Vol20 Issue3 TheColumn March2024 Europe

Cover Story
21 H
ow Analytics & Mass Spec Became the Driving
Force Behind Biotherapeutic Drug Development
Kelly Broster, Thermo Fisher Scientific, Waltham, Massachusetts, USA
This
article explores the key challenges analytics and mass
spectrometry solve within the biopharmaceutical industry.
Features
5 The State of UK Separation Science
Tony
Edge and Paul Ferguson, The Chromatographic Society (ChromSoc),
United Kingdom
06 March 2024 Volume 20 Issue 3 What
is the state of separation science in UK and what steps can be taken
to promote the importance of the subject?
9 N
ative Anion Exchange Chromatography Coupled to Mass
Spectrometry for the Charge Variant Analysis of IgG4-based
Monoclonal Antibodies
Ann Marie Rojahn and Dr. Daniel Eßer, YMC Europe, Dinslaken, Germany
The
advantages of a native anion exchange method coupled to mass
spectrometry for charge heterogeneity analysis of immunoglobulin G4
(IgG4)-based mAbs are described.
16 N
ovel Automated Method for Sample Preparation for
Peptide Mapping
Leslie Napoletano, Waters Corporation, Milford, Massachusetts, USA
This
article explores how automation can enhance laboratory productivity
and reduces errors in peptide mapping.
24 Rapid Method to Characterize Adeno-Associated Virus (AAV)

Therapies using SEC–FLD
Tran
Pham, Phenomenex, Torrance, California, USA
The
benefits of a fast and sensitive SEC technique incorporating a wide pore
stationary phase with fluorescence detection is described.
Regulars
2 News
In The Driving Seat 27

The latest research news and news in brief.
HPLC 2024 Preview
The importance of analytics and mass spectrometry Susan

A
Olesik, Program Chair, HPLC 2024
look at what’s in store for chromatographers who attend HPLC 2024 in
in biopharmaceutical drug development Denver, Colorado, USA from 20–25 July 2024.
News
From the CEO Profiling VOCs in Whisky

Welcome to the March issue of The Column. The first quarter of 2024 is certainly flying by
and I am sure everyone is looking forward to the clocks changing and enjoying a bit more
with GC×GC–MS
sunshine. Therapies using biopharmaceutical drugs represent some of the most imporant Researchers from Austria, Greece, and Italy conducted a study to analyze volatile organic
accomplishments in modern healthcare and analytical techniques are in some ways the compounds (VOCs) present in Irish and Scotch whiskys using solid-phase microextraction (SPME)
unsung heroes of modern healthcare, playing an important role throughout the drug Arrow with comprehensive two-dimensional gas chromatography coupled to mass spectrometry
development process. (GC×GC–MS) to examine the organoleptic characteristics that influence the taste of spirits. The
This is highlighted in our cover story where Kelly Broster from Thermo Fisher Scientific study sourced whisky samples from Vienna’s local market, and the results were published in the
explores the important role that analytics (and mass spectrometry in particular) play in the Journal of Chromatography A (1).
development of biopharmaceiutical therapies. The researchers from Aristotle University of Thessaloniki in Thessaloniki, Greece, the
Adeno-associated virus (AAV) is a versatile viral vector technology that can be engineered University Campus Bio-Medico of Rome in Rome, Italy, the University of Messina in Messina,
for very specific functionality in gene therapy. Speed of analysis is obviously crucial in any Italy, and Vienna University of Technology in Vienna, Austria, purchased samples of Irish whisky,
stage of the drug development process, and Tran Pham from Phenomenex highlights a single malt Scotch whisky, and blended Scotch whisky from a local market in Vienna. Among
rapid size-exclusion chromatography (SEC) method incorporating a novel stationary phase the most abundant aromatic VOCs in these and other whiskys are esters, alcohols, aldehydes,
combined with fluorescence detection to analyse this important class of analytes. ketones, terpenes, and furanic and sulfur compounds.
Automation is a hot topic in all laboratories and sample preparation can often be the An SPME Arrow method was used to extract VOCs from the headspace of whisky samples to
bottleneck in any chromatographic method. Leslie Napoletano from the Waters Corporation concentrate the compounds of interest for subsequent analysis by GC×GC–MS. This combination
highlights the importance of automating and developing the sample preparation workflow offered a high degree of resolution in separating complex mixtures, allowing for the identification of
for peptide mapping. various aromatic VOCs found in different whiskys, including esters, alcohols, ketones, terpenes, and
Continuing the biopharmaceutical theme, Anne Marie Rojahn and Daniel Eßer from YMC sulfur compounds, according to the researchers.
Corporation highlight the advantages of native anion exchange chromatography for the charge The researchers concluded that the improved sensitivity and repeatability of the SPME Arrow
heterogeneity analysis of immunoglobin G4 (IgG4)-based monoclonal antibodies, another method compared to traditional techniques offers significant promise for applications in quality
important group of compounds that are being studied in this constantly evolving sector. control and authenticity assessment within the whisky industry to provide valuable insights into the
One of the highlights in the chromatography calendar this year is HPLC 2024, which takes composition and characteristics of distilled spirits. —Patrick Lavery
place in Denver, Colorado, USA from 20–25 July 2024. Program Chair Susan Olesik offers
a glimpse of what’s in store to entice you to register. The LCGC International team will be Reference
there to cover the event so please stop by our booth to let us know about any hot topics we 1. Ferracane, A.; Manousi, N.; Tranchida, P. Q.; et al. Exploring
Igor Normann - stock.adobe.com

the Volatile Profile of Whiskey Samples Using Solid-Phase
should be covering.
Microextraction Arrow and Comprehensive Two-Dimensional Gas
Mike Hennessy Jr., Chromatography–Mass Spectrometry. J. Chromatogr. A 2022,
President and CEO, MJH Life Sciences 1676, 463241. DOI: 10.1016/j.chroma.2022.463241
2 News 5 Edge and Ferguson 9 Eßer and Rojahn 16 Napoletano
2
21 Broster 24 Pham 27 HPLC 2024 Preview 29 Training and Events
The Column www.chromatographyonline.com News
New Tool Improves MS/MS Analysis

of Single Cell Proteomics Data
Phenomenex Appoints New CEO
Kaveh Kahen, PhD, has been appointed A study authored by Benjamin C. Orsburn, reducing analysis time (1). To their knowledge, medicines that detect protein biomarkers is
president of Phenomenex, the Torrance, a professor of pharmacology and molecular they said, no prior tools have been able to expected to intensify.
California-based separation science sciences at Johns Hopkins University in judge data quality in advance of processing. The researchers reported that when it
technology company, according to a press Baltimore, Maryland, and Conor Jenkins, Both the DIDAR GUI and DIDAR itself were came to assessment and filtering of single cell
release. Kahen succeeds Matt Turner, currently a PhD student at the University designed to be compatible with all modern proteomics data, the DIDAR script was able
who moved on from the Phenomenex of Maryland, attempted to improve upon computer operating systems. to flag entire liquid chromatography–mass
presidency to pursue other opportunities, assessment of quality prior to processing Single cell proteomics by mass spectrometry spectrometry (LC–MS) runs that experienced
the company said. of single cell proteomics data, describing a (which has been previously abbreviated in the technical issues, either to be further evaluated
Phenomenex operates within the Life “lightweight” script of fewer than 100 lines literature as SCoPE-MS) has been growing in or removed from analysis altogether (1). Peptide
Sciences group of Danaher Corporation, of code written in the programming language popularity, Orsburn and Jenkins said, because spectral match level data was reportedly lost
making this a homecoming of sorts Python (1). Using this, the authors sought to it allows existing technology to be reused during filtering, but this was found not to have
for Kahen. After obtaining his PhD in accelerate the quantitation of reporter ion in innovative ways (1). SCoPE-MS features a a major effect on the final data set that was
analytical chemistry from The George peaks within each tandem mass spectrometry carrier channel of isobarically labeled peptides, processed. In the future, the authors seek to
Washington University, he began his (MS/MS) spectrum in a given file. Python at a relatively higher concentration, for the cut down on the number of necessary MS/MS
professional career at Sciex, another is a simplified programming language that purpose of resuspending labeled peptides from scans to be collected by the chosen instrument
Danaher Life Sciences holding. provides developers with a set of libraries, single cells, thus leveraging the additive signal in this case, a trapped-ion mobility time-of-
Following his tenure at Sciex, Kahen held tools, and frameworks for building custom of the multiplexed peptide samples to amplify flight mass spectrometer. Real-time DIDAR
various leadership posts at PerkinElmer, software applications for data analysis, web the single cell peptide signals more effectively. filtering may facilitate this, they said, allowing
according to the release, and more recently development, and machine learning. Demand for techniques that deal with the instrument to sequence only MS/MS spectra
was CEO at Advion Interchim Scientific. Their report, published in the Journal of proteomics has increased over the last which possess a predetermined number of
Kahen designed and developed new the American Society for Mass Spectrometry, several years because of their roles in disease single cell reporter ions. —Patrick Lavery
technologies and instruments early in his refers to the script as the Diagnostic Ion Data diagnosis, particularly the early detection of
career, and later as an executive gained a Analysis Reduction (DIDAR) graphical user cancer. But proteomics-based solutions also Reference
reputation for driving above-market growth interface (GUI), which the authors said was reach farther than just cancer detection and 1. Jenkins, C.; Orsburn, B. C. Simple Tool for Rapidly
in the fields of life sciences, analytical capable of simply producing summary reports treatment; as the frequency of other chronic Assessing the Quality of Multiplexed Single Cell
testing, and laboratory equipment, which identify single cell samples that fail illnesses such as diabetes and heart disease Proteomics Data. J. Am. Soc. Mass Spectrom. 2023,
according to the release. —Patrick Lavery to pass a certain quality threshold, thereby increase worldwide, the need for customizable 34 (12), 2615–2619. DOI: 10.1021/jasms.3c00238
3
The Column www.chromatographyonline.com News
Peaks of the Month News In Brief

• LCGC Lifetime Achievement and Emerging Leader Awards: Wolfgang F. Lindner and Martina
Peppygraphics - stock.adobe.com
Catani are the winners of the 17th annual LCGC Lifetime Achievement and Emerging Leader in Peak Scientific Acquires Noblegen
Chromatography Awards, respectively. These awards honor the work of talented separation scientists Dusty TLP Ltd., the owner of Peak Scientific,
at different stages in their career. The awards were presented in February in an oral symposium at
has acquired Wirac Automation Ltd, a company
the Pittcon 2024 conference in San Diego, California. Read Here>>
that trades as Noblegen and specialises in the
design, development, and manufacture of
specialist gas and cryogenic solutions. This is
• Trends in Biopharmaceutical Analysis: Biopharmaceutical analysis is a rapidly evolving field that
the latest step in the Dusty TLP board’s vision
requires the development of new technologies and methods to keep pace with the increasing
TensorSpark - stock.adobe.com
complexity of biologics. One of the most promising areas of research is the use of single-cell omics to expand its portfolio and develop its range of
and microfluidic chips for the analysis of biopharmaceuticals. Read Here>> products to better serve customers around the
world, according to a press release issued by
Peak Scientific.
Noblegen will remain a separate brand
November/December 2023 | Volume 36 Number s10
www.chromatographyonline.com
• Advances in Biopharmaceutical Analysis Supplement: This special issue offers a comprehensive from Peak Scientific, and maintain its own
Advances in
Biopharmaceutical
Analysis
AN LCGC EUROPE SUPPLEMENT
overview of contemporary analytical trends and recent advances in biopharmaceutical analysis, identity. Noblegen will continue to operate
aiming to provide valuable insights into the analytical workflows and characterization strategies independently, with the added support of
associated with biopharmaceutical products and new modalities. Read Here>> the parent company. Paul Hudson, newly
appointed General Manager for Noblegen,
Find podcasts, webinars,
and expert interviews at
chromatographyonline.com
commented, “I am thrilled to take up this
exciting opportunity with the team at
Noblegen to continue bringing their existing
customers the excellent products and
• Biopharmaceutical Perspectives: This short tutorial article is to review the terms and official customer service they have come to expect
nomenclatures for size-exclusion separations and to provide some guidance and recommendations from Noblegen.”
Faiza - stock.adobe.com
for practicing chromatographers. The interconversion between the different metrics is explained and Bill Johnston, founder of Wirac Automation Ltd,
some examples are presented. Read Here>>
who will continue working at Noblegen in the role
of Technical Director, added, “I’d like to thank all of
our customers and suppliers who have been part
of the business’s success over the years and I firmly
believe that under the new ownership even greater
Like us Join us Follow Us successes are to be had in the years ahead.”
4
The State of UK
Separation Science
Tony Edge and Paul Ferguson, The Chromatographic Society (ChromSoc), United Kingdom
The scientific community relies heavily on the analytical sciences to

support new ideas and theories, with separation science being a key
component. However, the UK has seen a steady decline in this scientific
discipline relative to other parts of the globe. To address this, the learned
communities came together to discuss the current state of the nation and
to initiate a plan to address this.
Chromatography in the UK began in earnest inevitably impacts research-led teaching,

in the 1950s and saw the development of the which in turn impacts industry being able to
theory for partition chromatography by Martin recruit suitably qualified candidates. The list
and Synge, who were both awarded Nobel of pure chromatographers from the UK today
Prizes for their contributions to chemistry in is not so extensive and filled with many grey
1952. Since then, the UK has seen significant hairs. There is healthy research in many areas
contributions to the development of the that use chromatography; however, without
theory and practice of chromatography from the practice of good chromatography, these
homegrown world leaders. other disciplines will struggle to develop
Separation science in the UK has seen effectively. To address this, the Community
some incredible times and some incredible for Analytical Measurement Scientists (CAMS-
practitioners. It delivers a large component UK), the Royal Society of Chemistry (RSC)
of measurement science in the UK, and has Separation Science Group (SSG), and The
grown from a technique that was only used Chromatographic Society (ChromSoc) came
akkash jpg - stock.adobe.com
in one or two industries to one that is used together and organised a meeting to identify
very widely. However, the UK is no longer where separation science is currently in the UK,
recognised as an innovative research leader and to understand what the future needs of
on the global separation science stage, which industry and academia may be. The first half of
5
The Column www.chromatographyonline.com Edge and Ferguson
the meeting looked at the state of separation if there is time left to initiate change. He said
science in research, teaching, and in industry there was also a need for better collaboration
and the second half looked to answer three and networking, as well as a need for HILICON
key questions: consistent regulations and standards to govern iHILIC
®
1. What does industry need in terms of (a) the use of separation science.
Advancing HILIC Separations in UHPLC and HPLC
type and depth of separation science skills Langley also stated that there was a
from graduate and postgraduate recruits, need to better understand and adopt
and (b) separation science technology sustainability and green chemistry, giving
innovation to help it deliver on future the example of sub-2 µm particles and
ambitions and remain competitive? supercritical fluid chromatography (SFC) as
2. Where could UK Research and Innovation ways that chromatographers have had an ®
iHILIC -Fusion ®
iHILIC -Fusion(+)
Silica based Silica based
(UKRI) investment in separation science impact, albeit not by design. He stressed
have the most significant impact in the that it was important to understand where
next 10 years with respect to societal UK separation science fits into the general
challenges and strategic priorities? landscape in the UK, and to also look at other
3. If UKRI were to fund research and countries to see what has happened there.
innovation in separation sciences, which Langley then summarised his presentation ®
iHILIC -Fusion(P)
®
iHILIC -(P) Classic
Polymer based Polymer based
approaches would be the most impactful stating that it was necessary to identify what
and accessible for the community to the destination is for this journey, and also to
engage and co-invest in? identify who to target so that the maximum Zwitterionic charge-modulated amide/diol HILIC
columns
The one day event was kicked off by Melissa return could be sought.
Complementary selectivities for separation of polar
Hanna-Brown, chair of the executive board for compounds
CAMS-UK. Session Two: Do We Still Need Excellent durability and ultra-low column bleeding
Separation Science in the UK? Universal for LC-MS based ”Omics” studies and
other applications
Session One: Setting the Scene The next presenter was Paul Ferguson of
iHILIC®-Fusion and iHILIC®-Fusion(+):
John Langley of the University of Southampton AstraZeneca and ChromSoc, who initially 1.8, 3.5, and 5 µm; pH 2-8
and the RSC SSG was the first speaker and posed the provocative question “Are ® ®
iHILIC -Fusion(P) and iHILIC -(P) Classic:
asked the audience if there is a problem, and separation science specialists relevant in 5 µm; pH 1-10
why should we be looking for a solution now? today’s analytical landscape?” He asked a
He stated that the community needed to series of questions to the audience regarding HILICON AB
understand how separation scientists ended the relevancy of separation science in industry Mail: info@hilicon.com | Web: www.hilicon.com
®
©2024 HILICON AB. All rights reserved. | iHILIC is a registered trademark of HILICON AB, Sweden
up in their current situation, and determine and academia and if the technology could
6
be simply outsourced. Ferguson finished Session Four: Separation Science in

by saying that in his experience, it was very the UK Academic System
difficult to recruit separation scientists with the The next presentation was delivered by
appropriate level of experience, that there is an Andrew Pitt of the University of Manchester
ever-increasing need for separation scientists and focussed on separation science in UK
in the UK, and that industry also had to play academia. Pitt started the presentation off by
a role in terms of the retention of separation giving anecdotal evidence that teaching and
scientists, ensuring that better career paths research funding was limited in universities.
were developed. He gave evidence that there was no depth in
the chromatography that is taught, using a
Session Three: Where Are figure of 12 h (maximum) and 3 h (average)
the Needs in the Future? for a three-year degree course, but suggested
The next presentation was delivered by that the ubiquitous nature of chromatography - Screening - - Optimization - - Validation -
Melissa Hanna-Brown of Pfizer and CAMS- in industry would lead it to be taught in a

UK, who focused on three main future wider range of subject areas such as physics,
needs: speed, resolution and, increasingly, engineering and materials science. The greater
an environmental focus. She spoke about breadth of departments would also help to
the general drive to increase the level of ensure that consistency across the topics
automation, portability and miniaturization was also maintained. Pitt also stated that the
within the laboratory, with focuses on practical use of chromatography was very
sample preparation and increased use of limited and there tended to be a focus on
predictive approaches to bring efficiencies the output related to the sample, and not
to all aspects of assay development. The to the actual quality of the chromatogram. The fine art of method development
requirement for an enhanced selectivity From the teaching, Pitt moved onto the levels
Get a perfect complement for the Nexera Method Optimization:
space with respect to stationary phase of funding for analytical science from UKRI. Scouting System: LabSolutions MD, the first software Design of experiments offers accurate retention
that integrates all phases of HPLC method develop modelling, even with limited input data, to identify
chemistry and more innovation with novel The data showed that separation science was ment. Thanks to an Analytical Quality by Design the most robust analytical conditions.
approach, processes become easier, safer and more
materials including tunable phases was also not a key component in any recent research efficient, thus saving time and minimizing errors. Validation:
Benefit from automated batch creation, statistical
addressed. In summing up, Hanna-Brown funding, and even the strategic plan for Screening: evaluation and intuitive reporting, all in a LabSolutions
The software offers automated screening of various database, ensuring data integrity.
spoke about the increasing body of work analytical science only had one mention of method parameters, according to a chosen multi
factorial experimental design.
in the literature on assessing ‘greenness’ of the technique. However, it has since been
methods and guidance for developing green determined that separation science is a critical www.shimadzu.eu/fine-art
Learn more about
LabSolutions MD!
sample prep and separation methods. skill set that is in decline in UK research.
SHIMAEU-2022-023_LabSolutionsMD_Ad_184x211_RZ_mali.indd 1 13.07.22 16:28
7
Session Five: Recruiting Separation one of the foremost academic separation and be more vocal about their impact challenges to industry and academia.
Science in the UK scientists in the world, who delivered a and contribution to advancement in other There was time spent analysing what was
Simon England from recruitment company presentation on his reflection on separation scientific areas. not working, but substantially more time
VRS spoke, stating that recruiting separation science in the Netherlands and the formation and effort was spent identifying possible
scientists at a graduate level was the easiest of the Top Institute for Comprehensive Session Seven: Separation Science solutions, and ultimately what separation
as there were plenty of roles to fill, but as Analytical Science and Technology (TI- in Belgium science could do to better the UK economy.
more experience is required, it gets harder COAST), the body on which CAMS-UK The final presenter of the first session was This included highlighting the potential for
to recruit. In general, separation scientists was modelled. Schoenmakers started his Deirdre Cabooter of the University of Leuven, better networking, improved and more
earn less money than other chemists, with presentation by stating that the Netherlands Belgium. The first aspect that she approached dedicated training, and also application
the example of entrance-level computational was small, with many of the separation was the dispersion of universities around the driven research and development to
scientists who earn more than graduate science research groups concentrated in Flanders region of Belgium. She then went support the new challenges that are being
chemists starting roles. Brexit was another Amsterdam. He then went on to describe through the universities and stated that all of set in the many application areas in which
challenge since barriers, specifically visas, TI-COAST and funding mechanisms in The these universities are very close to each other, chromatography is used.
have been put in place restricting movement Netherlands. Schoenmakers did state that as well as having a range of large organisations The organisers are planing to assemble
from Europe, although England did note that getting funding for pure fundamental research including Janssen, Pfizer, Organon, P&G, MSD, the feedback from the participants on
industrial placement and hands-on research is difficult; however, projects would normally GSK and UCB in close proximity. In fact, all of the day with a view to preparing a ‘White
projects added a lot to the attractiveness of a be performed in such a way as to ensure that these organisations are within a 50 km radius. Paper’ outlining the situation for this key
graduate candidate for a separation scientist some fundamental research was undertaken This helps the establishment of collaborations scientific discipline in the UK. This paper
role—however, there were not enough of as part of the project. and setting up industrial placements, and also will be shared with UKRI as the first step in
these being offered. In terms of teaching, in Schoenmakers’ ensures that postgraduates find very well-paid highlighting the importance of separation
opinion 12 h was not enough to teach jobs easily, whilst supporting local industries. science to UK industry, and outline
Session Six: Reflection on Separation separation science, and at Amsterdam Cabooter reviewed research funding opportunities to address the current malaise.
Science in the Netherlands and the they do typically 70 h. He suggested that mechanisms which were not so different from
Formation of TI-COAST universities should advertise the number and the UK, but it was evident that Belgium had Tony Edge is President of The
The initial set of presenters highlighted some types of jobs in industries to encourage more greater success in accessing funding for the Chromatographic Society (ChromSoc).
of the challenges and possible solutions, students to take up chromatography. In terms separation science, along with the potential Paul Ferguson is Honorary Secretary of
however the last two speakers came from of access to instrumentation, he said the access to EU Horizon funding as well. ChromSoc.
environments where these challenges had administrators were more expensive than the
been successfully addressed, and they offered instrumentation, and that he focussed on the Conclusions
some suggestions as to possible ways forward. knowledge that is not available in Google. The morning session highlighted the many E-mail: enquiries@chromsoc.com
.com
The first speaker was Peter Schoenmakers of Schoenmakers also said it was important that challenges that separation scientists face, Website: www.chromsoc.com
www.com
the University of Amsterdam, The Netherlands, separation scientists stop being so modest, as well as the consequences of these
8
Native Anion Exchange
Chromatography Coupled
to Mass Spectrometry
for the Charge Variant
Analysis of IgG4-Based
Monoclonal Antibodies
Ann Marie Rojahn and Dr. Daniel Eßer, YMC Europe, Dinslaken, Germany
Ion exchange chromatography is a standard method for characterising

monoclonal antibodies (mAbs) and ensuring their efficacy. This article
discusses the advantages of a native anion exchange method coupled to
mass spectrometry for charge heterogeneity analysis of immunoglobulin
G4 (IgG4)-based mAbs, currently being studied more intensively. It also
explains why cation exchange chromatography is better suited for the more
common IgG1-based mAbs, but less suitable for the IgG4-type mAbs.
Monoclonal antibodies (mAbs) are of different haematologic, immunologic,

immunologically active proteins, usually oncologic and infectious diseases.
based on immunoglobulin G (IgG) A wide variety of therapeutic antibodies are
molecules. mAbs target certain antigens available on the market, and several hundreds
that are found on, for example, cancer cells more are in research and development (2). To
Naeblys - stock.adobe.com
(1). This stimulates the immune system to ensure the efficacy of mAbs, quality attributes
attack those targets. mAbs are becoming have to be strictly controlled. These include
increasingly important for the treatment charge heterogeneity, which usually arises
9
The Column www.chromatographyonline.com Eßer and Rojahn
Figure 1: Native AEX–MS charge variant analysis of three different IgG4-based mAbs and
the IgG1-based NISTmAb, shown at basic (B) and acidic (A) variant peaks as well as the main
species (M), 5 μg injection using a non-porous BioPro IEX QF column (2).
Figure 2: Comparison of (a) CEX-TIC and (b) AEX-TIC of an IgG4 mAb (mAb-IV, pI=6.8) using a
strong CEX column, BioPro IEX SF, and the strong AEX column, BioPro IEX QF (2).
from post-translational modifications. Ion available mAbs. They are often IgG1-based
exchange chromatography (IEX) and capillary and possess a high isoelectric point (pI)
electrophoresis (CE) are commonly used to of usually ≥ 8 (3). Therefore, CEX is the
characterize the overall charge heterogeneity. traditional approach; also, coupling of CEX to
Cation exchange chromatography (CEX) mass spectrometry (MS) has been successfully
is an excellent method of characterizing the described (4). In contrast, anion exchange
charge heterogeneity for most commercially chromatography (AEX) has only been used for
10
Figure 3: Native AEX–MS analysis of mAb-II (left) and mAb-V (right) before (blue) and after PNGase well as the NISTmAb (pI=9.2) were analysed
F-treatment (black) (2).
(Table 1) using a strong anion exchange column
(SAX) with non-porous particles.
Experimental
Native AEX–MS Method Conditions: The
combination of AEX and MS requires a special
setup in which a stainless-steel T-piece after
the column divides the flow. Most of the
flow is directed to the UV detector, while the
remaining sub-microlitre per minute flow is
Figure 4: Native AEX–MS analysis of the PNGase F-treated and IdeS digested mAb-II Fc fragments directed to the nanoelectrospray ionization
at different temperatures: 25 °C (blue), 35 °C (orange) and 45 °C (red) (2). mass spectrometer (NSI-MS). NSI is used
because it can tolerate high salt concentrations
of up to 600 mM ammonium acetate. To
AZURA® P 8.1L –
further improve the spray stability, isopropanol High Performance
is used as a dopant, modified desolvation gas. UHPLC Pump
In addition to the MS detection, the general
method conditions were kept constant as • Increase sensitivity with ultra-low
dispersion
follows: Flow rate 0.4 mL/min and 10 µg mAb
• Ideal for MS applications
sample injection unless stated otherwise. A
• Performance and reproducibility
non-porous 100 × 4.6 mm, 5 μm strong AEX
without sacrificing robustness
column was used (BioPro IEX QF, YMC). A salt
• Unsurpassed reliability made in
gradient from 10 mM ammonium acetate (pH Germany
6.7 unadjusted) (A) to 300 mM ammonium
visit us at
acetate (B) (pH 6.8 unadjusted) is used to
Please
relatively acidic proteins such as human serum The successful application of an AEX method analyse IgG4-based mAb samples (in-house
albumin (5) or ovalbumin (6). However, for for charge heterogeneity analysis of IgG4-based mAbs from Regeneron).
IgG4-based mAbs, which are becoming more mAbs coupled to MS was achieved by Liu and The gradient started at 0 %B and was held think LC. think KNAUER.
important as therapeutic candidates, AEX colleagues from Regeneron (7). Their approach for 2 min; the ratio of B was then increased
Find out more at
may be an alternative approach. They possess is discussed here: Five different IgG4-based to 100% in 16 min and again held for 4 min. www.knauer.net/UHPLC
a pI < 8 and therefore CEX is less suitable (3). mAbs with different pIs (between 6.1–7.3) as The temperature was set to 45 °C, as a result
11
Figure 5: AEX–MS analysis of the PNGase F and IdeS-treated mAb-V Fc fragments (a) without
stress T=0, and (b) thermally stressed (T=6M @ 25 °C) (2).
Make Critical Decisions Faster
with the Next Generation of SIFT-MS
Next Gen
SIFT-MS
Chromatography-free,
real-time solutions for
volatile compound analysis:
of a previous temperature screening using an the IgG1-based NISTmAb. Here, only 5 µg

One configuration for
IgG4-based mAb at 25 °C, 35 °C and 45 °C. mAb sample is injected. It is shown that the
multiple, diverse analyses
Significant improvement of the variant separation AEX–MS method is suitable for IgG4-based
as well as sharper peaks was obtained at the mAbs with moderate pIs, but not for IgG1- Syft Tracer
No column changeover or
highest temperature (not shown). based mAbs with higher pIs. The separation other chromatographic
delays
Native CEX–MS Method Conditions: improves as the pI gets lower. mAb-III has a pI
mAb-IV with a pI of 6.8 was used to compare of 7.3, which is higher than the mobile phase Next Gen Solution Analyze >200 samples /
AEX–MS and CEX–MS. For the CEX analysis the pH, but sufficient separation still occurs. This for 21 CFR Part 11 Compliant Workflows
day with headspace
same chromatographic conditions were applied suggests that it is the surface charge rather automation
as for AEX except for the column and eluents. than the intrinsic charge that causes the AEX- Easy to use & interpret
A non-porous 100 × 4.6 mm, 5 μm strong based separation. In general, the acidic variants data
CEX column (BioPro IEX SF, YMC) was used. separated correlate with those commonly
Designed for use in the
The eluents used for CEX were the following: observed by CEX such as deamidation,
field, mobile settings, or in
(A) 20 mM ammonium acetate, pH adjusted glycation and sialic acid (Neu5Ac)-containing the lab
to 5.6 with 20 mM acetic acid and (B) 150 mM species. Deamidated variants were found
ammonium acetate (pH 6.8). in several peaks. The additional abundant
peak A1 of mAb-I contains deamidation that
Analysis of mAbs Using Native correlates with a known deamidation site in its
AEX–MS Conditions complementary determining regions (CDR). Simply. Faster.
Syft Tracer
These conditions were applied to various However, the other mAbs tested have no Pharm11
IgG4-based mAbs (Figure 1) as well as to deamidation sites in their CDRs, so these two
12
Table 1: Evaluated mAbs in this study. mAbs contain an Asn residue in the Fc region Tris-HCl buffer (pH 7.5) at 37 °C for 90 min to
mAb Type pI of each of the two heavy chains, up to two release the Fc and F(ab’)2 subunits. Since the
mAb-I IgG4 6.1 Asn to Asp conversions can be expected. pI of F(ab’)2 fragments is relatively high, these
Therefore, the mAbs were treated with fragments are poorly retained and are not
mAb-II IgG4 6.6
PNGase F at 1 IUB milliunit per 10 µg sample taken into account further.
mAb-III IgG4 7.3
in 100 mM Tris-HCl buffer (pH 7.5) at 45 °C In contrast to previous results, a lower
mAb-IV IgG4 6.8
for 1 h. temperature of 25 °C showed improved peak
mAb-V IgG4 6.9
Figure 3 shows the differences between the shape and charge variant separation for the
NISTmAb IgG1 9.2 untreated and the PNGase F-treated mAbs Fc fragment analysis compared to analysis at
mAb-II (pI=6.6) and mAb-V (pI=6.9). The higher temperatures, as shown in Figure 4
acidic peaks are probably due to site-specific including partially (B1) and non-glycosylated PNGase F-treatment decreased the pI to 6.4 for mAb-II. Four basic and two acidic variants
deamidation in the fragment crystallizable mAb (B2) species and 1 (B3) or 2 unprocessed for mAb-II and to 6.6 for mAb-V. Due to the can be identified. Both the main peak as well
(Fc) region. The basic variants observed can C-term K (B4) in addition to two acidic peaks. reduced pI, the overall retention as well as as the B1 peak show tailing shoulder peaks,
be identified as unprocessed C-terminal lysine However, A1 consists of a deamidated and the variant separation and peak sharpness are with identical mass to the corresponding peak,
(C-term K) and mAb species with different glycated variant and A2 contains another improved for both mAbs. After the PNGase which could be conformational isomers.
numbers of Fc N-glycans. In addition, the deamidated species. The use of AEX–MS F-treatment of mAb-II, the retention time of In addition to the NG B3 and the PG B1
glycoforms Man5/Man5 with unprocessed probably provides the possibility to separate the FG main species increases by about 1 min peak, B2 is identified as a fully glycosylated
C-term K and G0F/G0F-GlcNAc are observed site-specific deamidation variants. The overall due to the elevated acidity induced by the now species with one unprocessed C-term K while
for mAb-I. mAb-II demonstrates that this separation using AEX–MS is better as the two Asp residues. The retention time of the B4 is identified as a partially glycosylated
AEX–MS method is very sensitive to the method provides sharper peaks and additional PG species B1 shifts by only about 0.5 min, species with one unprocessed C-term K. These
macroheterogeneity of Fc N-glycosylation. The information about the basic variants. while the retention time of the NG species B2 findings were confirmed by peptide mapping.
main fully glycosylated (FG) species is separated remains unchanged. In the case of mAb-V, an
from the partially glycosylated (PG) peak B1 and Further Improvement of the Basic additional minor variant (B1a) can be detected Identification of Site-Specific
the non-glycosylated (NG) species B2, which are Variant Separation and identified as a partially glycosylated species. Deamidations After Thermal Stress
eluted earlier. Since mAbs with a lower pI are better mAb-V was thermally stressed to achieve
separated, a test was performed to see Monitoring Critical Fc Quality Attributes higher levels of deamidation and to facilitate
Comparing AEX–MS and CEX–MS whether the separation can be improved by This AEX–MS method can also be used for fractionation after IdeS digestion and
From the CEX–MS method, two acidic peaks lowering the pI through PNGase F (peptide:N- subunit analysis of mAbs after digestion with deglycosylation. Therefore, mAb-V was
can be observed and identified as deamidated glycosidase F)-mediated deglycosylation. This IdeS (immunoglobulin G-degrading enzyme of incubated at 25 °C for 6 months. The basic
(A1) and glycated (A2) variants (see Figure 2). reaction removes N-glycans and simultaneously Streptococcus pyogenes) protease. The already Fc variants of mAb-V are also assigned to the
No basic peaks were detected. In comparison, converts the glycan-bearing asparagine (Asn) deglycosylated mAbs were treated with IdeS 1 unprocessed C-term K and Fc N-glycosylation
the AEX–MS analysis shows four basic peaks residue to aspartic acid (Asp). Since all IgG4 IUB milliunit per 1 µg mAb sample in 100 mM macroheterogeneity (see Figure 5).
13
These basic variants did not change after Conclusions

thermal stress while an increase in peaks for The AEX–MS method described is very suitable
the acidic variants was observed, which can for characterising charge heterogeneity in
be completely attributed to deamidation. IgG4-based mAbs. Compared to CEX–MS,
Analysis of Fc fragments improved the AEX–MS shows overall better separation of
The Selectivity Company
resolution of separated deamidated variants. these mAbs with moderate pI and provides
Four acidic peaks are resolved from the additional information. The resolution of the Your Experts in
analysis of the thermally stressed mAb. Since glycosylated variants can be further improved by
there are only a few deamidation sites present PNGase F-mediated deglycosylation. AEX–MS
Reproducible Analyses
in the IgG4 Fc region, it is likely that site- methods are suitable for the Fc critical quality
Specifically designed BioLC columns
specific deamidations can be separated by attribute monitoring of IgG4-based mAbs, For proteins/peptides, mAbs, ADCs and oligonucleotides
AEX–MS. Possible deamidation sites are NG while CEX remains the better option for F(ab’)2
at VVSVLTVLHQDWLNGK; NK at VSNK; NG subunit analysis. With this AEX–MS method the Fully bioinert (U)HPLC
Coated, PEEK and PEEK-lined columns
and NN at GFYPSDIAVEWESNGQPENNYK. Fc critical quality attributes can be monitored
Therefore, the fractions of the deamidated without peptide mapping, saving time, reducing Superior performance and reproducible separations
variants were identified by peptide errors and, even more important, providing Reliable results, high throughput and long column lifetimes
mapping. Fraction A1a mainly contains reproducible results.
the GFYPSDIAVEWESNGQPEDNYK
peptide while A2 mainly contains the References
GFYPSDIAVEWES(isoD)GQPENNYK peptide. 1. Monoclonal Antibodies (mABs). World Health
It is also noticeable that A1b and A1c both Organization. https://www.who.int/teams/health-
contain the deamidated VSNK peptide, but product-policy-and-standards/standards-and-
A1b mainly contains the isoAsp form, whereas specifications/monoclonal-antibodies (accessed
A1c primarily contains the Asp form. It is 01/15/2023).
nich
striking that this AEX–MS method is capable 2. Kinch, M. S.; Kraft, Z.; Schwartz, T. Monoclonal Visit us in Mu
405
of separating site-specific deamidation Antibodies: Trends in Therapeutic Success and Hall A1, Booth
products at the Fc level, even at isoform Commercial Focus. Drug Discovery Today 2023, 28
resolution, so that these attributes can be (1), 103415. DOI: 10.1016/j.drudis.2022.103415
monitored without the need for peptide 3. Goyon, A.; Excoffier, M.; Janin-Bussat, M.- C.;
YMC’s Expertise Portal will always keep you up to date
mapping. This saves time and reduces sources Bobaly, B.; Fekete, S.; Guillarme, D.; Beck, A.
of error such as deamidation artefacts that Determination of Isoelectric Points and Relative www.ymc.eu | support@ymc.eu | +49 2064 427-0
can occur in peptide mapping. Charge Variants of 23 Therapeutic Monoclonal
14
Antibodies. J. Chromatogr. B: Anal. Technol. laboratory assistant at the University of

Biomed. Life Sci. 2017, 1065–1066, 119–128. DOI: Applied Sciences in Krefeld, Germany.
10.1016/j.jchromb.2017.09.033 After receiving a bachelor’s degree,
4. Yan, Y.; Liu, A. P.; Wang, S.; Daly, T. J.; Li, she pursued a master’s programme
N. Ultrasensitive Characterization of Charge in chemistry at the University of
Heterogeneity of Therapeutic Monoclonal Düsseldorf, Germany, with a focus on
Antibodies Using Strong Cation Exchange organic chemistry. Since 2019, she has
Chromatography Coupled to Native Mass worked for YMC Europe in Dinslaken
Spectrometry. Anal. Chem. 2018, 90 (21), 13013– as a product specialist in the analytical
13020. DOI: 10.1021/acs.analchem.8b03773 chromatography team.
5. Leblanc, Y.; Bihoreau, N.; Chevreux, G. Daniel Eßer studied chemistry at the
Characterization of Human Serum Albumin Isoforms University of Applied Sciences Bonn
by Ion Exchange Chromatography Coupled On-Line Rhein-Sieg, in Rheinbach, Germany,
to Native Mass Spectrometry. J. Chromatogr. B: with a focus on pharmaceutical and
Anal. Technol. Biomed. Life Sci. 2018, 1095, 87–93. analytical chemistry. He received his
DOI: 10.1016/j.jchromb.2018.07.014 PhD in pharmaceutical and medicinal
6. Füssl, F.; Criscuolo, A.; Cook, K.; Scheffler, K.; chemistry at the University of
Bones, J. Cracking Proteoform Complexity of Düsseldorf, Germany. During his postdoc
Ovalbumin with Anion-Exchange Chromatography– at the Institute of Pharmaceutical and
High-Resolution Mass Spectrometry under Native Medicinal Chemistry of the University of
Conditions. J. Proteome Res. 2019, 18 (10), 3689– Düsseldorf, he established a nanoLC–MS
3702. DOI: 10.1021/acs.jproteome.9b00375 system. In 2013 he joined YMC Europe
7. Liu, A. P.; Yan, Y.; Wang, S.; Li, N. Coupling in Dinslaken, Germany, as a product
Anion Exchange Chromatography with Native specialist for analytical chromatography.
Mass Spectrometry for Charge Heterogeneity Since 2017, he has been responsible
Characterization of Monoclonal Antibodies. Anal. for YMC’s analytical (U)HPLC column
Chem. 2022, 94 (16), 6355–6362. DOI: 10.1021/ portfolio as the product manager for
acs.analchem.2c00707 analytical chromatography.
Ann Marie Rojahn studied chemistry E-mail: d.esser@ymc.eu

and biotechnology in combination Website: www.ymc.eu
with an apprenticeship as a chemical
15
Novel Automated
Method for Sample
Preparation for
Peptide Mapping
Leslie Napoletano, Waters Corporation, Milford, Massachusetts, USA
Peptide mapping is considered a critical quality attribute (CQA) for the

identification of a protein’s primary structure and confirmation of any
peptide modifications. Peptide mapping can be streamlined with a
comprehensive kit and an autolysis-resistant trypsin for high-efficiency
digestions. Automation enhances laboratory productivity and reduces
errors in peptide mapping, making it advantageous to implement in a
biopharmaceutical laboratory.
If you have been tasked with developing and genetic stability (1). Many scientists
a peptide mapping method to further think of a peptide map as a “fingerprint”
characterize your protein, what is peptide of a protein that provides a comprehensive
mapping and where do you begin? Peptide understanding of the protein being
mapping is the analysis of chemical analyzed (1).
or enzymatic digests of a protein to The entire peptide mapping workflow
focus on specific peptide regions and can be broken down into three major steps:
Quardia Inc.- stock.adobe.com
modifications (1). It is an important assay sample preparation, separation by reversed-

in biopharmaceutical characterization phase liquid chromatography, and, lastly,
because it helps identify the primary protein data processing. Each step presents its own
structure to confirm process consistency set of challenges, with sample preparation
16
The Column www.chromatographyonline.com Napoletano
Figure 1: Schematic of desalting by SEC.
being highly laborious, complex, and prone the entire sample preparation method can
to variability. be more than 8 h. These long digestion
Traditionally, there are five key steps times often also introduce artificial
involved in preparing samples for peptide peptide modifications, such as asparagine
mapping: denaturation, reduction, deamidation and methionine oxidation (2).
alkylation, desalting, and digestion. It This process can be laborious and
is easy to imagine the many ways that overwhelming—which is why it is
error and variability could be introduced no surprise that many are looking to
in each step when developing a robust automated processes to streamline this
sample preparation method. Among the work. The use of an automated liquid
considerations that must be evaluated are handler for increasing throughput and
reagent reliability, desalting inconsistencies, alleviating the time spent on routine
digestion completion, and enzyme autolysis. pipetting is of great interest to scientists,
Beyond these, it is important to consider especially for sample preparation for
the time required to perform the peptide complex assays such as peptide mapping.
mapping sample preparation workflow. Historically, this type of automation
Depending on the length of digestion, has proven difficult, requiring large
17
Figure 2: High enzyme-to-protein ratios (1:5) are faster, but typically lead to high autolysis. the use of microcentrifuge tube-based 1:5 enzyme:protein ratio with minimal
RapiZyme Trypsin’s unique autolysis resistance and low missed cleavage unlocks efficient 30 min
devices can be more cumbersome and autolysis and low missed cleavages as
digestions versus standard 3 h digestions (using 1:20)
difficult to automate as a centrifuge is shown in Figure 2.
Remicade™ (infliximab) digestion, 1:5 Enzyme: Protein Ratio, 30 min. often required to process the samples.
Cartridge-based desalting devices Method
Trypsin Autolysis (% TIC Response) Missed Cleavage (% TIC Response)
rely on gravity for elution, but use of NIST Monoclonal Antibody (NISTmAb)
6.4%
4.2%
these devices is difficult to automate Reference Material 8671 was digested
and integrate with high-throughput using a comprehensive peptide mapping
workflows. Recent advances in the design kit, PeptideWorks Tryptic Protein Digestion
0.9% and development of desalt cartridges Kit, which includes RapiZyme Trypsin.
0.1%
allow for use of a multichannel pipette NISTmAb reference tryptic digest samples
Leading Competitor RapiZyme Trypsin Leading Competitor RapiZyme Trypsin
in a 96-well stand for ease-of-use and were prepared both manually and with
Digestion details: 37 °C, pH 7.5. Average of n=6 digestion replicates per enzyme vendor – 2 batches of each enzyme, 3 digestion replicates per batch. amenability to automation platforms (4). automation on the Andrew+ Pipetting
Last but not least is the daunting Robot with Extraction+ Connected
task of developing a robust enzymatic Device. NISTmAb samples (10 mg/mL)
investments of both time and money, as prior to digestion (3). Size-exclusion digestion procedure. Trypsin, the most were denatured and reduced in a solution
well as introducing new skill sets such as chromatography (SEC), also known as common enzyme for peptide mapping, containing 5 M guanidine hydrochloride
expertise in Python scripting. However, gel filtration, is a common desalting cleaves proteins on the C-terminal side (GuHCl) and 5 mM dithiothreitol (DTT)
recent advances in the development of mechanism where analytes are separated of lysine and arginine (5). The elevated for 30 min at room temperature.
new, automated solutions with intuitive based on their size in solution. Larger temperature and alkaline pH used in Iodoacetamide (IAM) was then added to
software can enable total, “hands-free” molecular weight analytes pass through tryptic digestions can induce artificial a final concentration of 10 mM and the
solid-phase extraction. the chromatographic bed and are eluted peptide modifications, complicating data samples were incubated for 30 min at
Protein denaturation, reduction, and first while alkylating reagents are trapped analysis (5). Tryptic digestions are further room temperature in the dark. Samples
alkylation are performed to provide the in the pores of the desalting device, which complicated by missed cleavages (under- were desalted with Sep-Pak SEC Desalting
proteolytic enzyme access to digestion allows the larger denatured, reduced, and digestion), non-specific cleavages (over- Cartridges and buffer exchanged with
sites for a more complete digestion (3). The alkylated protein to flow through first, as digestion), and autolysis (when an enzyme digestion buffer (10 mM CaCl2 and 100
most common denaturant used in peptide depicted in Figure 1. starts to digest itself). mM Tris HCl, pH 7.5). The concentration
mapping is guanidine hydrochloride. If Desalting devices can be in a 96-well A novel, autolysis-resistant, homogenously of desalted samples was measured with a
trypsin is used to cleave peptide bonds, plate, microcentrifuge tube, or cartridge methylated recombinant porcine trypsin UV plate reader and normalized to 0.1 mg/
guanidine can significantly interfere with format. While easy to automate, plate- was recently introduced to help overcome mL using the digestion buffer as a diluent.
its activity (3). Therefore, a desalting step is based desalting devices suffer from the challenges in tryptic digestion (6). This RapiZyme Trypsin was added to each
needed to effectively remove the guanidine limited loading capacity. On the contrary, trypsin enables fast 30-min digestions using sample at a 1:5 enzyme:protein ratio and
18
Figure 3: Relative abundance of missed and non-specific cleavages for NISTmAb digests prepared
using the manual and automated PeptideWorks. Workflows and a leading immobilized trypsin kit.
Error bars represent one standard deviation. PeptideWorks Tryptic Protein Digestion Kit delivers a
complete and specific digestion.
34th International Symposium

on Chromatography
digestion proceeded for 30 min at 37 °C. on published results and shown in red in
Finally, the reaction was quenched with 1% Figure 3. The digestion kit used yields a 93%
formic acid to a final concentration of 0.1% reduction in missed cleavages and 55%
The International Symposium on
and injected onto the LC–MS system. reduction in non-specific cleavages compared
Chromatography (ISC) represents the oldest
to the immobilized trypsin digest kit (7).
conference series focussing on separation
Results BPI chromatogram overlays of NISTmAb
Figure 3 displays the relative missed and non- digests prepared using the manual and science. ISC symposia have been organised since
specific cleavage results for NISTmAb digests automated workflows (left) and the relative 1956 in each even year. ISC is internationally
prepared using manual and automated. The abundance of select peptide modifications recognised as one of the premier meeting series
automated workflow used delivers NISTmAb for three batches (right) on the pipetting
isc2024.org for discussion of all modes of chromatography
digests with less than 5% missed and non- robot are shown in Figure 4. These results and separation science with a broad coverage of
specific cleavages, indicating high digestion show that a comprehensive peptide techniques and applications.
Photos ©Marketing Liverpool
efficiency without over-digestion of the mapping kit provided fast tryptic digests
protein. Missed and non-specific cleavage without sacrificing digestion completion To sponsor, exhibit or for more information,
results for NISTmAb digests prepared using or inducing high levels of method-induced please contact isc2024@sasevents.co.uk
an immobilized trypsin digest kit are based deamidation or oxidation. The optimized
19
Figure 4: The relative abundance of select peptide modifications determined by LC–MS for three 2. Jiang, P.; Li, F.; Ding, J. Development of an Shiner, S., Trudeau, M. Quick and Robust
batches of NISTmAb digests prepared using the automated PeptideWorks Workflow. Results and
Efficient LC–MS Peptide Mapping Method Sample Preparation for Tryptic Peptide Mapping
BPI chromatograms for NISTmAb digests prepared with the manual workflow are also shown
to demonstrate consistency between the manual and automated sample preparation. Error bars Using Accelerated Sample Preparation for with the PeptideWorks Kit using Simple,
represent one standard deviation. Monoclonal Antibodies. J. Chromatogr. Automatable Workflows; Waters Application
B 2020, 1137, 121895. DOI: 10.1016/j. Note 720008019; Waters Corporation: Milford,
Manual Sample Prep
jchromb.2019.121895 MA, 2023. https://www.waters.com/nextgen/
3. Mouchahoir, T.; Schiel, J. E. Development of us/en/library/application-notes/2023/quick-
an LC–MS/MS Peptide Mapping Protocol for robust-sample-preparation-fortryptic-peptide-
the NISTmAb. Anal. Bioanal. Chem. 2018, 410, mapping-with-the-peptideworks-kit-using-
Automated Sample
Prep 2111–2126. DOI: 10.1007/s00216-018-0848-6 simple-automatable-workflows.html (accessed
4. Sep-Pak SEC Desalting Cartridges, 1cc 5K 2024-02-20).
MWCO Care and Use Manual. Waters User
Manual 720007983EN; Waters Corporation: Leslie Napoletano is a Principal Product
Milford, MA, 2023. https://www.waters.com/ Marketing Manager within Waters
automation protocol generates reproducible productivity and reduce errors, especially webassets/cms/support/docs/720007983en.pdf Consumables & Lab Automation Group.
results over time and provides an increase in in a labor-intensive method, such as (accessed 2024-02-21). She has been with Waters since 2012
productivity with simple, robust automation peptide mapping. 5. Ren, D.; Pipes, G. D.; Liu, D.; et al. An Improved providing support and education to
alleviating potential pipetting errors (5). Trypsin Digestion Method Minimizes Digestion- scientists in New Jersey with Waters
Acknowledgment Induced Modifications on Proteins. Anal. chemistry consumables. More recently
Conclusion Special thanks to Bill Warren, Caitlin Hanna, Biochem. 2009, 392 (1), 12–21. DOI: 10.1016/j. in 2021, Leslie has moved into the
Developing a robust sample preparation Meagan Callis and Stephan Koza for your ab.2009.05.018 Product Marketing group focusing on
method for peptide mapping can be contributions. 6. Ippoliti, S.; Zampa, N.; Yu, Y. Q.; Lauber, M. bringing on new consumable products
laborious and filled with many variables. A. Versatile and Rapid Digestion Protocols for protein characterization. Prior to
Faster digestions often yield incomplete References for Biopharmaceutical Characterization joining Waters, Leslie worked as a
digests with additional autolysis species 1. USP. Biotechnology-Derived Articles–Peptide Using RapiZyme Trypsin; Waters Application scientist in protein characterization
making data analysis more complex. Mapping <1055>; USP32–NF27 Page 496, Note 720007840; Waters Corporation: in the pre-clinical development of
Peptide mapping can be streamlined Interim Revision Announcement: USP32–NF27 Milford, MA, 2023. https://www.waters.com/ monoclonal antibodies at Merck and
using a comprehensive kit with lot tracible No. 1 Page 41, Pharmacopeial Forum: Volume nextgen/us/en/library/application-notes/2023/ prior to that at Teva as a R&D chemist.
reagents and a novel autolysis-resistant No. 32(2) Page 571; Rockville, MD, 2016. versatile-and-rapid-digestion-protocols-for-
high purity trypsin allowing for high- https://www.usp.org/sites/default/files/usp/ biopharmaceutical-characterization-using-
E-mail: Leslie_Napoletano@waters.com
efficiency 30 min digestions. The use of document/harmonization/biotechnology/b05_ rapizyme-trypsin.html (accessed 2024-02-21). Website: www.waters.com
automation can further enhance laboratory pf_ira_35_1_2009.pdf (accessed 2023-12-11). 7. Hanna, C. M.; Danaceau, J. P.; Koza, S. M.;
20
How Analytics and
Mass Spec Became the
Driving Force Behind
Biotherapeutic
Drug Development
Kelly Broster, Thermo Fischer Scientific, Waltham, Massachusetts, USA
Biopharma discovery and development scientists are achieving new

levels of structural insight into therapeutic proteins. By utilizing
biopharmaceutical analytical technology and mass spectrometry,
laboratories are now empowered to reveal ultra-low-level modifications
and determine site-specific critical quality attributes (CQAs) and granular
information—delivering more confidence when progressing candidates
throughout the development pipeline, whilst ensuring drug efficacy and
patient safety. This article explores the key challenges analytics and mass
spectrometry solve within the biopharmaceutical industry.
In the dynamic world of biopharmaceutical allowing them to delve deeper into the
discovery and development, scientists structural intricacies of biotherapeutic proteins
are constantly striving to advance their and create new drugs.
understanding of therapeutic proteins. These innovations empower laboratories to
paisorn - stock.adobe.com
Breakthroughs in biopharma analytical uncover ultra-low-level modifications, determine

technology and mass spectrometry (MS) site-specific critical quality attributes (CQAs),
have opened new avenues for researchers, and obtain granular information pivotal to the
21
The Column www.chromatographyonline.com Broster
drug development process. This article will dive development, pharmacokinetics, and process
into the critical role that analytics and mass monitoring. Its high sensitivity, specificity,
spectrometry play in addressing key challenges and ability to handle complex samples make
within the biopharmaceutical industry. it indispensable in the biopharmaceutical powered by
industry. As drug development techniques
GC OPERATOR
The Evolution of Mass Spectrometry advance and quality control demands increase,
in Drug Development mass spectrometry’s role in later stages of
Traditionally, mass spectrometry was primarily development will only increase and become
associated with the early stages of drug more critical. The fundamental concepts of GC explained. From system set-up to shutdown.
discovery and was commonly used to assess
the purity of raw materials and identify Real-Time Insights and Enhanced • How to set-up a GC system SCAN HERE
FOR MORE
compounds of interest. Quality Control • Basics of the chromatographic process
However, the pharmaceutical landscape The technology behind mass spectrometry • Sample introduction
has undergone a seismic shift in recent not only aids in compound identification but
• Columns and temperature programming
years and laboratories now recognize the also offers real-time insights into the stability
• Measuring and optimizing chromatographic parameters
importance of mass spectrometry throughout and degradation of pharmaceutical products
drug development. It is no longer confined throughout the drug development journey.
to the initial stages but is integral to quality Continuous monitoring allows laboratories
control and safety assessments at every step, to detect deviations from specifications
and has become the driving force behind promptly, ensuring the final product adheres
biotherapeutic drug development and is to regulatory standards at all stages. This
enhancing quality by design (QbD). real-time monitoring capability accelerates
Mass spectrometry is an invaluable tool in research and development timelines,
the rapid identification and characterization of enabling laboratories to make informed
compounds and has been since its inception decisions based on accurate data. Speed-to-
in the early 20th century. The technique’s market advantages are particularly crucial in
high-resolution capabilities empower early- addressing urgent medical needs, such as
stage laboratories to analyze complex samples during the COVID-19 pandemic.
with unparalleled accuracy. It plays a vital Mass spectrometry’s sensitivity and
role in biopharmaceutical development and specificity make it a reliable method for www.chromacademy.com/gc-operator
manufacturing by providing detailed insights safeguarding product integrity and upholding
into protein structure, quality control, biosimilar the reputation of drug manufacturers for
22
The Column www.chromatographyonline.com Broster
biotherapeutics. The importance and value spectrometry is their ability to uncover ultra- throughout the development demands, analytics and mass spectrometry
of mass spectrometry for biotherapeutic low-level modifications in biotherapeutic process is a paramount concern in accelerate the development process while
development is demonstrated by the work drugs. These modifications, which were biopharmaceuticals. Analytics and mass ensuring drug efficacy and patient safety. The
of the MAM Consortium, an industry-wide, previously challenging to detect, can spectrometry can facilitate quality integration of these tools into the entire drug
nonprofit organization with the mission to now be identified with precision. This control and offer real-time insights development journey enables pharmaceutical
advance multi-attribute method (MAMs) and capability provides invaluable insights into to promptly address deviations from companies to stay competitive and deliver
other LC–MS applications in pharmaceutical the structural integrity and stability of required specifications, allowing for safe and effective medications to patients
and biotechnology companies for product therapeutic proteins, enhancing the overall greater consistency and reliability in more swiftly than ever before.
characterization, in-process control, and GMP quality of drug candidates. product quality. As the biopharmaceutical industry continues
release and stability testing (1). Additionally, analytics and mass • Regulatory Demands: The to evolve, analytics and mass spectrometry
spectrometry enable researchers to pinpoint biopharmaceutical industry operates will remain indispensable, paving the way for
Reducing Contamination Risk and site-specific CQAs in biotherapeutic drugs. in a highly regulated environment. groundbreaking discoveries and innovations in
Regulatory Compliance This granular information is essential for Meeting regulatory demands is essential the field of biotherapeutic drug development.
Mass spectrometry is well aligned to help ensuring drug efficacy and patient safety. for bringing new therapies to market. There is still enormous untapped potential
pharmaceutical companies comply with By identifying specific attributes that may Analytics and mass spectrometry aid in to be realized for the implementation of
regulatory requirements, as the technology impact a drug’s performance, scientists can compliance by providing robust data mass spectrometry in biopharmaceutical
provides robust analytical data. This data make informed decisions and refine drug and documentation required for development and manufacturing that will
encompasses drug purity, acceptable dosage development strategies. Mass spectrometry regulatory approval. bring life-changing treatments to the
levels, and the necessary documentation enables another critical level of information market faster.
required as therapies progress from research through the process of building quality in A New Era in Biotherapeutic Drug
to clinical application. The inclusion of each design step of the drug development Development Kelly Broster, PhD, is Senior Manager
mass spectrometry at all stages of drug process. Key challenges it addresses include: In the ever-evolving landscape of of Pharma & Biopharma Market
development significantly impacts the • Structural Complexity: Biotherapeutic biotherapeutic drug development, analytics Development and Collaborations
transition time from laboratory research to drugs are inherently complex, and and mass spectrometry have emerged as at Thermo Fisher Scientific.
clinical applications, ensuring adherence understanding their intricate structures the driving force behind structural insights
to regulatory standards throughout is vital for drug development. Analytics and quality control. These technologies Reference
the process. and mass spectrometry provide the tools empower researchers to uncover ultra-low- 1. MAM Consortium. https://mamconsortium.org
needed to navigate this complexity, level modifications, identify site-specific (accessed 2024-03-04).
Analytics and Mass Spectrometry as allowing scientists to unravel the nuances critical quality attributes, and navigate the
Biotherapeutic Game Changers of these molecules. complexities of biotherapeutic molecules. By E-mail: Kelly.broster@thermofisher.com
One of the remarkable achievements of • Quality Control & Consistency: addressing key challenges, such as structural Website: www.thermofisher.com/uk/
en/home.html
biopharmaceutical analytics and mass Maintaining rigorous quality control complexity, quality control, and regulatory
23
Rapid Method
to Characterize
Adeno-Associated
Virus (AAV) Therapies
Using SEC–FLD
Tran Pham, Phenomenex, Torrance, California, USA
Adeno-associated virus (AAV) gene therapies have created analytical

challenges for drug developers. Current technologies and consumables
are not well-suited for the already low sample volume used to determine
the critical quality attributes (CQAs) of AAVs. Stationary phases with novel
pore and column dimensions and inert particle chemistry demonstrate
increased efficiencies and robustness for AAV analysis. These advances can
enable researchers to make critical analytical decisions with confidence and
ultimately, ensure drug time-to-market.
Adeno-associated virus (AAV) gene therapies and robustness for AAV analysis. These
have created analytical challenges for drug advances can enable researchers to make
developers. Current technologies and critical analytical decisions with confidence and
consumables are not well-suited for the already ultimately, ensure drug time-to-market.
low sample volume used to determine the The rapid growth of AAV gene-based
Dr_Microbe - stock.adobe.com
critical quality attributes (CQAs) of AAVs. New therapies is catalyzed by several factors such
tailored chromatography methods with novel as AAVs’ wide range of tropism for increased
pore and column dimensions and inert particle personalized medicine efficacies and their
chemistry demonstrate increased efficiencies ability to carry large amounts of genome (the
24
The Column www.chromatographyonline.com Pham
the analytical challenges of this small-volume, processing, formulation, and storage conditions.
Figure 1: Zoomed-in chromatograms of AAV serotypes on a Biozen 3 µm dSEC-7, 300 × 4.6 mm
column demonstrating consistent recovery of AAV monomer and impurities across tested serotypes. advanced therapeutics industry. The gold standard for AAV aggregate analysis
The good news is that chromatographic has long been analytical ultracentrifugation
3 10 consumables and solutions are rapidly (AUC). However, AUC requires sample sizes up
4
2.5 8
3
evolving to address these unmet needs while to 400 mL, takes an average 90 min of run time
0
6
maintaining, or even increasing, data quality. to complete, and has a high learning curve.
LU
LU
LU
1.5 2
4
1 1 Coupled with existing instrumentation, these Size-exclusion chromatography (SEC) is
2
0.5
0 0
0
different approaches enable the analysis of recognized as an orthogonal method to
3
2 3 4 5 6
Time (min)
7 8 9 2
3
3 4 5 6
Time (min)
7 8 9
2
2 3 4 5 6
Time (min)
7 8 9
multiple AAV critical quality attributes (CQAs), AUC and is more efficient. On average, AAV
2.5 2.5
1.75 which help to mitigate sample consumption. aggregate analysis with SEC consumes only
1.5
2 2
1.25 These approaches ultimately increase AAV about 20 μL of sample, and analysis for one
LU
LU
LU
1
drug development efficiencies and ensure sample can be completed in as little as 14 min.
1.5 1.5
0.75
1 1
0.5 0.5
0.5
0.25
time-to-market. But this is still not ideal. Researchers are seeking
2 3 4 5 6
Time (min)
7 8 9 2 3 4 5 6
Time (min)
7 8 9 2 3 4 5 6
Time (min)
7 8 9 to achieve faster turnaround times for analysis
1 1.2
0.9 Improving Aggregate Analysis while consuming even less sample volume—all
1
0.8
0.7 0.8
The most intense and high volume of analysis without compromising data quality.
LU
LU
0.6
0.5 0.6
occurs during the AAV characterization stages. Current SEC technology research is proving
0.4
0.3
0.4 Early comprehension of the vector’s CQAs is that an inert, 700 Å pore size particle provides
0.2
2 3 4 5 6 7 8 9
0.2
2 3 4 5 6 7 8 9
critical for drug candidate selection as well robust and reliable baseline separation
Time (min) Time (min)
as for process and formulation development between monomer and aggregate, as well as
and optimization. Robust and reliable better peak identification between aggregates.
drug) to ensure dosing optimization. Currently, Common chromatographic methods used for analytical methods used to characterize AAVs Coupled with smaller inner diameters, an SEC
there are more than 235 AAV therapies in established modalities such as monoclonal are therefore required. The importance of column with these dimensions holistically
various clinical trial phases, and twelve AAV- antibodies (mAbs) are applicable to analyzing robustness and reliability of these methods mitigates high sample consumption without
based therapies have been approved since AAVs. The problem is that the manufacturing is even further emphasized when “fit-for- sacrificing data analysis efficiency and quality of
2012. The global market for AAVs, currently scale of viral vectors is a lot smaller than it is purpose” consumables are used. AAV aggregate analysis.
estimated to be worth more than $767.7 for mAbs. The AAV preclinical manufacturing An essential CQA of AAVs is aggregate
million, is expected to expand at a compound sizes range from hundreds of microliters to 50 identification and quantification. Aggregation Demonstrating an Optimized SEC
annual growth rate of 22.5% through 2030 (1). L, while mAb production volumes have typically can cause AAV-based therapeutics to lose Column for AAV Analysis
With this growth comes significant challenges been over 500 L. Some consumables are not efficacy or impact immunogenicity, which Research was performed in 2023 on AAV
to ensure the viral vectors’ efficiencies currently optimized to handle smaller volumes. negatively impacts drug and patient safety. serotypes 1, 2, 3, 4, 5, 6, 8, 9 and rh10 using
throughout the drug development stages. Therefore, there is an unmet need to support Aggregation is often driven by suboptimal a Biozen 3 µm dSEC-7 (Phenomenex). Two
25
The Column www.chromatographyonline.com Pham
column dimensions were tested: 150 × 4.6 with the achievable resolution demonstrated heat exposure, the smaller species decreased, References
mm and 300 × 4.6 mm. For AAV8-CMV- that a suitable particle size was chosen. resulting in more of the largest species. 1. Adeno Associated Virus Vector Manufacturing
GFP, five samples at concentrations ranging The column was found to be suitable for Market Size, Share & Trends Analysis Report By Scale
from 2×1011 viral genomes per mL (vg/mL) separating the monomer from its aggregates Conclusion Of Operations (Clinical, Commercial), By Method, By
to 2×1012 vg/mL were prepared in 0.2 μm and impurities for serotypes 1, 2, 3, 5, 6, The analytical capabilities of SEC columns Application, By Therapeutic Area, By Region, And
filtered 1x phosphate buffered saline with 8, 9 and rh10 with a 5-microliter injection specifically designed for AAVs have been Segment Forecasts, 2023 - 2030. Adeno Associated
0.001% Pluronics F68 into HPLC vials. For volume. Different serotypes displayed demonstrated. This technology, with its optimized Virus Vector Manufacturing Market Report,
serotype AAV studies on the 300 × 4.6 mm different retention times (Figure 1). Resolution column and pore sizes, promotes reproducible 2030. Grand View Research, 2023. https://www.
column format, separate dilutions of 4×1011 between monomer and aggregate remained separations and recoveries for various AAV grandviewresearch.com/industry-analysis/adeno-
vg/mL were prepared and centrifuged at consistent when the volume was increased serotypes. An SEC method coupled with a associated-virus-vector-manufacturing-market-report
10,000 relative centrifugal field for 5 min prior to 10 μL Therefore, doubling the viral load fluorescence detector (FLD) using a 150 × 4.6 mm (accessed 2024-03-04).
to injection on the LC system (Agilent 1260 on the 300 × 4.6 mm column did not column can be completed in 8 min at a flow rate
Infinity with Agilent FLD and UV). affect the percentage of monomer recovery of 0.5 mL/min per on a normal HPLC system. As Tran Pham is a Global Market
determination. It is possible that even higher a result of the reproducible analytical performance Development Manager for BioPharma at
Results and Discussion resolution separations could be achieved of this column, it could be used for stability studies Phenomenex. She has more than 15 years
The study demonstrated that the SEC using a 300 × 4.6 mm format and a flow rate to determine the purity and quantity of the of industry experience that encompasses
technology produced highly reproducible of 0.25 mL/min. monomer left in the solution. It can also enable analytical development, training, sales,
peak areas, retention time, and percentage The study also demonstrated that the SEC the determination of the critical quality attributes in and marketing.
monomer. The running pressures for the 150 column could be used to examine the effects drug substances and drug products. Furthermore,
× 4.6 mm and 300 × 4.6 mm columns at a of forced degradation on AAVs. Upon exposing it could be used to drive the development of other
E-mail: tranp@phenomenex.com
flow rate of 0.5 mL/min were 78 and 155 bars, AAV1 to heat, two new hydrodynamically analytical methods such as AAV capid load and Website: www.phenomenex.com
respectively. These running pressures combined larger species were formed. With continued titer quantity as well as IgM aggregation.
26
HPLC 2024 Preview
A look what’s in store for chromatographers who attend HPLC 2024
in Denver, Colorado from 20–25 July 2024.
Susan Olesik, Program Chair, HPLC 2024
It is my pleasure to invite you to Additionally, the applications of artificial

participate at the 52nd International intelligence and machine learning for
Symposium on High Performance method development will be described.
Liquid Phase Separations and Related Plenary and keynote presentations
Techniques (HPLC 2024) which will be by leading scientists in the field will be
held from Saturday through Thursday, provided. Most presentations, oral and
20–25 July 2024, in Denver, Colorado, poster, will be from abstracts submitted by
USA. The HPLC symposium series is participants, which allow new innovations to
known as the internationally leading be presented across the large span of liquid
conference on liquid separations and separations. Short courses, tutorials, and a
related technologies. large exhibition along with vendor seminars
This year the program will span will provide participants the opportunity to
separation science in liquids and see state-of-the-art technology.
supercritical fluids, innovations in The program is designed to provide
sample preparation, column technology, insightful presentations that will be
theoretical insights on chromatographic appealing to analytical scientists in
improvements, hyphenated and the pharmaceutical and chemical
multidimensional methods, and electrically industries, as well as academics and
driven methods. Cutting-edge microscale environmental studies.
separation devices and their applications The annual HPLC conference hosts a
for disease detection and biomarker number of award presentations and a
discovery will be highlighted. State-of- recently formed competition. The Csaba
the-art methods for applications, such as Horváth Award recognizes his contributions
eunikas - stock.adobe.com
nucleic acid separations and detection, to HPLC and his strong interest in fostering
proteomics, glycomics, and of course careers of young scientists and engineers
multi-omics will be discussed. working in the field of separation science.
27
The Column www.chromatographyonline.com Event Preview: HPLC 2024
The Uwe D. Neue Award recognizes HPLC Tube is a scientific contest for the to highlight their current science and Susan Olesik is Program Chair,
scientists that have made and continue best video in which authors present the instrumentation. Don’t miss the conference HPLC 2024 and Distinguished
to make significant contributions to the impact of their research on societal needs. gala at the Ellie Caulkins Opera House. University Professor at the Department
field of separation science, in honor of the Please see the conference website for more Registration for the conference is of Chemistry and Biochemistry,
legacy of Uwe D. Neue, late scientist and details on each of these. open and we are accepting abstracts for Ohio State University, Ohio, USA.
Waters Corporate Fellow. Awards for the Join us in the mile high city where HPLC presentations. Look for more details on our
top ten best posters will also be provided. 2024 is driving the field to new heights! website at https://hplc2024-symposium.
E-mail: olesik.1@esu.edu
Finally, the HPLC Tube competition is a Take part in unique opportunities for org. I look forward to LCGC readers joining
Website: www.hplc2024-symposium.org
recent addition to the programming. The colleagues to interact and for vendors us at HPLC 2024!
A horse and carriage on the promenade in

Denver, Colorado, where the 2024 HPLC
conference will take place.
28
The Column www.chromatographyonline.com Training and Events
Training Courses
GC Comprehensive GC Hardware LC–MS Introduction MISCELLANEOUS
GC Introduction Training (Agilent GC) Onsite training Introduction to Infrared (IR)
Website: www.chromacademy. 18 March 2024 Website: www.chromacademy. Spectroscopy
com/channels/gc-training-courses/ The Open University, com/channels/lc-ms/principles/lc-ms- Online webcast from
principles/gc-introduction Milton Keynes, UK introduction CHROMacademy
Website: https://www.anthias. Website: www.chromacademy.
GC Troubleshooter co.uk/training-courses/2-day- com/channels/infrared/principles/
comprehensive-gc-hardware-training- introduction-to-infrared-spectroscopy
Website: www.chromacademy. Absolute Basics of Deconvolution
com/channels/gc-training-courses/ agilent-7890-gc
15 March 2024
troubleshooting/gc-troubleshooter Online Applied Stability Indicating
HPLC/LC–MS Methods
Website: https://www.anthias.co.uk/
Understanding HPLC 11 March 2024
Operating and Understanding training-courses/basics-deconvolution
Website: www.crawfordscientific. The Open University,
GC
com/training-consultancy/hplc- Milton Keynes, UK
Website: www.crawfordscientific. training/hplc-fundamentals Website: https://www.anthias.co.uk/
com/training-consultancy/ SAMPLE PREPARATION training-courses/Applied-Stability-
gc-training/operating-and- HPLC Troubleshooter Fundamentals of Solid-Phase Indicating-Methods
understanding-gc Website: www.chromacademy. Extraction (SPE) Mechanisms
com/channels/hplc-training-courses/ Online training
troubleshooting/hplc-troubleshooter Website: www.chromacademy.
GC Headspace com/channels/sample-preparation/
Website: www.crawfordscientific. Fundamentals of LC–MS technique/fundamentals-of-spe-
com/training-consultancy/ Website: www.chromacademy. mechanisms
gc-training/gc-headspace com/channels/lc-ms/principles/ Please send your event and training course
fundamentals-of-lc-ms-video-training- information to Alasdair Matheson
AMatheson@mjhlifesciences.com
course
29
The Column www.chromatographyonline.com Training & Events
Event News
9–12 April 2024
Analytica 2024
Messe München GmbH, Munich, Germany
Email: info@analytica.de
Website: https://analytica.de/en/munich/
28–31 May 2024

18th International Symposium on Hyphenated Techniques in Chromatography and Separation Technology (HTC-18)
Leuven, Belgium
Email: info@htc-18.com
Website: https://htc-18.com
20–25 July 2024

HPLC 2024
Denver, Colorado, USA
Email: olesik.1@osu.edu
Website: https://hplc2024-symposium.org/
15–18 September 2024

3rd Sample Preparation Conference/2nd Green and Sustainable Analytical Chemistry Conference
Crete, Greece
Email: epsillakis@tuc.gr
Website: https://www.eusp-gsac2024.tuc.gr/en/home
30
Contact Information
Group Publisher Executive Editor Headquarters: Custom Projects
Oliver Waters Alasdair Matheson 2 Commerce Drive
owaters@mjhlifesciences.com amatheson@mjhlifesciences.com Cranbury, NJ 08512, USA
Robert Alaburda Shannon Stolz
Sales Manager Managing Editor Director Senior Editor
Europe
Liz Mclean Kate Jones Jeanne Linke Northrop Terri Somers

lmclean@mjhlifesciences.com kjones@mjhlifesciences.com Managing Editor Senior Editor
Sales Operations Executive Megan Manzano
Sarah Darcy Senior Editor
sdarcy@mjhlifesciences.com
Executive Vice President, Vice President, Content Creative Director, Publishing Corporate
Healthcare and Industry Sciences Alicia Bigica Melissa Feinen
mfeinen@mdmag.com President & CEO Senior Vice President, Content
Brian Haug abigica@mjhlifesciences.com
Mike Hennessy Jr Silas Inman
bhaug@mmhgroup.com Associate Editorial Director Senior Art Director
Caroline Hroncich Gwendolyn Salas Chief Financial Officer Vice President,
Vice President, Industry Sciences
chroncich@mjhlifesciences.com gsalas@mjhlifesciences.com Neil Glasser, CPA/CFE Human Resources & Administration
Todd Baker
Senior Graphic Designer Shari Lundenberg
tbaker@mjhlifesciences.com Senior Technical Editor Chief Operating Officer
Jerome Workman Helena Coppola Beth Beuhler Senior Vice President, Mergers &
Associate Publisher hcoppola@mjhlifesciences.com
jworkman@mjhlifesciences.com
North America
Edward Fantuzzi Acquisitions, Strategic Innovation

Chief Marketing Officer
efantuzzi@mjhlifesciences.com Managing Editor Phil Talamo
Administration and Sales Offices Brett Melillo
John Chasse Woodbridge Corporate Plaza, Executive Creative Director,
National Account Manager jchasse@mjhlifesciences.com 485F US Highway One South, Suite 210, Chief Data Officer
Michael Howell Creative Services
Iselin, New Jersey 08830, USA Terric Townsend
mhowell@mjhlifesciences.com Editor Jeff Brown
Tel: +1 732 596 0276 | Fax: +1 732 647 1235
Will Wetzel Executive Vice President,
National Accounts Associate wwetzel@mjhlifesciences.com Corporate Office, Global Medical Affairs &
Claudia Taddeo 641 Lexington Ave., 8th Floor, Corporate Development
ctaddeo@mjhlifesciences.com Editor New York, NY 10022-4503, USA Joe Petroziello
Patrick Lavery
plavery@mjhlifesciences.com
MIKE HENNESSY SR
Assistant Editor Founder | 1960–2021
Aaron Acevedo
aacevedo@mjhlifesciences.com
Mission Statement
The Column (ISSN 2050-280X) is the analytical chemist’s companion within the dynamic world of chromatography. Interactive and accessible, it provides a broad understanding of technical applications and products while
engaging, stimulating, and challenging the global community with thought-provoking commentary that connects its members to each other and the industries they serve.
Whilst every effort is made to ensure the accuracy of the information supplied, MultiMedia Healthcare LLC accepts no responsibility for the opinions and statements expressed.
Custom Reprints: Contact Brian Haug at MJH Life Sciences. E-mail: bhaug@mmhgroup.com
© 2024 MultiMedia (UK) LLC Limited all rights reserved. No part of the publication may be reproduced in any material form (including photocopying or storing it in any medium by electronic means and whether or
not transiently or incidentally to some other use of this publication) without the written permission of the copyright owner except in accordance with the provisions of the Copyright Designs & Patents Act (UK) 1988 or
under the terms of the license issued by the Copyright License Agency’s 90 Tottenham Court Road, London W1P 0LP, UK.
Applications for the copyright owner’s permission to reproduce any part of this publication outside of the Copyright Designs & Patents Act (UK) 1988 provisions, should be forwarded in writing to Permission Dept.
e-mail: ARockenstein@mjhlifesciences.com. Warning: the doing of an unauthorized act in relation to a copyright work may result in both a civil claim for damages and criminal prosecution.
31

Vol20 Issue3 TheColumn March2024 Europe

Uploaded by

Copyright:

Available Formats

You might also like

Vol20 Issue3 TheColumn March2024 Europe

Uploaded by

Document Information

Original Description:

Original Title

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

Vol20 Issue3 TheColumn March2024 Europe

Uploaded by

Copyright:

Available Formats

Cover Story

24 Rapid Method to Characterize Adeno-Associated Virus (AAV)

In The Driving Seat 27

From the CEO Profiling VOCs in Whisky

Igor Normann - stock.adobe.com

2 News 5 Edge and Ferguson 9 Eßer and Rojahn 16 Napoletano

New Tool Improves MS/MS Analysis

2 News 5 Edge and Ferguson 9 Eßer and Rojahn 16 Napoletano

Peaks of the Month News In Brief

2 News 5 Edge and Ferguson 9 Eßer and Rojahn 16 Napoletano

The scientific community relies heavily on the analytical sciences to

Chromatography in the UK began in earnest inevitably impacts research-led teaching,

2 News 5 Edge and Ferguson 9 Eßer and Rojahn 16 Napoletano

2 News 5 Edge and Ferguson 9 Eßer and Rojahn 16 Napoletano

be simply outsourced. Ferguson finished Session Four: Separation Science in

Melissa Hanna-Brown of Pfizer and CAMS- in industry would lead it to be taught in a

2 News 5 Edge and Ferguson 9 Eßer and Rojahn 16 Napoletano

2 News 5 Edge and Ferguson 9 Eßer and Rojahn 16 Napoletano

Ion exchange chromatography is a standard method for characterising

Monoclonal antibodies (mAbs) are of different haematologic, immunologic,

2 News 5 Edge and Ferguson 9 Eßer and Rojahn 16 Napoletano

2 News 5 Edge and Ferguson 9 Eßer and Rojahn 16 Napoletano

2 News 5 Edge and Ferguson 9 Eßer and Rojahn 16 Napoletano

of a previous temperature screening using an the IgG1-based NISTmAb. Here, only 5 µg

2 News 5 Edge and Ferguson 9 Eßer and Rojahn 16 Napoletano

2 News 5 Edge and Ferguson 9 Eßer and Rojahn 16 Napoletano

These basic variants did not change after Conclusions

2 News 5 Edge and Ferguson 9 Eßer and Rojahn 16 Napoletano

Antibodies. J. Chromatogr. B: Anal. Technol. laboratory assistant at the University of

Ann Marie Rojahn studied chemistry E-mail: d.esser@ymc.eu

2 News 5 Edge and Ferguson 9 Eßer and Rojahn 16 Napoletano

Peptide mapping is considered a critical quality attribute (CQA) for the

modifications (1). It is an important assay sample preparation, separation by reversed-

2 News 5 Edge and Ferguson 9 Eßer and Rojahn 16 Napoletano

Figure 1: Schematic of desalting by SEC.

2 News 5 Edge and Ferguson 9 Eßer and Rojahn 16 Napoletano

2 News 5 Edge and Ferguson 9 Eßer and Rojahn 16 Napoletano

34th International Symposium

2 News 5 Edge and Ferguson 9 Eßer and Rojahn 16 Napoletano

2 News 5 Edge and Ferguson 9 Eßer and Rojahn 16 Napoletano

Biopharma discovery and development scientists are achieving new

Breakthroughs in biopharma analytical uncover ultra-low-level modifications, determine

2 News 5 Edge and Ferguson 9 Eßer and Rojahn 16 Napoletano

2 News 5 Edge and Ferguson 9 Eßer and Rojahn 16 Napoletano

2 News 5 Edge and Ferguson 9 Eßer and Rojahn 16 Napoletano

Adeno-associated virus (AAV) gene therapies have created analytical

2 News 5 Edge and Ferguson 9 Eßer and Rojahn 16 Napoletano

2 News 5 Edge and Ferguson 9 Eßer and Rojahn 16 Napoletano

2 News 5 Edge and Ferguson 9 Eßer and Rojahn 16 Napoletano

Susan Olesik, Program Chair, HPLC 2024

It is my pleasure to invite you to Additionally, the applications of artificial

2 News 5 Edge and Ferguson 9 Eßer and Rojahn 16 Napoletano

A horse and carriage on the promenade in

2 News 5 Edge and Ferguson 9 Eßer and Rojahn 16 Napoletano

2 News 5 Edge and Ferguson 9 Eßer and Rojahn 16 Napoletano

Messe München GmbH, Munich, Germany

28–31 May 2024

24 Rapid Method to Characterize Adeno-Associated Virus (AAV)