Accepted Manuscript

Title: Ultra-high-performance liquid chromatography for the characterization

of therapeutic proteins

Author: Szabolcs Fekete, Davy Guillarme

PII: S0165-9936(14)00183-6

DOI: http://dx.doi.org/doi: 10.1016/j.trac.2014.05.012

Reference: TRAC 14303

To appear in: Trends in Analytical Chemistry

Please cite this article as: Szabolcs Fekete, Davy Guillarme, Ultra-high-performance liquid

chromatography for the characterization of therapeutic proteins, Trends in Analytical Chemistry

(2014), http://dx.doi.org/doi: 10.1016/j.trac.2014.05.012.

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1

Ultra-high-performance liquid chromatography for the

characterization of therapeutic proteins

Szabolcs Fekete *, Davy Guillarme School of Pharmaceutical Sciences, University of Geneva, University of Lausanne, Boulevard d’Yvoy 20, 1211

Geneva 4, Switzerland

HIGHLIGHTS

UHPLC conditions were successfully applied to characterize therapeutic proteins

High-resolution separations are of prime interest in biopharmaceuticals analysis

IEX and SEC columns packed with sub-2-µm particles are commercially available

HILIC columns packed with small particles are useful for glycan profiling

The coupling of UHPLC with MS is highly informative

ABSTRACT

The pharmaceutical market has markedly changed over the past few years, and there are today

an increasing number of therapeutic drugs produced from biological sources. These

biopharmaceuticals include recombinant peptides, proteins, and monoclonal antibodies

(mAbs). Their detailed characterization could be difficult and time consuming, so it requires

powerful chromatographic and spectroscopic methods. In this context, the use of columns

packed with sub-2-µm particles at very high pressure, also known as ultra-high performance

(or pressure) liquid chromatography (UHPLC) has been reported as successful. Various

modes of chromatography are compatible with UHPLC columns and conditions, including

reversed-phase liquid chromatography (RPLC), size-exclusion chromatography (SEC), ion-

exchange chromatography (IEX) and hydrophilic interaction chromatography (HILIC).

Keywords:

Biopharmaceutical

Characterization

Hydrophilic interaction chromatography (HILIC)

Ion-exchange chromatography (IEX)

Mass spectrometry (MS)

Monoclonal antibody (mAb)

Reversed-phase liquid chromatography (RPLC)

Size-exclusion chromatography (SEC)

Therapeutic protein

Ultra-high-performance liquid chromatography (UHPLC)

* Tel.: +41 22 37 963 34; Fax: +41 22 379 68 08.

E-mail address: szfekete@mail.bme.hu (S. Fekete)

1. Introduction

The large majority of traditional pharmaceuticals are chemically-synthesized small-

molecule compounds. In addition to these “chemical” substances, there are a number of

substances that are produced from biological sources (i.e., biological systems or biological

molecules). These “biopharmaceuticals” include recombinant peptides, proteins or

glycoproteins [1,2]. The pharmaceutical potential of numerous proteins (e.g., interferons,

interleukins, and growth factors) that are naturally produced in the body was originally

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demonstrated more than 40 years ago. These molecules have obvious advantages, including

high efficacy, high specificity, wide therapeutic range, limited side effects, and exceptional

chemical and biological diversity. The clinical use of therapeutic proteins has enabled the

treatment of a wide range of life-threatening diseases, which were considered incurable or

untreatable only a few decades ago. Dozens of new drugs for the treatment of cancer, AIDS

and arthritis are on the market or are very close to regulatory approval [3]. Today, the global

protein therapeutics market is worth over $100b, thereby evolving towards a total

pharmaceutical market share of 20% [4,5]. We expect that, within the current decade, more

than 50% of the new drug approvals will be biologics [4,6,7].

Therapeutic proteins are large, heterogeneous and subject to a variety of enzymatic and

chemical modifications during expression, purification and long-term storage [5]. These

changes include several possible modifications, such as oxidation, deamidation,

glycosylation, aggregation, misfolding, or adsorption, leading to a potential loss of therapeutic

efficacy or unwanted immune reactions.

Because the development of biopharmaceuticals and biosimilars is especially complex,

regulatory agencies, such as the United States Food and Drug Administration (FDA) and

European Medicines Agency (EMA), require a demonstration of detailed characterization

(e.g., verifying primary structure and appropriate post-translational modifications, secondary

and tertiary structure), lot-to-lot and batch-to-batch comparisons, stability studies, impurity

profiling, glycoprofiling, determination of related proteins, excipients and protein aggregates

[8]. For this purpose, a single analytical technique is generally not sufficient, and a variety of

orthogonal methods are required to characterize such a complex sample fully, as summarized

elsewhere [9].

The primary structure of proteins can be identified with two reference techniques, namely

mass spectrometry (MS) and Edman degradation/sequencing [10]. The use of liquid

chromatography (LC) with tandem MS (LC/MS/MS) in de nova sequencing dominates the

process of sequencing proteins, and with peptide-mass fingerprinting combined with MS/MS

became the preferred techniques. Numerous spectroscopic techniques are available to assess

protein secondary and tertiary structure, such as X-ray crystallography, nuclear magnetic

resonance (NMR), UV/Vis spectrophotometry, fluorescence, circular dichroism (CD),

dynamic light scattering (DLS), static light scattering (SLS), differential scanning calorimetry

(DSC) and infrared spectrophotometry (IR) [11,12].

In addition to these methodologies, electrophoresis is also a key technique for protein

analysis, and different modes can be employed in slab gel or capillary format. Isoelectric

focusing (IEF) is able to distinguish charge differences (isoelectric point) among proteins

through the use of a pH gradient [13]. Capillary zone electrophoresis (CZE) has several well-

established attractive features for the characterization of such complex samples, including its

high resolving power and throughput. High-performance LC (HPLC) is another option for the

detailed characterization of proteins [14].

The three most common modes of chromatography are size-exclusion chromatography

(SEC), which separates proteins based on their size or hydrodynamic volume, ion-exchange

chromatography (IEX), able to separate proteins based on their charge, and reversed-phase

chromatography (RPLC), where separation occurs on the basis of the hydrophobicity of the

proteins. This last strategy offers a higher resolving power than SEC and IEX.

Today, one of the most widely-used analytical methods for therapeutic protein

characterization is LC, probably due to the impressive developments of the past few years,

enabling a new level of chromatographic performance. Recent developments in LC columns,

such as ultra-high-performance LC (UHPLC), packed with wide-pore superficially porous

particles (SPPs) and organic monolith columns allow a dramatic increase in the efficiency and

the resolution of protein separations, even with large, intact molecules.

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The aim of this article is to review the current trends in UHPLC and the potential for

UHPLC strategy for the characterization of therapeutic proteins. In this review, we focus

solely on unidimensional separations and particle-based, stationary-phase formats for routine

UHPLC applications.

2. Need for high kinetic performance in protein analysis

Higher separation efficiencies and throughput have always been of great interest in LC.

The pharmaceutical industry is interested in using rapid, efficient procedures for qualitative

and quantitative analyses to cope with the large number of samples and to reduce the time

required to deliver results [14]. When dealing with protein analysis, selectivity is often limited

so the only way to improve separation is to increase chromatographic efficiency. Moreover,

high-molecular-weight compounds, such as intact proteins, may have numerous

conformations, post-translational modifications, or multiple isoforms that can cause

broadened peaks and shifted retention times in the chromatograms. Another reason for

broadened peaks is the slow molecular diffusion of these compounds due to their large size.

Typical routine tasks are the separation of oxidized, deamidated or reduced forms of intact

proteins. Because the differences in molecular structure are small, similar retention behaviors

of the different forms are expected. In many cases, the selectivity cannot be improved and, as

a result, enhancement of separation efficiency must be considered. The stationary-phase

dimensions and morphology, and the mobile-phase temperature are the two most relevant

parameters for improving the efficiency of protein separations. Thanks to the latest stationary-

phase technologies, such as sub-2-μm porous particles, superficially porous particles (SPPs),

or wide-pore monolithic columns, the separation power was significantly increased in recent

years. In addition, it is also possible to extend the column length (e.g., coupling columns in

series) to achieve the required plate count or peak capacity. The use of elevated mobile-phase

temperature in the range 60–90°C further improves performance. At higher temperature, the

viscosity of the mobile phase decreases and the diffusivity of large proteins increases, leading

to sharp chromatographic peaks. The other benefit of elevated mobile-phase temperature is

reduction in adsorption of undesired proteins at the surface of the stationary phase.

3. Reversed-phase liquid chromatography (RPLC)

In RPLC, solute retention is predominantly mediated through the hydrophobic interactions

between the non-polar amino-acid residues of the proteins and the bonded n-alkyl ligands of

the stationary phase. Proteins are thus eluted based on their hydrophobicity. Large molecules

possess a so called “on-off” retention mechanism. Their retention strongly depends on minor

variations in the solvent strength, and a small change (<1%) in the organic modifier content

can lead to a significant retention change. For this reason, isocratic conditions are impractical,

and gradient elution mode is recommended. The efficiency of RPLC is superior to other

chromatographic modes and its robustness makes it well suited for use in a routine analysis

environment [5]. Mobile phases typically consist of water, acetonitrile or methanol and 0.1%

trifluoroacetic acid or formic acid (TFA, FA). The separation mechanism is based on a

combination of solvophobic and electrostatic interactions, the latter governed by the ion-

pairing interaction of TFA with basic side chains of a few amino acids (i.e., arginine, lysine,

histidine) and the N-terminus amino groups [14].

It was recognized very early that one of the best approaches to improving intact protein

RPLC separations would be to use small-particle packing material [15]. It came into practice

in the late 1990s, when very fine particles (sub-2-μm or sub-μm) could strongly improve the

separation performance, but required a huge increase in system pressure. To overcome the

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pressure limitations of conventional HPLC with a pressure limit of 400 bar, the research

groups of Jorgenson [16,17] and Lee [18] constructed prototype instrumentation and

experimental columns packed with very small non-porous material and performed analyses at

very high pressures (up to 7200 bar). New nomenclatures have appeared to describe this

higher back-pressure requirement, including ultra-high-pressure liquid chromatography, ultra-

high-performance liquid chromatography or very high-pressure liquid chromatography (i.e.,

UHPLC, UPLC, VHPLC or vHPLC).

The first commercial system for ultra-high pressure operation was released in 2004. It was

able to operate at pressures as high as 1000 bar, and the system was known as ultra-

performance liquid chromatography (UPLC). Since then, UHPLC systems compatible with

pressures up to 1200–1400 bar have become available from most HPLC manufacturers.

The use of such UHPLC systems and columns packed with highly-efficient sub-2-μm

particles became popular for the separation of small molecules very soon after their

introduction. At the beginning, only columns packed with standard pore-size particles (e.g.,

80–130 Å) were available, so the UHPLC approach was not amenable to separation of larger

proteins. In 2006, columns packed with wide-pore (300 Å) 1.7-µm particles were introduced

and opened up new avenues for protein analysis. These columns packed with sub-2-µm, fully

porous, hybrid particles (Acquity BEH300) were used with great success in protein and

peptide separations. Due to the intrinsic chemical stability of this hybrid-particle technology,

a wide pH range (pH 1–12) and high-temperature operation (up to 80°C) can be employed,

enabling a versatile, robust separation technology for method development. Since then,

several new generations of superficially porous, wide-pore materials and organic monoliths

have also been commercialized.

3.1. Non-porous versus fully porous particles

Non-porous and porous particles are the two main types of spherical packing materials

used for fast HPLC separation of biomolecules. The major difference between these two types

of particles is that the porous particles show higher resistance to the transparticle mass-

transfer process in the stagnant mobile phase inside the pores. Issaeva et al. demonstrated an

extremely high-speed separation of proteins and peptides using 1.5-µm non-porous silica

particles [19]. Non-porous particles can provide lower mass-transfer resistance and higher

efficiency than porous particles, but they have significantly lower surface area and sample-

loading capacities.

According to Wu et al., the loading capacity of 1.7-µm Acquity C18 porous particles is

~16.5 times larger than that of Micra C18 non-porous 1.5-µm particles [20].

Another issue is the very low retention achieved on non-porous particles compared to

totally porous particles. In spite of the early success of non-porous particles in protein

analysis, most of the applications are currently performed with sub-2 µm, fully porous

particles or using the new generation of superficially porous particles (SPPs). Today, two

providers (Waters and Agilent) offer sub-2-µm, fully porous, wide-pore (300 Å) materials for

reversed-phase protein separations. Those packings are available with C3, C4, C8, C18 and

diphenyl chemistries, offering a broad range of selectivity. There is another stationary phase

packed with 1.9-µm particles and possessing pore size of 175 Å (ThermoFisher Scientific)

that may also be of interest for the separation of proteins possessing molecular weights below

20 kDa [21].

A recent study systematically compared the peak capacity achieved on conventional 3–5-

µm wide-pore packings against 1.7-µm, fully porous, and a new generation of 3.6-µm, SPP,

wide-pore particles [21]. The last two types of column provided higher separation power,

especially for large proteins (e.g., myoglobin, bovine serum albumin, and filgrastim).

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Another study showed the possibilities of performing very fast separation (1.5–2 min) of

therapeutic protein variants using short columns of 50 mm packed with sub-2-µm porous

particles [22]. UHPLC was also used to separate a mixture of 10 protein standards. The

optimized method yielded improved chromatographic resolution, enhanced sensitivity, and a

three-fold increase in throughput. Subsequent cell-lysate analysis demonstrated no

compromise in chromatographic or mass spectral data quality at one-third of the original run

time [23].

A possible complication of UHPLC relates to the effect of pressure and mobile-phase

velocity (frictional heating) on the retention properties of large molecules. Dramatic changes

of retention with pressure were reported in a recent study [24]. An increase of retention factor

of around 150% for peptides (~ 1.3 kDa), 800% for insulin (~ 6 kDa) and up to > 3000% for

myoglobin (~ 17 kDa) was observed when increasing the pressure from 100 bar to 1100 bar.

The important effect observed for the isocratic elution of proteins probably relates to

conformational changes of the protein in addition to the effect of pressure on molecular size.

In gradient-elution mode, the pressure-related effects on protein retention were found to be

less pronounced, but still readily observable (an increase of apparent retention factor between

0.2–2.5 was observed).

3.1.1. Intact protein analysis (“top-down” approach)

In RPLC, it is possible to analyze therapeutic proteins in their intact forms. This approach

is often considered for impurity profiling or heterogeneity evaluation. This can be useful in

the biopharmaceutical field, to evaluate the stability and the purity of therapeutic protein

samples, as a release test, or to compare the chromatographic profile of biosimilar products.

Typically oxidized, reduced and deamidated forms of the native protein can be separated and

quantified in RPLC conditions.

Fig. 1 illustrates the analysis of native filgrastim (18.8 kDa) and its related

impurities/degradants (oxidized and reduced forms). As shown in Fig. 1, the baseline

resolution of these different forms can be obtained in less than 1.4 min using a 50-mm

column, at 80°C, and a reasonable flow rate. The resolution of these peaks was excellent,

demonstrating that high-throughput analysis can be carried out effectively for these

biomolecules.

In another study, the separation of the related oxidized and reduced forms of interferon α-

2A (19.2 kDa) were successfully performed in 5 min on a 150-mm long column packed with

1.7-µm fully porous particles [25].

UHPLC is useful for not only fast separations, but also performing high-resolution

separations by using long columns. Fig. 2 shows the gain in resolution upon conversion of a

conventional HPLC method on a column packed with 5-µm particles to a UHPLC method

using columns packed with sub-2 µm particles [5]. All the pre-eluting and post-eluting

variants of this 30-kDa protein were better resolved in the UHPLC method with a comparable

analysis time.

Coupling columns packed with sub-2-µm particles in series was recently applied to

improve peak capacity for the characterization of a 150-kDa intact monoclonal antibody

(mAb) [26]. In this work, a 150-mm-long column generated a peak-capacity value of nc = 62,

while the 30-cm and 45-cm-long columns provided nc = 95 and nc = 117, respectively.

Despite the resolution power being doubled with the 45-cm-long column, the baseline

resolution of the mAb from a few variants eluted prior to the main isoform could not be

achieved. Nevertheless, the separation cannot be further improved by simply increasing the

column length or gradient time because the experiments were performed at 80°C to limit

adsorption at the surface of the stationary phase. A gradient time longer than 20 min may be

detrimental for the stability of intact mAbs and should be employed with caution as

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documented elsewhere [27]. Finally, this improved analysis of intact mAbs could be useful

for comparing the chromatographic profile of between batches or with biosimilar products.

3.1.2. Analysis of protein fragments (limited proteolysis, “middle-down” approach)

Complete characterization of a large protein cannot be accomplished by the analysis of the

intact protein alone, so various enzymes, such as pepsin, papain, Lys-C and IdeS, are often

used to obtain relatively large fragments to facilitate the investigation of their micro-

heterogeneity. This limited proteolysis approach – or “middle down” – was used recently for

the characterization of mAbs [13]. Papain is primarily used to cleave mAbs into three

fragments, namely one crystallizable fraction (Fc) and two identical antigen-binding

fragments (Fab) of ~50 kDa each However, pepsin and IdeS generate F(ab′)2 fragments of

~100 kDa.

The reduction of disulfide bonds is also commonly used to produce two light-chain (LC)

and two heavy-chain (HC) fragments of 25 kDa and 50 kDa, respectively. To prepare only

25-kDa fragments, the reduced mAb (HC and LC) can be further digested with papain. This

process yields three main fragments of ~25 kDa, namely the native LC, the single-chain Fc

(sFc), and the Fab portion of the HC (Fd). This approach (generating ~ 25-kDa domains) is

generally known as the combinatory strategy (combination of intact-mAb reduction and

papain digestion) and is useful for the characterization and comparison of mAbs by RPLC

[28]. In most cases, the limited proteolysis approach can determine the heterogeneity of mAb

related to the different parts of the molecule.

Fig. 3 shows an example of a fast analysis of 25-kDa mAb (IgG1) fragments obtained after

limited proteolytic digestion approach. Several variants of the LC, sFc and Fd fragments can

be separated on a 150-mm-long column, applying a 6-min gradient at elevated temperature.

The inset on Fig. 3 displays the variability of the mAb related to the Fd domain. A generic

computer-assisted method-development approach was recently proposed for the RPLC

analysis of mAb fragments on sub-2-µm fully or superficially porous particles [29].

Outstanding separations within relatively short analysis times (typically 6–10 min) were

demonstrated on rituximab, bevacuzimab and panitumumab employing gradients of 30–40%

acetonitrile, temperatures above 70°C and 0.1% TFA as ion-pairing additive [29].

Fast, efficient separation of mAb-fragment variants in UHPLC conditions were reported in

several studies [5,8,25–29], suggesting that UHPLC is indeed a very powerful, promising tool

for characterization of mAbs. A recent study showed the advantages of the middle-down

approach versus the top-down approach to mAb analysis [30]. To improve structural analysis

of mAbs, a middle-down UHPLC-MS protocol was suggested using fast sample preparation

(about 1 h) followed by subsequent analysis of 25-kDa fragments. The reported results

showed that the application of this middle-down approach doubled the mAb-sequence

coverage previously obtained with top-down MS [30].

3.1.3. Peptide mapping (“bottom-up” approach)

Peptide mapping is commonly undertaken to determine the primary structure of proteins

after cleavage into proteolytic peptides. The power of peptide mapping lies in the large

number of site-specific molecular features that can be detected. When using one digestion

enzyme (e.g., trypsin), peptide mapping is typically carried out for protein identification.

When using multiple enzymes, peptide mapping is applied for the confirmation of a complete

amino-acid sequence, e.g., when confirming the amino-acid sequence of a biosimilar and

comparing it with the originating molecule. Historically, this approach is certainly the most

important one in RPLC analysis of proteins. However, peptide mapping is highly demanding

in terms of chromatographic performance, since samples are very complex, containing

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hundreds of peaks [5,31]. So far, many papers have been published on UHPLC peptide

mapping, but we discuss only a few key references in this review.

As an example, the determination of methionine oxidation in mAb – by peptide mapping –

was performed using BEH300 C18 1100 x 2.1 mm, 1.7-µm column [32].

In a similar study, peptide fragments generated after tryptic digestion were analyzed to

characterize the oxidation of mAb samples [33]. UHPLC coupled with electrospray ionization

time-of-flight MS (ESI-TOF/MS) and multivariate analysis were used to detect alterations in

the conformational states of the protein. For this study, a (100 x 1 mm) C18 column packed

with 1.7-µm particles was used.

Quantification and characterization of mAb deamidation was performed by Wang et al.

using UHPLC-MS after tryptic digestion [34].

An alanine replaced with a serine sequence variants was separated and identified in an

IgG4 monoclonal antibody by UHPLC-MS [35].

A new UHPLC peptide-mapping approach using ammonia as an mobile-phase additive

was reported by Liu et al. [36]. Compared to traditional methods using acidic mobile phase

additives (trifluoroacetic acid or formic acid), this method exhibited excellent overall

performance. It was particularly advantageous over the traditional approaches in terms of the

UV and MS sensitivities, and sequence coverage. Due to significant differences in the

chromatographic selectivity, supplemental peptide-mapping analysis could potentially be

carried out using basic mobile-phase conditions yielding “orthogonal” separations.

The most popular and widespread stationary-phase chemistries in peptide-mapping

analysis are C18, C8 and C4. The new CSH (Charged Surface Hybrid) phenyl-hexyl phase

was studied as an alternative bonded phase in reversed-phase peptide separation [37]. Peak

capacities of 150–200 were achieved on a 100-mm-long column in 8 min. Moreover, a

possible mathematical treatment of peptide maps was evaluated to improve resolution.

Applying power functions on raw chromatographic data can be a neat tool to improve peak

recognition and resolution [37].

3.2. Superficially porous particles as an alternative to fully porous and non-porous particles

Columns packed with fully porous particles have constraints in separation speed and

efficiency because of limitations in the stationary-phase mass transfer, resulting from the

relatively long diffusion times required for proteins to cross the porous structure [38]. The

concept of SPPs (often named shell, core-shell, fused-core, or partially porous) was first

applied by Horváth in the late 1960s [39,40]. They were initially intended for the analysis of

macromolecules, such as peptides and proteins. SPPs are made of a solid, non-porous silica

core surrounded by a porous shell layer having similar properties to that of the fully porous

materials conventionally used in HPLC. Later, Kirkland showed that 30–40-μm diameter

SPPs provided much faster separations compared with the large, fully porous particles that

were used earlier in LC [41]. The rationale behind this concept was to improve the column

efficiency by shortening the diffusion path that the analyte molecules must travel and to

improve their mass-transfer kinetics.

Nowadays, columns packed with the latest generation of sub-3-µm SPPs rival the

efficiency of columns with sub-2-µm fully porous particles, but at only half the operating

pressure. To improve the separation efficiency further, columns packed with sub-2-µm SPPs

(1.3–1.7 µm) were recently commercialized and showed impressive efficiency [42–45]. SPP

materials have been successfully applied in many fields of application, including the

characterization of large biomolecules under their intact, fragmented or digested form.

Columns packed with wide-pore (300 Å) 3.6-µm SPPs (Aeris Widepore) showed

significant gains in analysis time and peak capacity compared to fully porous materials for

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intact protein analysis [21]. Fig. 4 shows the separation of intact Interferon α-2A-related

impurities (oxidized and reduced forms) within 5 min applying a 150-mm-long column

packed with 3.6-µm SPPs [25]. In another recent study, the same column was used to separate

N-methionylated variants of Interferon α-2A in a drug substance and finished product, and for

the analysis of variants in untreated, oxidized and slightly degraded samples [46].

A column packed with 400-Å SPPs of 3.4-µm (Halo Fused-core) was also used for the

efficient separation of large intact proteins. This study reported the fast separation of intact-

protein mixtures, and very high-resolution separations of mAbs and associated variants [47].

In another work, the efficiency and the possible analysis time of 1.7-µm SPPs and fully

porous materials were compared for peptides and large intact proteins [48]. This study

suggests a two-fold increase in terms of achievable peak capacity and analysis speed for large

proteins when using SPPs, compared to those achieved with fully porous materials of the

same particle size.

For the separation of peptides and moderate size proteins, a 160-Å packing was introduced

in 2010 by Advanced Material Technology and Supelco [49,50]. An average pore size of 160

Å allowed unrestricted access of molecules up to ~15 kDa, depending on their molecular

conformation [51].

Recently introduced 1.3-µm and 1.6-µm SPPs were applied to peptide mapping of mAb

samples [44,45]. By combining long columns (200–300 mm) with long analysis time, peak

capacity around 1000 could be reached using 1.3-µm SPPs for 0.5–2-kDa peptides. When

using 50-mm-long columns and analysis time of 10 min, peak capacities of 200–300 can be

readily achieved.

4. Size exclusion chromatography (SEC)

SEC is a traditional technique widely employed for the detailed characterization of

therapeutic proteins. It can be considered a reference technique for the qualitative and

quantitative evaluation of aggregates. The main advantage of this approach is the mild

mobile-phase conditions that permit the characterization of proteins with minimal impact on

the conformational structure and local environment.

There is now a wide variety of porous packing materials with different particle sizes

available for the separation and characterization of macromolecules by SEC [52]. As an

example, UHPLC technology, originally developed for RPLC applications, is available for

SEC operation using 1.7-µm BEH silica particles with varying pore sizes of 125–900 Å [53].

Using the recently introduced sub-2-µm particles in SEC (aqueous mobile phase, 150-mm

column length), very high pressures (up to 500–600 bar) can be generated. Thermal effects

and shear forces might therefore become critical for temperature- or pressure-sensitive

proteins [54,55]. These effects might also cause some changes in protein structure, and there

is an additional risk of on-column protein denaturation and aggregation.

Nevertheless, very fast separations of peptides, myoglobulin and insulin aggregates were

demonstrated with 1.7-µm SEC columns [56]. These very efficient columns were also applied

to characterize recombinant mAbs and aggregates within 3–4 min [57].

5. Ion-exchange chromatography (IEX)

IEX is widely used for profiling the charge heterogeneity of proteins resulting from

enzymatic or chemical modifications [5]. The technology is used for characterization and

release testing. Currently, cation exchange (CEX) is considered the gold standard for the

analysis of mAbs, given their basic nature. In IEX, the sample is loaded onto a column under

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mobile-phase pH where the net charge of the protein is opposite to the charged ligand of the

stationary phase. Elution is then achieved using a salt gradient and/or a pH gradient [5].

Since electrostatic/ionic interactions are energetically strong, the IEX mechanism is

considered a slower mass-transfer process, so non-porous materials are typically used for IEX

separations of proteins to eliminate the contribution of trans-particle mass-transfer resistance

that strongly contributes to band broadening of large biomolecules. Usually 5–10-µm non-

porous particles are used in routine IEX applications. However, particle size also plays a role

in band broadening with non-porous material, but its impact is certainly less important than

for fully porous or SPP particles. Indeed, particle size mostly affects the Eddy dispersion of

columns packed with non-porous particles. This is probably the reason why UHPLC has not

yet been considered for IEX separations. Currently, only two manufacturers offer IEX

columns of 1.7-µm non-porous particles (Agilent BioMab and Sepax Technologies’

Antibodix).

6. Hydrophilic interaction chromatography (HILIC)

HILIC combined with fluorescence detection is a key technique to analyze N-glycans

originating from biopharmaceuticals [58–62].

This mode of chromatography is often performed on columns packed with sub-2-µm

particles. This approach enables the very efficient separation of glycans and allows structural

isomers to be resolved. The method has also been applied successfully to the measurement of

sialic acid and phosphate-containing glycans that provide increased retention over their

neutral counterparts [58–62].

Ahn et al. separated N-linked glycans by HILIC utilizing a 1.7-µm glycan column and 2-

aminobenzamide (2-AB) to label glycans [60]. Similar to the RPLC mode, the HILIC column

packed with 1.7-µm amide sorbent improved significantly the peak capacity compared to 3-

µm packing. The optimal peak capacity was achieved at a flow rate of 0.2–0.5 mL/min.

Excellent resolutions were achieved for the high-throughput separation of 2-AB-labeled

glycans released from fetuin, RNase B, and human IgG.

Bones et al. applied UHP-HILIC as a second dimension of a multidimensional separation

[61]. The combined IEX x HILIC allowed the rapid, comprehensive analysis of complex N-

glycosylation profiles of therapeutic glycoproteins.

The combination of UHP-HILIC and capillary electrophoresis with laser induction

fluorescence detection (CE-LIF) was applied in another study for the identification of

oligosaccharides on recombinant β-glucuronidase [62].

7. Combination of UHPLC and MS

MS has become increasingly popular for the characterization of peptides and proteins. The

success of this technique is related to the introduction of two highly sensitive and “soft”

ionization techniques [i.e., electrospray ionization (ESI) and matrix-assisted laser desorption/

ionization (MALDI)], enabling the transfer of intact proteins into the gas phase without

fragmentation, and to the continuous improvements of MS instruments that can detect high

m/z ratios at elevated sensitivity [63]. In addition, modern MS instruments can discriminate

proteins with close m/z ratios because of their very high spectral resolution {e.g., maximal

resolution of 50,000 for time-of-flight (TOF), up to 500,000 for Orbitrap [64] and up to

several millions with Fourier-transform ion-cyclotron resonance (FT-ICR) [65]}, in

comparison with quadrupole-based instruments (resolution 1000).

ESI and MALDI can be readily combined with RPLC and HILIC, while the other

chromatographic approaches (SEC or IEX) are compatible with MALDI (more tolerant to

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non-volatile salts) and have limited on-line compatibility with ESI. The combination of RPLC

and MALDI is primarily off-line and consists in the collection of spots originating from the

RPLC column. In contrast, ESI is a flow-based on-line approach, where the liquid sample is

transformed into an aerosol via a nebulization process and ionized to produce gas-phase ions.

The main application of LC-MS in the analysis of therapeutic proteins is identification of

the protein primary structure and impurities or degradation products. Nowadays, de novo

sequencing for the rapid determination of protein sequence can be achieved by peptide

mapping using RPLC-ESI/MS/MS. Alternatively, MALDI/MS of collected RPLC peaks is

also feasible, albeit slower and more labor intensive. LC-MS-based peptide mapping of mAbs

typically covers over 98% of the sequence determination and the confirming protein

identification [5]. Implementing tandem MS systems obtains fragmentation data on the

peptides and further enhances confidence in peptide identity, sequence and modification sites

[5].

Enzymatically-derived peptide fragments of a mAb were generated using a dedicated

sample-preparation protocol, then separated by RP-UHPLC and detected by ESI-MS/MS

using a bottom-up approach [66]. The MS/MS was able to sequence co-eluting peptides.

UHPLC was also combined with QqTOF/MS in a detailed study of mAb deamidation

under extreme conditions to achieve chromatographic resolution of the deamidated products,

while maintaining relatively short analysis time [67].

The on-line LC-MS approach can also be applied to separation and identification of HC

and LC species in SEC by adding organic modifiers (20% acetonitrile) and a mixture of TFA

and FA into the mobile phase, albeit at the cost of separation power [68].

The feasibility of direct coupling of IEX methods applying low ionic strength pH gradient

with MS was recently studied for the separation of mAb variants [69]. By applying an

ammonium-hydroxide buffer system and methanol in the mobile phase, an improvement in

the ionization efficiency was observed.

Using an additional make-up pump (switching valve) between the column and the MS inlet

to increase the organic solvent content of the mobile phase can also improve ionization

efficiency and sensitivity. This approach is sometimes applied to the analysis of small

molecules in RPLC and supercritical fluid chromatography (SFC). Such make-up pumps,

splitters and switching valves are available from different vendors. However, applications

using SEC and IEX have not been found in the literature for protein analysis.

8. Conclusion

As shown in this review paper, there are some significant advantages in using UHPLC

columns packed with sub-2-µm particles to enhance the throughput or resolving power of

regular HPLC. The advantages observed in UHPLC conditions with small molecules are still

valid in most cases with large biomolecules, at least in the RPLC mode, though a few

modifications of the mobile and stationary phases may be required.

For a successful analysis of intact proteins, mAbs (150 kDa) or mAb fragments of 25–100

kDa (top-down or middle-down approaches), the RPLC mobile-phase conditions have to be

adjusted. It is advisable to add an ion-pairing reagent (0.1% TFA) and to thermostat the

column at T > 70°C. In terms of stationary phases, sub-2-µm C18 or C4 bonded phases on

fully porous or SPP wide-pore particles appear to be ideal. Under these conditions, a peak

capacity of more than 100 can be achieved in about 10–20 min, with relatively long UHPLC

columns of 150–450 mm. In the case of peptide mapping (bottom-up approach), there is no

need for a very high temperature (50°C can be sufficient) and the column pore size can be in

the range 130–200 Å. Under such conditions, a peak capacity of 300–600 can be obtained in 1

h, using a 150-mm column packed with sub-2-µm particles.

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Other than their use for RPLC, columns packed with sub-2-µm particles can also be employed

with other chromatographic modes, such as HILIC, SEC or IEX. HILIC is generally

employed for profiling glycans. In SEC, caution should be exercised to minimize the

artifactual aggregates under UHPLC conditions. For IEX, the interest in columns packed with

sub-2-µm particles is less obvious, and non-porous particles are generally used, as the kinetic

advantage of UHPLC-type columns is much more limited in IEX.

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Fig. 1. Fast separation of filgrastim-related proteins. Column: Acquity BEH300 C18 50x2.1 mm, 1.7

µm. Mobile phase A: 0.1% TFA in water, mobile phase B: 0.1% TFA in acetonitrile. Gradient: 47–62

% B, Temperature: 80°C, Detection FL: 280–350 nm. Injected volume: 1 µL. Peaks: 1,2: oxidized

filgrastim, 3: filgrastim, 4: reduced filgrastim. (Adapted from [22], with permission).

Fig. 2. Separation of a 30-kDa therapeutic protein on a reversed-phase HPLC column packed with 5-

µm (a) and sub-2-µm particles (b). Both columns were operated on the same UHPLC system under

identical gradient conditions at 60°C using mobile phases containing water, acetonitrile and 0.1%

TFA. The 280-nm UV traces are displayed. (Adapted from [5], with permission).

Fig. 3. Fast analysis of 25-kDa mAb (IgG1) fragments obtained after limited proteolytic digestion.

Column: Acquity BEH300 C18 150x2.1 mm, 1.7 µm. Mobile phase A: 0.1% TFA in water, mobile

phase B: 0.1% TFA in acetonitrile. Gradient: 31–40 %B in 6 min, Temperature: 80°C, Detection FL:

280–360 nm. (Unpublished data from the authors’ laboratory).

Fig. 4. Representative chromatograms of Interferon α-2A related proteins. Conditions: mobile-phase

A: 0.1% TFA in water, mobile phase B: 0.1% TFA in ACN, gradient profile: 35–60% B in 5 min,

followed by 2 min re-equilibrating, flow rate: 0.3 mL/min, T = 60°C, injected volume = 5 µL, λ = 210

nm. Column: Aeris WP C18, 150 x 2.1 mm, 3.6 µm. (Adapted from [25], with permission).

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