the quest for highly effective resolution: therapeutic protein … · 2018-01-24 · the global...

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THE QUEST FOR HIGHLY EFFECTIVE RESOLUTION: THERAPEUTIC PROTEIN CHARACTERIZATION VIA SEPARATIONS-COUPLED NATIVE MS

INTRODUCTIONThe biopharmaceutical industry has grown tremendously in less than four

decades. The first commercial biologic therapeutic, the recombinantly expressed

human insulin Humulin, made by Eli Lilly & Co. and Genentech, was launched in

1982. Today, biologics dominate annual blockbuster drug sales lists, and by 2020

the global market is expected to exceed $300 billion.1 New types of biologically

derived molecular therapies are now being developed at an unprecedented rate.

The rapid growth of biologic drugs has been underpinned by several significant

advancements in the analytical instruments used to characterize them. The

biopharmaceutical industry depends on these tools to speed up drug discovery

and development and to ensure new medicines meet increasing demands for

safety and efficacy.

An analytical platform that combines a separation technique such as liquid

chromatography or capillary electrophoresis (CE), interfaced with mass spectrometry,

is the main way new biologics are characterized. Modern therapeutic protein

characterization is usually accomplished using multiple MS-based workflows. Each

workflow offers a unique perspective to identify and measure certain molecular

features of the drug. Researchers can take the complementary data from this suite of

workflows to construct detailed models of highly complex proteins.

The principal characterization workflow, intact mass analysis, uses an MS system

to measure the mass of the whole, intact biologic. Ideally, intact mass analysis

minimally perturbs the drug substance and therefore offers a quantitative bird’s-

eye view of the different protein isoforms that compose a product. As samples

become heterogeneous, however, intact mass data quality suffers tremendously.

To keep pace with current and future analytical challenges, platform peak

capacity—defined here as the capacity of an entire analytical platform to separate

peaks and identify individual protein isoforms—must be maximized through

conscientious optimization. Several new methods for intact protein analysis have

recently been developed that improve platform peak capacity. These methods,

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covered in detail below, include using MS methods and hardware capable of high

effective resolution, adopting native MS techniques for high spectral separation,

and coupling solution separations directly to MS.

AVOIDING UNNECESSARY COMPLEXITIES While the measured mass is the primary piece of information used to identify a

protein isoform, mass accuracy—the closeness of a measured mass to that of a

hypothesized theoretical mass—represents the confidence of that identification.

The success of an intact mass workflow depends on its ability to resolve individual

isoforms to attain highly accurate mass measurements.

Analytical platforms are usually discussed in terms of the theoretical resolution

achievable by each subsystem (such as LC, CE, and MS); however, it is the actual

measured—or effective—resolution that determines the analytical performance.

In mass spectrometry, resolution is defined by the International Union of Pure and

Applied Chemistry as M/∆M, where ‘M’ is the measured mass-to-charge (m/z)

ratio of an ion and ‘∆M’ is the minimum separation (m/z) between two adjacent

peaks which can be possible in a given spectrum. Although it might be possible

to emulate a system’s theoretical resolution using surrogate small-molecule

standards such as cesium iodide, the measured resolution of an intact protein raw

mass spectrum is often significantly lower than the theoretical value.

Several factors, impacting various points of the workflow, can contribute to

MS peak broadening and reduced effective mass resolution compared to a

platform’s theoretical performance. Modern MS instruments incorporate two

basic components: an ionization source to convert solution-phase proteins into

gas-phase ions and a mass analyzer to separate and detect ions according to mass

versus charge.2 Detailed characterization is typically performed using electrospray

ionization (ESI) coupled directly to a mass analyzer such as time of flight (TOF),

Orbitrap, or Fourier transform ion cyclotron resonance (FTICR). On the vast

majority of available instruments, analysts use ESI MS systems to determine an

average mass to characterize large intact proteins such as monoclonal antibodies

(mAbs). In such an experiment the MS resolution is sufficient to resolve different

charge states but not the individual isotopes which make up each charge state.

For an intact mAb, the “natural” peak width caused by the isotope distribution is

approximately 25 Daltons (figure 1).3

Figure 1. Zhang et al. simulated MS peak width of a mAb (M + H)+ ion using increasing resolution (R = M/ΔM). A resolution of 20,000 produces a peak width that is a very close fit of the natural peak width shown at 200,000 in which individual isotopes are clearly resolved. Figure was reproduced from Zhang et al., 2008 with permission from John Wiley and Sons.3


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Since biological samples always exhibit some degree of heterogeneity, broad

MS peaks can be, and often are, a reflection of the sample itself. As the role

of the analyst is to measure the existing heterogeneity of a sample, sample

heterogeneity is not an aspect that analysts can usually control; however,

minimizing artifact species is quite helpful in producing high effective MS

resolution. Sample degradation due to pH- or temperature-related stress

can lead to the addition of several possible low-mass posttranslational

modifications (PTMs), such as deamidation (+ 1 Da), disulfide cleavage (+ 2

Da), and oxidation (+ 16 Da). Many artificial PTMs result in mass changes that

are either less or approximately equal to the natural peak width value, which

may be at best only partially resolvable by MS alone. The increased presence

of near-isobaric isoforms resulting from low-mass PTMs will increase the

apparent width of an MS peak.

Desalting is a major prerequisite for MS analysis of intact proteins by ESI MS and

often presents an opportunity for workflow optimization. The ESI process allows

proteins to transition from being in solution in the liquid phase to becoming

ions in the gas phase for MS detection. By aerosolizing protein solutions from

a metal needle with a high applied voltage in a heated environment, proteins

immersed within the shrinking droplets are coaxed into becoming individual

protein ions. High concentrations of nonvolatile salt during the ESI process will

suppress or altogether eliminate the ionization of proteins. Low concentrations

of nonvolatile salt during ESI can often generate interpretable data but will

reflect strongly bound noncovalent salt adducts on proteins. Both sodium (+

22 Da) and potassium (+ 38 Da) are common metals that often form low-mass

adducts which approximate the natural peak width of a mAb and thereby

broaden MS peaks of large intact proteins such as mAbs.

Similar to desalting, poor desolvation of protein ions can diminish effective

resolution and cause MS peak broadening. During ESI and throughout the

ion transmission process, protein ions must undergo complete desolvation

to disrupt weak interactions of nonspecifically bound molecules that will

otherwise result in apparent MS peak broadening. In this case, water (+ 18 Da)

or other common mobile-phase components, such as acetonitrile (+ 41 Da) and

formic acid (+ 46 Da), can act in a similar manner to the examples noted above

in which low-mass chemical artifacts approximate a protein natural peak width.

ESI mass spectrometers should be tuned to apply a modest amount of in-source

collisional dissociation for protein analysis to sufficiently address the issue of

desolvation without applying so much energy as to cause proteins to fragment.

EFFECTIVE RESOLUTION CURRENTLY DRIVING MS HARDWARE EVOLUTIONOver the past 30 years, the ionization methods and mass analyzers that analysts

prefer have evolved to meet the needs of the biopharmaceutical industry. ESI

TOF MS has historically been the most frequently used platform for intact mass

analysis. ESI TOF MS has been proved to effectively scan across a broad mass

range and is capable of measuring even very large, MDa-scale compounds.

Resolution is commonly understood to remain constant across the mass-to-


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charge ratio (m/z) range with TOF MS systems. However, TOF MS has some

drawbacks. Aberrant behaviors of protein ions are known to cause artificial

broadening of MS peaks. In these cases, the resulting effective resolution is

significantly inferior to the theoretical isotopic peak width or that observed

for single charged calibration ions used to set resolution specifications for the

instrument. For intact proteins specifically, sources of artificial peak broadening

within TOF instrumentation have been identified and attributed to mass-

dependent variations of the initial kinetic energies of protein ions within the

source region and collision cross section dependencies occurring in the drift

tube.4,5 Both issues impart Gaussian-like distributions on the time of arrival of

ions in TOF systems and manifest as MS peak broadening.

As a result, increasing numbers of analysts are shifting to new mass analyzer

technologies. The mass-dependent effective resolution limitations of TOF MS

are avoided with FT MS (Orbitrap and FTICR) detectors. Mass analysis in this

case involves directly measuring the frequencies of ions oscillating within

a trapping device so as to interpret m/z. FT MS theoretical mass resolution

decreases with increasing m/z. Unlike TOF, FT MS systems produce intact

protein MS peak widths that match the expected widths calculated based on

the resolution settings used and natural isotopic peak width (figure 2).6 Mass

resolution of FT MS systems is not affected by the molecular weight of ions but

rather only by their m/z.

Orbitrap MS has become a popular choice for therapeutic protein

characterization. Orbitrap MS was originally optimized for small molecules and

tryptic peptides and, for several years, was not a viable choice for intact protein

Figure 2. Raw data of a single charge state of a mAb acquired on TOF and Orbitrap platforms. Peak widths were expressed in “Thomsons” (Th) which also represent m/z ratio. Two different Orbitrap resolution settings (transient lengths) indicate that there are multiple isobaric species that are not resolved using TOF MS. A high Orbitrap resolution setting, which utilizes a long transient (t = 256 milliseconds), yielded an ion peak width that is nearly identical to the ‘natural’ (“isotopic”) theoretical peak width. Figure was reproduced from Thompson et al., 2014 with permission from Taylor & Francis.6


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characterization. It left only FTICR as a legitimate alternative to TOF MS. FTICR MS

systems have historically offered very high performance, but these instruments

are not often used in the biopharmaceutical industry because of their high price

tag and maintenance costs. Orbitrap MS systems offer FT MS high-resolution

data at significantly lower cost and require less physical laboratory space. Newer-

generation systems have been purpose built for measuring intact proteins.7

Orbitrap MS is now a compelling alternative to high-end TOF MS systems for

intact mass analysis capable of high effective resolution.6,8–11 Reports over the

past five years have demonstrated state-of-the-art intact protein analysis using a

variety of experimental conditions coupled to ESI Orbitrap MS.

NATIVE MS KEEPS IT SIMPLENative MS, an analytical technique designed to minimally perturb protein

structure and preserve noncovalent interactions, is another option for

improving intact protein analysis. Native MS is performed using mobile phases

that mimic aqueous physiological conditions.12 Native MS solutions typically

include highly pure volatile salts (such as ammonium acetate) limited to low

or moderate concentrations to maintain ESI sensitivity.13 Preserving protein

quaternary structure is a requirement for some types of biotherapeutics to

assess characteristics that are otherwise not possible in denaturing conditions

such as reversed-phase (RP) LC/MS. Notable examples are determination of the

stoichiometry of protein-ligand complexes as well as drug-to-antibody ratio

(DAR) of noncovalently associated subunits in cysteine-linked antibody-drug

conjugates (ADCs).14,15

Native MS can have many advantages for protein analysis. As isoform mixtures

become more heterogeneous and span a greater mass range, there is an

increased likelihood that two isoforms will produce multiple unique electrospray

ions that interfere at a given m/z value. This can happen when two or more

species have truly isobaric masses (m) or when two or more species with

different masses (m) and charge states (z) coincidentally interfere at the same

m/z value. As the distribution of charge states decreases, ions are observed at

increasingly higher m/z values at greater distances between successive charge

states. An important, albeit less-appreciated, characteristic of native MS is that

by preserving protein folding in solution, the protein ion average charge state is

significantly decreased and the total ion current is distributed across a smaller

number of charge states. Native MS approaches can provide simplified mass

spectra because of fewer ions interfering at the same m/z values.

This quality of native MS can be exploited to dramatically increase platform

peak capacity over denaturing MS methods that produce ion distributions of

relatively higher charge states. Such a comparison was performed by Marcoux

and colleagues in the analysis of a lysine-linked ADC, trastuzumab emtansine.9

The phenomenon of charge reduction and increased spectral separation

inherent to native MS is illustrated in figure 3.16 This report confirms native MS

is an excellent tool for characterizing any type of heterogeneous intact protein

mixture, including both noncovalently bound ADCs and those with interchain

disulfide bonds intact.


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Until recently, the high spectral separation afforded by native MS has been

appreciated only by a small number of practitioners. This is likely due to the

historical difficulty involved in attaining high-quality ESI spectra from 100%

aqueous solution. It can also be argued that adoption of native MS has been

impeded somewhat by the low effective resolution of the TOF MS systems

generally used for intact protein analysis.

The latest MS technologies avoid this problem. A new Orbitrap-based mass

spectrometer, the Thermo Scientific Exactive Plus EMR system, introduced in

peer-reviewed literature approximately five years ago and then commercially a

year later, allowed the theoretical advantages of high spectral separation to be

substantiated on a benchtop MS system capable of high effective resolution.17

Orbitrap-based intact mass analysis using native MS techniques is often referred

to as “high resolution” native MS. In a period of five years Orbitrap technology has

spawned a new generation of native MS applications.18–20

In practical terms, adopting a native MS approach is an easy way to systematically

reduce potential artifacts. Using relatively gentle solution-phase and gas-phase

conditions, native MS minimizes the risk of workflow-related PTM artifacts.

Storing and analyzing proteins in physiological conditions puts minimal stress on

biologics and largely avoids degradative nonenzymatic PTMs, such as asparagine

deamidation and aspartic acid isomerization, which can result from high

temperature or excessively high or low pH.

GREATER THAN THE SUM Analytical systems that directly interface solution separation with mass

spectrometry offer further options for maximizing platform peak capacity for

intact protein analysis.

Figure 3. Native MS analysis of intact proteins allows improved separation of mass peaks at higher m/z range. (A) An ESI MS spectrum from reversed-phase LC, which is a denaturing technique, yields a charge state distribution of relatively higher charge at lower m/z ranges, while a native MS spectrum of the same mAb yields a charge state distribution of relatively lower charge at higher m/z ranges. (B) A detailed view shows that two to three sequential charge state envelopes overlap in RPLC/MS spectrum compared to an overlap of zero to one charge state envelopes in the native SEC/MS spectrum. Figure adapted from Bailey et al., 2016.


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Historically, direct coupling of MS to solution separations has been limited to

RPLC, size exclusion chromatography (SEC), and CE. Typically, these separation

techniques are all performed using similar denaturing-type conditions (water/

organic, acidic pH) for LC mobile phases or CE background electrolyte (BGE)

that are chosen to attain high sensitivity ESI MS of proteins. In an industrial

setting, RPLC/MS has been adopted as the most common means to perform

intact mass analysis for proteins of all sizes, including large protein species such

as mAbs. This choice benefits from a collective industrial expertise developed

over many years using RP to efficiently separate smaller compounds (such as

peptides and small molecules) on the basis of hydrophobicity as well as many

years of optimizing the integration with ESI MS. This configuration has been

demonstrated for mAbs to leverage separation of isobaric isoforms that differ

by only a single C-terminal lysine residue, allow sequential elution, and thereby

avoid interference at the MS level.21

In practice, however, actual separation of large intact protein isoforms can fail

to materialize. But by taking several factors into account, analysts can obtain

useful intact protein mass analysis data from RPLC/MS.

Typically, RPLC is skewed for steep gradients and shorter run times, which

deprioritizes actual isoform separation. With conscious efforts to optimize

an LC method, it is possible to prevent the peak tailing that can otherwise be

dramatic enough to preclude any separation of subtle structural differences of

isoforms. Strong modifiers or ion-pairing reagents, such as trifluoroacetic acid

(TFA), are required to achieve optimal LC performance. While this approach is

effective, adoption of TFA-based mobile phases for LC/MS remains controversial

in therapeutic protein characterization because the marked decrease in ESI

sensitivity. Additionally, carryover can impede many types of small-molecule

analyses when performed later using the same equipment.

For routine purposes, mating separations to MS does, in fact, bring increased

productivity to the intact mass analytical platform in a somewhat roundabout

manner. In addition to separating protein isoforms, chromatography—and to

some extent CE—can efficiently separate proteins from the nonvolatile buffer

salts that plague ESI, and largely satisfies the requirement for protein desalting.

Lazar and colleagues showed several years ago that denaturing SEC provided

superior desalting in a head-to-head comparison with RP- and sieve-based

desalting methods upstream of intact mass analysis.22

LC/MS platforms also raise productivity simply through incorporating the use

of autosampler technologies. Running large batches of samples on a 24/7 basis

is routine in many lab environments and is simply not possible using a direct

infusion MS approach. But coupling chromatography with MS also means

that the typical MS tuning/optimization routine that is usually performed

by infusion can instead be performed with a sample queue using a matrix of

tuning/method conditions. In this case the overall method design space can be

thoroughly and systematically tested.


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Intact mass analysis by LC/MS can be further improved by the seamless

integration of ultraviolet or other optical detection. This configuration is ideal

for providing an orthogonal means for straightforward protein detection in

addition to MS analysis. Dual detection using LC/UV/MS can bolster protein

quantitation and monitor autosampler accuracy, and it can be helpful

for observing proteins outside the tuned or optimized detection ranges.

Using a detection system that applies only a single narrow wavelength to

proteins (variable wavelength detection)—rather than a broad spectrum

of wavelengths (photo diode array)—is recommended in order to minimize

potential photooxidation artifacts on methionine and tryptophan residues. This

recommendation holds for peptides and intact proteins alike.

SOFTWARE CATCHING UP WITH THE TIMES Intact proteins are reflected in ESI mass spectra as Gaussian-like distributions

of highly charged ions spanning ranges of more than 1,000 m/z units. The final

step of an intact mass workflow is to interpret the masses of proteins using

the numerous redundant m/z data. Purified proteins can usually be interpreted

by hand by a knowledgeable analyst, but even subtle heterogeneity can

demand the use of deconvolution software that models and reports the masses

and abundances of individual isoforms. Deconvolution algorithms that use

maximum entropy modeling have provided deeply insightful interpretation of

complex intact protein spectra in which numerous MS peaks may be partially

or completely overlapping.23 Software-enhanced resolution is used routinely

and to great effect; however, automatically generated deconvolution results

can make it difficult for an analyst to judge whether or not the software

accurately models the raw data. The raw MS data and deconvolution results

can be cross-compared to ensure that noise peaks and harmonic series are not

interpreted as false positive protein species. Maximum entropy approaches

require some degree of prior knowledge for protein samples; false positives

can easily be produced when grossly misguiding the algorithm. In spite of this

caveat, deconvolution software has expanded the field of intact mass analysis

to nonexperts. When modeling parameters are appropriate for a given sample,

deconvolution software can easily provide an answer.

Deconvolution can also be used as a form of relative quantitation when the

conventional relative quantitation based on the individual charge states is

not possible. Software-aided deconvolution of intact protein spectra can be

particularly helpful when spectra show extensive ion interferences. The vast

majority of intact mass analysis workflows call for deconvolution software

to analyze data resulting from LC/ESI MS platforms. But deconvolution

software packages have been developed using an approach that is actually

a better fit for direct infusion rather than separations. For intact mass

analysis, a mixture of isoforms from a moderately purified intact protein

often elutes more or less as a single LC peak. Data analysis typically involves

creating an averaged spectrum over a desired retention time (RT) range

followed by deconvolution of this single spectrum. Typically, spectra are

averaged according to the full width of the peak at half-maximum height


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(FWHM), though there is not a formal rule. Importantly, the specific

selection of the RT range for deconvolution can have significant influence

on the overall spectrum quality as well as the quantity of isoforms

represented. Determining FWHM can be a painfully subjective process when

chromatographic peaks deviate from the ideal. High-throughput intact mass

analysis requires either extremely stable RT over many runs or a dedicated

analyst to ensure the RT range for an average spectrum is correctly selected

for each LC/MS run. For high-end laboratories, this single step represents a

bottleneck that can require an expert to minimize the risk of false positives.

The average-over-RT approach described above is truly not appropriate for any

type of relative quantitation or isoform profiling based on deconvolution of the

LC/MS data. Commonly, in this process, the analysts make a basic assumption

that every identified isoform has also eluted identically within an identical

RT range, when almost always the opposite is true. Individual abundances

determined using a single, discrete RT range will not provide insight to ion

(isoform) intensity changing over time.

The Sliding Window algorithm in Thermo Scientific BioPharma Finder software

embraces a so-called time-resolved deconvolution approach. This deconvolution

method involves creating numerous averaged spectra over a larger RT range,

deconvolving each, and integrating LC peak area of the individual ion intensities

according to each isoform. Time-resolved deconvolution is valuable for any

instances of time-based quantity changes. Foremost, this includes LC and CE,

but it could be applied to infusion when, for instance, monitoring individual

species in kinetic reactions.

Time-resolved deconvolution was demonstrated by Gazis and Horn to solve the

issue of measuring DAR for intact ADC samples.24 ADCs are mixtures of isoforms

created by conjugating hydrophobic small-molecule compounds to an intact

mAb. This manufacturing strategy can generate numerous isoforms exhibiting

Figure 4. Chromatogram and elution profiles of nine components identified by thesliding window algorithm for an ADC sample. Adapted from Gazis et al., 2015.


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unique elution profiles. The authors used their Sliding Window algorithm in

combination with a dedicated deconvolution algorithm (ReSpect) to correctly

determine the DAR value from an ADC mixture analyzed using RPLC/MS. Their

approach showed that the conventional average-over-RT approach provides

an erroneously low DAR value, which reflects the fact that the conventional

approach does not account for the late-elution and tailed profiles of the highly

conjugated isoforms (figure 4).

The concept of platform peak capacity also pertains to deconvolution software

in the sense that the final results of the workflow aim to have the greatest

capacity for creating true-positive isoform identifications. Time-resolved

deconvolution offers a strategy to accomplish this in an automated fashion with

LC/MS data. Toward determining true positives, time-resolved deconvolution

data can be used to determine the individual elution profiles of each of the

identified isoforms. This presents an opportunity to filter the results to species

that show elution behavior consistent with actual protein elution and thus

reduce false-positive identification of lower abundance species that may

otherwise be in question. Furthermore, the deconvolution peak areas, also

known as extracted deconvolution chromatograms (XDC) can be utilized to

determine the relative quantitation of individual protein isoforms.

DEMOCRATIZATION AND ORTHOGONALITY IN THE NEAR FUTURE To maximize platform peak capacity for intact protein mass analysis, one appealing

avenue is to combine several of the separate enhancements discussed above into a

single system. Adding separation technologies, such as SEC, to native MS promises

to democratize state-of-art intact therapeutic protein characterization workflows.

Native SEC/MS is arguably the most adoptable among the variety of published

reports on this emerging area. It is possible to perform isocratic separation using

the same volatile-salt solution used in conventional, infusion-based native MS.

Even so, interfacing SEC with native MS requires special considerations. SEC does

not inherently provide a mechanism for on-column sample concentration during

loading and thus requires a threshold level of initial sample concentration to allow

detection. More importantly, SEC can be perturbed by secondary interactions,

whether hydrophobic or ionic in nature. Offline SEC/UV methods normally use

mobile phases containing organic or high concentrations of salt to disrupt potential

secondary interactions and encourage size-based separation. As native SEC/MS

often requires optimizing MS sensitivity by using low ionic strength aqueous

mobile phase, potential secondary interactions may arise between proteins and SEC

stationary phase. The latter is often the variable that can be readily changed.

Native intact mass workflows can be improved by adding SEC-based desalting

and automated sample injection. Valliere-Douglass and colleagues showed that

native SEC, coupled directly to an MS system, could efficiently desalt a cysteine-

linked ADC sample while preserving the noncovalently structured isoforms.15

The DAR value determined using the native SEC/MS method roughly matched

the value calculated by the traditional approach using hydrophobic interaction

chromatography and UV detection.


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A separate study also applied this native SEC (desalting)/MS strategy to

determine the DAR value of a trastuzumab emtansine sample. In this case,

highly conjugated forms eluted later, presumably because of secondary

interactions, and thus the DAR value was observed to change according to

retention time. Similar to Gazis and Horn, data analysis using ReSpect and

Sliding Window meant the correct DAR value could be determined regardless of

RT shift (figure 5).25

Native SEC/MS offers other advantages, too. ESI source-related artifacts related

to both aggregation and dissociation can impede infusion-based native MS data

analysis. Size separation of protein isoforms helps tremendously in assessing

the validity of noncovalent aggregated species. In their analysis of multimers

of bovine serum albumin, Muneeruddin and colleagues demonstrated that

native SEC/MS avoids source-related artifacts by allowing aggregate size to be

correlated with RT.26 This report also shows that MS systems may struggle to

provide quantitative data over a broad range of protein sizes. MS signal response

(sensitivity) decreases nonlinearly with increasing m/z ranges, and so UV

detection must be embedded in the platform.

Figure 5. Optimized sliding window deconvolution provides accurate DAR value. (A) Averaging spectra across the front, FWHM, or tail of an LC peak can influence the DAR value when isoforms do not perfectly coelute. (B) Higher drug-load isoforms can elute later in native SEC/MS, which may introduce error in DAR measurement. (C) Sliding Window analysis that encompasses entire chromatographic peak provides higher and more accurate DAR value than the FWHM averaged spectrum. The difference in DAR value reflects increased degree of tailing of high-drug-load ADC species, which is not correctly interpreted by the conventional approach. Adapted from Bailey et al., 2017a.


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Ion-exchange chromatography (IEC) can perform charge-based separations and has

also been successfully coupled to native MS. IEC is usually mated to UV detection

alone because conventional IEC mobile phases are not compatible with ESI MS.

Recently, however, analysts have used native IEC/MS for a growing list of cationic

protein types, including lysozyme, PEGylated interferon-ß, and mAb samples.

These examples show that native cation-exchange MS efficiently separates charge

heterogeneity and permits direct MS detection of the individual isoforms.27–29

Importantly, this strategy allows separation of many types of isoforms in solution,

the masses of which normally interfere at the MS level. In these examples, however,

the combined use of TOF MS and high concentrations of ammonium acetate

analytical gradients of 100–500 mM used to drive protein elution negatively affects

overall data quality. Both sensitivity and effective resolution were decreased to an

extent that low-abundance isoforms do not appear to be possible to interpret.

Bailey and colleagues introduced a high-performance version of native IEC/MS,

optimized for high effective resolution and high platform peak capacity. The

team used the platform for the intact mass analysis of trastuzumab. Isoforms

were eluted using a gradient of low to high pH, while mobile phases comprised

of extremely pure ammonium acetate in constant background concentration of

50 mM.30 This workflow highlights the use of both extracted-ion chromatogram

(XIC) as well as XDC plots in navigating the type of complex data that can arise

from a native IEC/MS characterization study (figure 6). Orbitrap MS and time-

resolved deconvolution were essential for attaining high peak capacity. Overall,

this configuration provided a dynamic range of three orders of magnitude of

confidently identified intact mAb isoforms, which exceeds previous reports using

any other strategy for intact mass analysis. Additionally, the authors showed that

separation of isobaric species combined with high effective MS resolution allows

accurate measurement of very subtle mass differences that are even significantly

less than the natural peak width of the protein ion.

Figure 6. Sliding window ReSpect deconvolution results of IEC/MS analysis of trastuzumab. (A) Base peak chromatogram (BPC) of a native IEC/MS analysis of trastuzumab shows two major chromatographic peaks corresponding to deamidated (†) and relatively unmodified (*) isoforms with very similar masses observed as expected. (B) Theoretical difference in mass of deamidated and unmodified forms is 6.6 ppm, which is equivalent to a 1 Da mass shift for a species of approximately 150 kDa molecular weight. (C) An extracted-ion chromatogram (XIC) plotted for deamidated and unmodified isoforms using a 3 ppm width shows that the raw MS data reflect a true difference in measured masses of these near-isobaric isoforms. (D) Sliding window extracted deconvolution chromatograms (XDC) are plotted for the top three glycoforms in both deamidated and unmodified forms. Adapted from Bailey et al., 2017b.


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A powerful alternative to IEC for separating charge heterogeneity is CE. A native

CE/MS method was reported recently by Belov and colleagues that incorporated

the use of a BGE consisting of low-strength ammonium acetate followed by

Orbitrap MS detection and time-resolved deconvolution using Sliding Window.31

Applying their method to the analysis of several types of intact proteins, the

researchers obtained high-quality results similar to the IEC-based data of Bailey

and colleagues. Low sample consumption and relatively high sensitivity were key

features of their method directly contributed by CE. The authors concluded that

separation improves the quality of native MS data over using infusion alone.

CONCLUSION Intact mass analysis requires sufficient resolution to correctly determine the

identity and quantity of isoforms in a given mixture. The need to perform deep

characterization of new heterogeneous therapeutic protein formats requires that

effective resolution is optimized at multiple levels to increase the overall peak

capacity of the analytical platform. Several new variations on conventional intact

mass analysis offer increased platform peak capacity: (1) using MS hardware

and methods capable of high effective mass resolution, (2) adopting native MS

techniques for high spectral separation, (3) coupling separations directly to MS,

and (4) analyzing data using time-resolved deconvolution software. We believe

that these intercompatible strategies should indeed be used in combination to

push the existing boundaries of performance and accessibility of intact mass

analysis workflows.

For more information on this subject, please visit

www.thermofisher.com/NativeMS.

ACKNOWLEDGMENTSThis white paper was written by Aaron O. Bailey and Jonathan L. Josephs at

Thermo Fisher Scientific and edited and produced by C&EN Media Group editors

and designers.

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