPLC and HPLC analyses.
30.00
10.00
12.00
14.00
16.00
18.00
20.00
22.00
24.00
26.00
28.00
64.00
24.00
28.00
32.00
36.00
40.00
44.00
48.00
52.00
56.00
60.00
Alliance™ HPLC System
XBridge Glycan BEH Amide
XP, 130Å, 2.5 µm,
3.0 x 225 mm total length (150 mm + 75 mm)
ACQUITY UPLC H-Class PLUS Bio System
ACQUITY UPLC Glycan BEH Amide, 130Å, 1.7 µm,
2.1 x 150 mm
Total Run Time = 55 min
Total Run Time = 121.3 min
Fluorescence (relative)
Fluorescence (relative)
Time (min)
1
2
3
4 5
6
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8
9 10
11
12
13
14 15
16
17
18
19
20 21
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Waters, The Science of What’s Possible, Alliance, ACQUITY, Auto•Blend, eCord,
Protein-Pak, Rapi Fluor-MS, UPLC, and XBridge are trademarks of Waters Corporation.
All other trademarks are the property of their respective owners.
©2020 Waters Corporation. Printed in the U.S.A. May 2020 720005992EN LM-PDF
www.waters.com/hclassbio
Waters Corporation
34 Maple Street
Milford, MA 01757 U.S.A.
Tel.: 1 508 478 2000
Fax: 1 508 872 1990
www.waters.com
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