epitope mapping of an antibody receptor using hydrogen...
TRANSCRIPT
Epitope Mapping of an Antibody Receptor Using
Hydrogen-Deuterium Exchange MS
Enhancing the Sensitivity of
Atmospheric Pressure Ionization GC–MS
Peptide Mapping of Monoclonal Antibodies
and Antibody–Drug Conjugates
Volume 16 Number 4 October 2018
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6 Current Trends in Mass Spectrometry October 2018 chromatographyonl ine .com
Articles
October 2018
Epitope Mapping of an Interleukin Receptor for Three Therapeutic Antibodies by Hydrogen-Deuterium Exchange Mass Spectrometry 8
Eric Largy, Caroline Cajot, and Arnaud Delobel
Among all the analytical techniques available for epitope mapping studies, hydrogen–deuterium exchange mass spectrometry (HDX-MS) is usually the fastest and easiest to carry out. We present here the epitope mapping of three distinct monoclonal antibody (mAb) candidates targeting the same antigen, an interleukin receptor. The goal is to establish the binding mode of these mAbs, and explain possible differences observed for in vitro binding and in vivo function.
Enhancing the Sensitivity of Atmospheric Pressure Ionization Mass Spectrometry Using Flow Modulated Gas Chromatography 15
Karl J. Jobst, John V. Seeley, Eric J. Reiner, Lauren Mullin, and Adam Ladak
The past decade has witnessed resurgent interest in coupling GC to atmospheric-pressure chemical ionization (APCI), which is suitable for the high column flows required for using flow modulation. This study assesses the use of GP-APCI with flow modulation for sensitive detection of selected trace organics.
Peptide Mapping of Monoclonal Antibodies and Antibody–Drug Conjugates Using Micro-Pillar Array Columns Combined with Mass Spectrometry 20
Koen Sandra, Jonathan Vandenbussche, Isabel Vandenheede, Bo Claerebout,
Jeff Op de Beeck, Paul Jacobs, Wim De Malsche, Gert Desmet, and Pat Sandra
The structural complexity of monoclonal antibodies (mAbs) challenges the capabilities of even the most advanced chromatography and mass spectrometry techniques. This study examines the use of micro-pillar array columns in combination with mass spectrometry for peptide mapping of both mAbs and antibody–drug conjugates (ADCs).
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Monoclonal antibodies (mAbs) constitute a major and fast-growing biotherapeutic class, thanks notably to their outstanding selectivity for specific targets.
Their physicochemical characterization is complex, because of their size (around 150 kDa), numerous variants caused by post-translational modifications, and their tendency to aggre-gate (1). Furthermore, there is a need for better understanding of how these aspects may alter the structure and function of mAbs, as observed in vivo or in vitro by techniques such as enzyme-linked immunosorbent assay (ELISA) or surface plasmon resonance (SPR). This need prompts the study of the precise nature of the interaction between mAbs and their antigens at the molecular level. Epitope mapping consists of the determination of the binding site of the antigen (coined epitope), and provides several very useful applications:• A way to screen mAb candidates and assist in the devel-
opment of new candidates (2)• A better understanding of the mechanisms of action (3–5)• An additional layer of intellectual property protection (6).
A number of methods can be used to map interactions between proteins. X-ray crystallography is probably the gold
standard for such endeavors (7,8), but it suffers from several drawbacks. Most notably, it is a lengthy process that relies on the amenability of the proteins to crystallize, and “only” yields a snapshot of one energetically accessible conformation. In that regard, solution-based techniques are best suited to study dy-namic systems because they take into account all attainable conformations. Nuclear magnetic resonance (NMR) experi-ments provide similar resolution but are often too difficult to carry out in this context (9). Other methods such as circular dichroism or mutagenesis usually lack in resolution or do not provide direct evidence, respectively.
The hydrogen–deuterium exchange mass spectrometry (HDX-MS) approach is much faster and easier to carry out than the aforementioned methods. Although it does not provide atomic resolution, the approach allows the determination of epitopes at the scale of a few amino acids (regardless of the com-plexity of the sample), requires smaller sample amounts, tolerates impurities and formulants (such as salts and buffers), and oper-ates in solution in near-physiological conditions. Furthermore, it provides insight into the structure and interaction dynamics on a large time scale (from a few seconds to several hours).
Eric Largy, Caroline Cajot, and Arnaud Delobel
The study of protein-protein interactions is critical in the development of biotherapeutics, to
develop new candidates, to understand modes of actions, and to protect intellectual property.
Among all the analytical techniques available for epitope mapping studies, hydrogen–deuterium
exchange mass spectrometry (HDX-MS) is usually faster and easier to carry out. We present here
the epitope mapping of three distinct monoclonal antibody (mAb) candidates targeting the same
antigen, an interleukin receptor. The goal is to establish the binding mode of these mAbs, and
explain possible differences observed for in vitro binding and in vivo function.
Epitope Mapping of an
Interleukin Receptor for Three
Therapeutic Antibodies by
Hydrogen–Deuterium Exchange
Mass Spectrometry
chromatographyonl ine .com October 2018 Current Trends in Mass Spectrometry 9
We describe below the epitope map-ping of three distinct mAb candidates targeting the same antigen, an interleu-kin receptor. The goal is to establish the binding mode of these mAbs, and ex-plain possible differences observed for in vitro binding and in vivo function.
Experimental
Principles of the Method
The analytical workflow for HDX-MS is summarized in Figure 1. Proteins in solution are diluted in an excess of deuterium oxide (D2O) so that hydro-gen atoms are exchanged for deuterium atoms on the amide nitrogen of the pep-tide bond (10,11). Side-chain H atoms also exchange, but at too fast a rate, and are consequently entirely back-ex-changed during the liquid chromatog-raphy–MS (LC–MS) analysis (that is, hy-drogen atoms replace deuterium atoms). After quenching the deuteration by low-ering both the temperature and the pH, the protein is digested online by a pro-tease. The resulting peptides are quickly desalted and separated at low tempera-ture. Finally, MS measures the uptake of deuterium using the difference in peptide mass resulting from the mass difference between exchanged deute-rium (2.0141 Da) and hydrogen (1.0078 Da). This exchange of D for H occurs at rates varying by a factor of as much as 108 depending on hydrogen bonding and solvent accessibility (Figure 2) (12). Schematically, hydrogen atoms from accessible regions and hydrogen atoms not involved in H-bonds exchange faster. By measuring hydrogen–deuterium ex-change rates, changes in protein struc-ture and dynamics are determined. Note that the exchange rate is also influenced
by experimental parameters such as the temperature and pH (13).
Concretely, binding a mAb on the antigen buries the amino acids from the binding sites, hence screening them from the bulk medium and ultimately slowing down the H-to-D exchange. In the approach described in this article, the proteins are digested online with a pepsin column, which generates a large number of partially overlapped peptides and allows the analysis of ex-change data at the scale of a few amino
D2O labeling
pH 7.0, 20 °C
Quench
pH 2.5, 1 °C
On-line pepsin
digestion
pH 2.5, 20 °C
Filtering and analysis Identification
LC–MS
separation and
detection,
0 °C
Figure 1: Analytical workflow of hydrogen–deuterium exchange mass spectrometry.
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acids. The epitope mapping can there-fore be conducted by a straightforward comparison of the exchange data of the antigen unbound and bound to the mAb for each peptide. Peptides for which a statistically significant decrease in deuterium uptake is found are likely to be part of the epitope. By combining the results of all peptides, the epitope can be more finely characterized.
Analytical Workflow
Brief ly, the experiments, which were replicated at least three times, can be de-
scribed as follows: The samples were di-luted ten-fold into a deuterated buffer for 30 min, after which they were quenched at a low pH (2.5) and a low temperature (1.0 °C) to minimize the amount of back exchange. Furthermore, to ease the on-line digestion of the proteins in subop-timal conditions, the quench buffer was complemented with high concentra-tions of a reducing agent that can oper-ate at acidic pH and a chaotropic agent, namely TCEP and guanidine.
Proteins were then injected in the LC system, where they were quickly digested
in an immobilized pepsin column, at 20 °C, then desalted on a reversed-phase cartridge, and separated on an analyt-ical reversed-phase column at 0.0 °C (again, to minimize the back exchange). The system was coupled to a high-res-olution electrospray ionization quadru-pole time-of-flight (ESI-Q-TOF) mass spectrometer for peptide identification and deuteration quantitation purposes, which were assisted by various software solutions (PLGS, UNIFI, and DynamX, all from Waters). Undeuterated peptides (no incubation in D2O) were used as ref-erence point and for MSE-based identi-fication, in which the sequence of the peptides was systematically confirmed by collision-induced dissociation (CI-D)-based fragmentation. Their observed retention times and theoretical isotope profiles were subsequently used to detect the corresponding deuterated peptides and quantify their deuteration.
Hardware
The HDX experiments were conducted using an HDX-2 system from Waters, consisting of an automated sample prepa-ration bench (which performs the incu-bation in deuterium, quenching and in-jection steps at controlled temperature), two pumps (supplying the mobile phases to the digestion, desalting, and analyti-cal columns). A module allows the tem-perature to be set independently for the digestion column on one hand, and de-salting and analysis on the other. Deuter-ation and digestion were performed at 20 °C, while the quench and desalting/sepa-ration at 1 and 0 °C, respectively. Samples and Buffers
The interleukin receptors unbound and bound to the antibody were prepared at around 25 μM in each partner, in an equilibration buffer composed of 10 mM sodium phosphate complemented with sodium chloride (100 mM) and adjusted to pH 6.8. Given that the KD of the mAb–antigen complex lies in the 10-10 M range, we can assume that more than 99.7% of 1:1 complex is formed at this concentra-tion, hence maximizing the observable effects without resorting to the use of an excess of mAb. Given that the epitope mapping is performed by studying pep-tides from the antigen, it is preferable to
100
120
2 3 4 5 6 7 8 10 11 129
100
80
60
40
20
0
8.38
Peptide 126-137n = 60.32%RSD
02.00
Cu
mu
lati
ve
de
tect
ed
pe
pti
de
s
4.00
Retention time
6.00 8.00 10.00 12.00 14.00 16.00
Time
%
Figure 3: Six overlaid LC–MS chromatograms of a randomly selected interleukin receptor peptide
(top), and cumulative peptides elution as a function of time (bottom).
D2O
HD
Figure 2: D-labeling of a protein in the presence of an excess of D2O.
chromatographyonl ine .com October 2018 Current Trends in Mass Spectrometry 11
5
60 65 70 75 80 85 90 95 100 105 110
120
180
137 peptides, 99.6% sequence coverage, 6.44 average redundancy
N-glycosylation site reported and observed by HDX-MS
N-glycosylation site unreported but observed by HDX-MS
Disulfide bridge (reported)N-glycosylation concensus sequence (no glycosylation reported nor obseved by HDX-MS)
185 190 195 200 205 210 215 220 225 230 235
125 130 135 140 145 150 155 160 165 170 175
115
10 15 20 25 30 35 40 45 50 55
Figure 4: Sequence coverage of the interleukin receptor. Peptides are depicted by blue bars, disulfide
bridges by green lines, and N-glycosylation sites by orange frames.
minimize the quantity of mAb peptides that may complicate the detection, iden-tification, and deuterium uptake quanti-tation of the antigen peptides, because of the large occurrences of peptide coelution resulting from the nonoptimal separation conditions (10 min gradient at 0 °C).
The deuteration buffer composition was matched to the equilibration buf-fer composition, but in D2O instead of water. The pH was also matched by ad-justing its apparent value at 6.4, given that pD = pH – 0.4 (14). The quench buffer was composed of 100 mM so-dium phosphate, 400 mM TCEP, and 4 M guanidine, and was adjusted at pH 2.3, hence ensuring that the post-quench pH of the solution was 2.5 by 1:1 mixing. Finally, mobile phases involved in the online digestion and analytical separation of the peptides were water and acetonitrile, adjusted to pH 2.5 with 0.2% formic acid.
Results
Analytical Separation
For peptide identification and deute-rium quantitation purposes, it is pref-
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chromatographyonl ine .com12 Current Trends in Mass Spectrometry October 2018
erable to limit coelution events as much as possible. Although a short gradient at low temperature cannot extensively separate the large number of peptides entering the column, it is still possible to ensure that there is no large skew-ing of peptide elution leading to a very large number of them being eluted in a short time range. Figure 3 illustrates the fairly linear elution of undeuterated peptides that was obtained for the inter-leukin receptor using our experimen-tal conditions, which limited the occur-rences of coelution. Furthermore, six injections of independently prepared samples exhibited very little retention time deviation for a randomly selected peptide (0.32% RSD). Calculated across 399 peptides, the average retention time (RT) standard deviation was as low as 0.009 min, which is critical to ensure proper identification of the deuterated peptides later in the experiment.
Sequence Coverage
The sequence coverage, peptide length, and average peptide redundancy per amino acid are important parameters regarding the quality of the results, notably in terms of confidence and resolution. It is, of course, preferable to cover the protein sequence as much as possible to avoid missing a binding site. Shorter peptides yield higher res-olution results, especially when com-bined with high redundancy where partially overlapped peptides allow narrowing the sequence of the binding site. A high redundancy also increases the confidence in the results because it provides a means to evaluate the con-sistency of the results.
An excellent sequence coverage, together with a very good average re-dundancy, was obtained (99.6%, 6.44 peptides per amino acid; see Figure 4) from 137 peptides identified using both the PLGS and UNIFI software. As ex-pected, the redundancy is not homo-geneous; the N-terminal half contains three disulfide bridges and is thus more difficult to digest than the C-terminal half, which does not have any. Hence, the average redundancies of the 8–90 and 178–225 regions are 4.7 and 8.2, respec-tively. All regions are covered sufficiently well to gather high-quality results.
Pooled SD
93—125
71—85
93—125
93—108
125—14167—93
67—85
10
Deute
rium
up
take
(D
a)
Deute
rium
up
take
(D
a)
Deute
rium
up
take
(D
a)
8
6
4
2
0
-2
8
6
4
2
0
-2
8
6
4
2
0
-2
Complex
mAb A
mAb B
mAb C
DifferenceIL
Figure 5: Deuterium uptakes of the interleukin receptor (blue) and complex (orange) peptides (from
N- to C-terminal), and corresponding difference (green). The pooled standard deviation is shown as
a mirrored grey area. Arbitrary thresholds at ±0.5 and ±1.0 Da are illustrated by dashed and plain
gray lines, respectively.
5
60 65 70
75 80 85 90 95 100 105 75 80 85 90 95 100 105 75 80 85 90 95 100 105
110 120
180 185 190 195 200 205 210 180 185 190 195 200 205 210 180 185 190 195 200 205 210
215 220 225 230 235
Relative fractional uptake (%)
No protection
mAb A mAb B mAb C
Maximum protection
215 220 225 230 235 215 220 225 230 235
125 130 135 140
145 150 155 160 165 170 175 145 150 155 160 165 170 175 145 150 155 160 165 170 175
115 110 120 125 130 135 140115 110 120 125 130 135 140115
10 15 20 25 30 35 5 10 15 20 25 30 35 5 10 15 20 25 30 35
40 45 50 55 60 65 7040 45 50 55 60 65 7040 45 50 55
Figure 6: Heatmap of the deuteration differences between the unbound and complexed interleukin
receptor (bottom). The inferred epitope site 1 (blue, red and orange) and site 2 (green) are shown
on the 3D structure (top).
chromatographyonl ine .com October 2018 Current Trends in Mass Spectrometry 13
mA
b B
mA
b A
mA
b C
70
SS
MD
SS
MD
SS
MD
50
30
10
-10
-30
-50
-70
8.0
6.0
4.0
2.0
-2.0
0.0
-4.0
-6.0
-8.0
3.5
2.5
1.5
0.5
-0.5
-1.5
-2.5
-3.5-2.5 -2.0 -1.5 -1.0 -0.5 0.0
Average Log2 fold change
1.0 1.5 2.0 2.50.5
-2.5 -2.0 -1.5 -1.0 -0.5 0.0 0.5 1.0 1.5 2.0 2.5
-3.0 -2.5 -2.0 -1.5 -1.0 -0.5 0.0 0.5 1.0 1.5 2.0 2.5 3.0
Figure 7: Dual-flashlight plot for the discovery of peptides with a statistically significant protection.
Epitope Determination
The interleukin receptor deuteration D was calculated as an average of the ex-perimental replicates and compared peptide per peptide between the two states (bound versus unbound). The differences between both states were calculated by equation 1, where IL and cplx refer to the interleukin receptor and complex, respectively:
ΔD=DIL
-Dcplx
[1]
This calculation means that the bind-ing of the mAb on a given peptide would result in a positive ΔD value for said pep-tide. For instance, such observations can be made for regions 71–85 and 93–125 of the antigen in presence of mAb A (Figure 5), where the difference is above 1.0 Da, an arbitrary threshold often used in the litera-ture (15). mAb B has an additional neigh-boring region above this threshold (region 125–141), whereas mAb C only has a few peptides barely above the limit, suggest-ing a lower binding strength, or possibly a different binding mode resulting in a de-creased shielding of the backbone hydro-gen atoms from the bulk medium. These regions, likely constituting the epitope, are clearly highlighted in a heatmap colored by ΔD (Figure 6). Large regions (regions 1–60, 140–190, and 201–236) show virtu-ally no difference between the two states, whereas other regions (regions 126–139 and 191–200) display slightly higher ΔD values. Incidentally, a secondary epitope site may be hypothesized in the 191–200 region of mAb A only, as also observed by orthogonal techniques (data not shown).
The statistical significance of uptake differences can be assessed by calculat-ing the corresponding pooled standard deviation SD
P, following equation 2,
where SD is the standard deviation in-ferred from the replicates.
SDp= +SD
ILSD
cplx√2 2 [2]
The hypothesized epitopes exhibit differences larger than their corre-sponding pooled standard deviation for several peptides, which suggests that there is a statistically significant binding event taking place within these peptides. A few peptides from the secondary site also ex-hibit this property for mAb A. There is no
chromatographyonl ine .com14 Current Trends in Mass Spectrometry October 2018
peptide displaying a significantly negative ΔD in any case, suggesting that no other change in protein conformation leading to more exchangeable hydrogen atoms is taking place upon binding.
To quickly screen all peptides for each complex, an alternative visualization of the results was set up, taking into ac-count both the magnitude of the change in deuteration and the pooled standard deviation. This approach allows the quick identification of the peptides with the most significant changes in deutera-tion upon binding, hence yielding a ro-bust epitope. This result was achieved by calculation of the SSMD (strictly stan-dardized mean difference), following equation 3.
SSMD = = —
DIL
−Dcplx
√2 2+
ΔD
SDpSD
ILSD
cplx
[3]
Each peptide’s SSMD was plotted against the deuteration change magnitude D
IL/D
cplx, expressed as a base 2 logarithm
(Figure 7). As expected, the best peptides (located in the top right-hand corner) are located on the hypothesized primary epi-tope site, for all three mAb candidates. Note that the experimental conditions used in our study (1:1 molar mixture of the two proteins), together with the excel-
lent KD
of the primary epitope, disfavor the discovery of secondary sites.
Finally, the changes in deuteration for the hit peptides are qualitatively evident from the corresponding mass spectra, which further strengthens the epitope determination (Figure 8).
Conclusion
Using HDX-MS, the epitope of three mAbs were straightforwardly inferred, in solution, for an interleukin receptor tar-get. Qualitative and quantitative analysis allowed the identification of the peptidic regions of the antigen that are protected from deuteration upon antibody binding. The resulting epitope location is analo-gous for all three candidates, although one mAb seems to also bind a secondary site.
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Eric Largy, Caroline Cajot, and Arnaud
Delobel are with Quality Assistance SA, in
Donstiennes, Belgium. Direct correspondence
about this article to arnaud.delobel@quali-
ty-assistance.be.
78-84
402.0 403.0 404.0 405.0 406.0
m/z
407.0 408.0 14.5 415.5 416.0 417.5
m/z m/z
418.5 419.5 667 669 670 671 672 673 674 675 676 677668
85-93 112-123
Undeuterated reference
Antigen
Complex
Figure 8: Examples of mass spectra of three peptides (peptides 78–84, 85–93, and 112–123) from
the undeuterated antigen reference (blue, top), the deuterated antigen (red, middle), and complex
(green, bottom) samples.
chromatographyonl ine .com October 2018 Current Trends in Mass Spectrometry 15
The exposome represents all environmental exposures over the course of a lifetime (1). The identities of most environmental contaminants and their roles
in causing chronic diseases are unknown. This is partly a result of the analytical challenges associated with biomon-itoring trace levels of environmental toxicants in small sample quantities of serum or tissue. It is estimated that standard gas chromatography–mass spectrometry (GC–MS) and liquid chromatography–MS (LC–MS) platforms are not sufficiently sensitive to identify approximately 90% of unknown contaminants (2).
Inspired by advances in comprehensive two-dimensional gas chromatography (GC×GC) (3), Patterson and coworkers elegantly showed that (targeted) sub-femtogram detection of environmental pollutants is achievable using a technique termed cryogenic zone compression (CZC) (4). With CZC, the 3–8 s width of a GC peak is compressed to approxi-mately 200 ms, resulting in a significant enhancement in signal intensity (4–6). This has opened the door to the pos-
sibility of monitoring environmental toxicants using less invasive and inexpensive sampling techniques, such as collection of dried blood spots (7). The cost of requisite hardware, however (such as a thermal modulator), and associated cryogens and refrigeration is a drawback.
Signal enhancement is also possible using a valve-based f low modulator, a technology pioneered by J.V. Seeley (Oakland University, Michigan, USA) (8,9). In contrast to thermal modulation, a f low modulator requires a high carrier gas f low (>10 mL/min) to transfer the column ef-f luent from the modulator into the mass spectrometer. The potential of f low modulation has not been fully re-alized, because of the perception that high column f lows are not compatible with most (electron ionization [EI] and chemical ionization [CI]) mass spectrometers. This is usually addressed by eff luent splitting at the cost of sensitivity. Improved pumping capacity of modern turbo-molecular pumps can mitigate the problem, but still this hyphenation is generally considered difficult (10,11).
Karl J. Jobst, John V. Seeley, Eric J. Reiner, Lauren Mullin, and Adam Ladak
Peak intensity enhancement is one highly desirable outcome of comprehensive two-dimensional gas
chromatography (GC×GC). When coupled to mass spectrometry (MS), such enhancement is usually
achieved with a thermal modulator using a technique called cryogenic zone compression (CZC).
Differential flow modulation is a simple and cost-effective alternative to thermal modulation, but the
requisite high flow rates are generally perceived as being incompatible with most (electron ioniza-
tion [EI] and chemical ionization [CI]) mass spectrometers. The past decade has witnessed resurgent
interest in coupling GC to atmospheric pressure chemical ionization (APCI), which requires high gas
flows to assist ionization. This article reports on the modification of a GC–APCI system with a flow
modulator and evaluates its potential to enhance the sensitivity towards selected trace organics.
Enhancing the Sensitivity of
Atmospheric Pressure Ionization
Mass Spectrometry Using Flow
Modulated Gas Chromatography
chromatographyonl ine .com16 Current Trends in Mass Spectrometry October 2018
(a)
(b)
Experimental
The experiments were performed using a Xevo G2-XS quadrupole time-of-f light mass spectrometer (Waters) coupled to a modified 7890B gas chro-matograph (18,19) (Agilent). In GC–APCI, the GC effluent is swept into the ion source by a 350 mL/min make-up flow (N2). A schematic of GC–APCI is shown in Figure 1(a).
Ions formed by a corona discharge needle set to 3 μA were transferred into the mass spectrometer, aided by 20 V potential and cone and auxil-iary gas f lows of 175 L/h and 100 L/h, respectively. In the charge transfer mode (Figure 1[b]), ionization was typically initiated by the formation of N2
•+ and Nn•+ cluster ions, which may
undergo charge exchange with ana-lyte molecules (M) provided the ion-ization energy (IE) of M is lower than the recombination energy (RE) of N2
•+. Non-reactive collisions between the incipient ions and the surround-ing N2 results in collisional cooling that significantly reduces fragmenta-tion and therefore also the detection limits for the molecular ions (M•+). Other ionization schemes are also possible by introducing dopants to the ion source. For example, H2O can be used to promote protonation. O2
•- is the primary charge carrier in negative ion mode APCI, which can result in selective ionization and structure diagnostic fragmentation of halogenated compounds (18,20). The technique of ionizing a sample of molecules by gas-phase ion-mol-
Figure 1: (a) The atmospheric pressure gas chromatography (APGC) ion source; (b) general scheme for positive mode ionization in dry ion source.
Figure 2: The flow modulator used in this study.
GC coupled to atmospheric pres-sure chemica l ionizat ion (APCI) (12–16) is, by contrast, ideally suited to high f lows. In GC–APCI, the GC eff luent is swept into the ion source, which is held at atmospheric pres-sure, by a high f low of nitrogen gas (>100 mL/min). There is a lso the additional benefit that the elevated pressures promote collisional cooling of the incipient ions. Consequently, very little fragmentation occurs, re-sulting in further signal enhance-ment of the molecular ion. With widespread adoption of atmospheric
pressure ionization techniques, such as electrospray ionization (ESI) and APCI, there is growing interest in coupling GC–MS using atmospheric pressure ionizat ion. This ar t icle reports on proof-of-concept exper-iments using a simple, cost-effec-tive f low modulator (17) coupled to a quadrupole t ime-of-f l ight mass spectrometer (QTOF-MS). The re-sults indicate that environmental contaminants can be detected at low femtogram levels using a full-scan-ning instrument capable of nontar-geted analysis.
chromatographyonl ine .com October 2018 Current Trends in Mass Spectrometry 17
Figure 3: (a) Comparison of pre- and post-modulation of 500 fg of TCDD; (b) CZC of 500 fg of
13C-TCDD and 2.5 fg of TCDD. Experiments were performed using a Zoex ZX2 thermal modulator.
Columns: 15 m × 0.25 mm, 0.1-μm db-5 (Agilent) and 0.18 mm × 0.18 μm, 40-m db-5 (Agilent)
were employed for experiments (a), and (b) and (c), respectively.
used to explore the potential of cou-pling f low modulation to GC–APCI. The construction, operation, and performance of this device has been described recently (17). Brief ly, the multimode modulator (17) of this study was constructed around a 1/8” outside diameter (o.d.) liner (standard PTV injector liner) (Figure 2).
A piece of tubing or capillary can also be used for primary eff luent col-lection, but the 1/8” o.d. liner provided the volume required (~200 μL) to ac-commodate a 4 s modulation period and 3 mL/min carrier gas f low. One end of the joining liner was attached to a cross union, or as shown in Figure 2, a modified tee. The exit of the pri-mary column (15 m × 0.25 mm, 0.1-μm db-5 [Agilent Technologies]) was in-serted through the cross and then into the joining liner, so that the column exit sat approximately 1 cm from the gooseneck of the liner. The opposite end of the liner was attached to a tee union. A 0.5 m × 0.53 mm Rxi Guard
ecule reactions is well studied (20), and, since its inception, an enormous number of studies have appeared on its applications in analytical MS.
A ZX2 thermal modulator (Zoex) was used for CZC experiments. A multimode modulator (17) designed and constructed by J.V. Seeley was
chromatographyonl ine .com18 Current Trends in Mass Spectrometry October 2018
column (Restek) that served as the transfer line to the MS was inserted into the tee and butted against the gooseneck. Auxiliary f low (125 mL/min) was delivered to the tee and cross unions via a two-way, three-port solenoid valve (not shown). A f low restrictor was connected to the cross connection. The operation of the multimode modulator for the purpose of signal enhancement is described below.
Results and Discussion
CZC relies on the same principles of operation as GC×GC, whereby a pulse of cooled nitrogen gas is used to condense and focus GC eff luent
(primary peak), followed by a pulse of heated gas for re-injection (secondary peak). The timing of the two pulses (modulation period) is configured so that the width of a GC peak is com-pressed. This is i l lustrated in Fig-ure 3(a), which displays a ~3 s wide peak prior to modulation and a nar-row (250 ms wide) peak post modula-tion. The tenfold enhancement in sig-nal intensity (Figure 3[a]) enables low femtogram detection of 2378-TCDD (tetrachlorodibenzo-p-dioxin) using QTOF, see Figure 3(c). Note that the identity of the native compound (Fig-ure 3[c]) is confirmed by comparison with the corresponding 13C-labelled compound (Figure 3[b]).
A flow modulator does not condense or focus the GC eff luent. Instead, the effluent is trapped in a sample loop of sufficient volume to accommodate the primary flow. The width of the second-ary peak can still be controlled by the secondary f low. Figures 4(a) and 4(b) show the flow modulator of this study in the collection and injection states, respectively. In the collection state, ef-fluent from the primary column (3 mL/min) backfills into the liner as primary carrier gas escapes via the flow restric-tor. In the injection state (Figure 2[c]), the secondary flow (125 mL/min) is di-verted through the sample loop, flush-ing the accumulated contents into the mass spectrometer.
From the ratio of the primary and secondary f lows, one anticipates a signal enhancement of approxi-mately 40×. As shown in Figure 5(a), the signal-to-noise ratios (S/N) of two polychlorinated biphenyls (101 and 118) are approximately 6:1. When the modulator is activated, this S/N in-creases by a factor of 20 to 124:1 (see Figure 5[c]). Note that the acquisition time of the mass spectrometer is nor-mally set to acquire ~10 data points across the peak. As shown in Figure 5(b), the S/N of the primary peaks in-creases to 24:1 when the acquisition rate is adjusted from 30 Hz to 4 Hz. Still, signal enhancement of approxi-mately 10:1 is evident from the over-laid chromatograms in Figure 5(d).
Summary
A f low modulator was constructed and evaluated using QTOF-MS. Like CZC, flow modulation produces sharp peaks (<200 ms wide) with a significant improvement in signal intensity com-pared to unmodulated peaks. Aside from the obvious implications to quan-titative analysis, the enhanced sensi-tivity also helps address the critical need for nontargeted identification of unknown environmental toxicants. The analytical platform described in this study is capable of acquiring high quality, full scan mass spectra of femtogram level toxicants and as such may be a promising tool in en-vironmental monitoring, biomoni-toring, and exposomic research (20).
Figure 4: The home-built flow modulator and its operation in the (a) collection and (b) injection
states.
chromatographyonl ine .com October 2018 Current Trends in Mass Spectrometry 19
Time (min)
Time (min)
Time (min)
Time (min)
References
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55 (2012).
Figure 5: Extracted ion chromatograms of PCB-101 and PCB-118 without modulation (a) 30 Hz
and (b) 4 Hz; and (c) with modulation; (d) overlaid chromatograms (a–c).
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Larsen, Anal. Chem. 77, 7826 (2005).
(17) J.V. Seeley, N.E. Schimmel, and S.K.
Seeley, J. Chrom. A 1536, 6–15 (2018).
(18) S. Fernando, M.K. Green, K. Organ-
tini, F. Dorman, R. Jones, E.J. Reiner,
and K.J. Jobst, Anal. Chem. 88, 5205
(2016).
(19) D. Megson, M. Robson, K.J. Jobst, P.A.
Helm, and E.J. Reiner, Anal. Chem. 88,
11406–11411 (2016).
(20) A.G. Harris, Chemical Ionization Mass
Spectrometry (CRC Press, Boca Raton,
USA, 1983).
(21) J.R. Sobus, J.F. Wambaugh, K.K. Isaacs,
A.J. Williams, A.D. McEachran, A.M.
Richard, C.M. Grulke, E.M. Ulrich, J.E.
Rager, M.J. Strynar, and S.R. Newton,
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(2017) doi: 10.1038/s41370-017-
0012-y.
Karl Jobst is a development scientist with
the Ontario Ministry of the Environment and
Climate Change (MOECC). His research in-
terests focus on the development and appli-
cation of mass spectrometric techniques for
the analysis of complex environmental sam-
ples. John Seeley is a professor of chemis-
try at Oakland University in Rochester, Mich-
igan, USA. For the past 20 years, his research
has largely focused on the development of
flow modulation GC×GC and its application
to separations of complex mixtures of vola-
tile organic compounds. Eric Reiner is an
adjunct professor at the University of To-
ronto and emeritus mass spectrometry re-
search scientist at MOECC. He has spent over
30 years at MOECC working on the develop-
ment of analytical methods for the analysis
of halogenated persistent organic pollutants
(POPs). Lauren Mullin is a principal scien-
tist at Waters Corporation. Her work focuses
on the development of liquid and gas chro-
matography–mass spectrometry methods
to address food, environmental, and various
small molecule research analyses. Adam
Ladak is a senior strategic scientific mar-
keting manager at Waters Corporation. He is
responsible for food and environmental mar-
ket development worldwide with a focus on
environmental research. Direct correspon-
dence about this article to Adam_Ladak@
waters.com.
chromatographyonl ine .com20 Current Trends in Mass Spectrometry October 2018
The discovery of monoclonal antibodies (mAbs) in the 1970s and the realization of their therapeutic potential in the following decades has been a key milestone in
medicine (1–3). Today, over 70 mAbs have received regula-tory approval in the US and Europe, of which over 20 display blockbuster status, and over 50 are in late-stage clinical de-velopment (4–6). With the top-selling mAbs evolving out of patent, there has been a growing interest in the development of biosimilars (5,7,8). In 2013, we witnessed the European approval of the first mAb biosimilars and in 2016, the first mAb biosimilar also reached marketing authorization in the US. Since then, a growing number of mAb biosimilars have reached approval in both Europe and the US. The successes of mAbs have furthermore triggered the development of var-ious next-generation formats. In oncology, antibody–drug conjugates (ADCs) are particularly promising, because they
synergistically combine a specific mAb linked to a biologi-cally active cytotoxic drug via a stable linker. The promise of ADCs is that highly toxic drugs can selectively be delivered to tumor cells, thereby substantially lowering side effects as typically experienced with classical chemotherapy. Currently two ADCs are marketed and over 30 are in clinical trials.
Together with a huge therapeutic potential, mAbs come with an enormous structural complexity. Opposed to small-molecule drugs, mAbs are large (ca. 150 kDa) and heterogeneous (as a result of the biosynthetic process and subsequent manufacturing and storage) making their analysis very challenging. Despite the fact that only a single molecule is cloned, hundreds of possible variants differing in post-translational modifications (N-glycosylation, asparagine deamidation, aspartate isomerization, methionine oxidation), amino acid
Koen Sandra, Jonathan Vandenbussche, Isabel Vandenheede, Bo Claerebout, Jeff Op
de Beeck, Paul Jacobs, Wim De Malsche, Gert Desmet, and Pat Sandra
Monoclonal antibodies are becoming a core aspect of the pharmaceutical industry. Together with
a huge therapeutic potential, these molecules come with a structural complexity that drives state-
of-the-art chromatography and mass spectrometry (MS) to its limits. This article discusses the
use of micro-pillar array columns in combination with mass spectrometry for peptide mapping of
monoclonal antibodies (mAbs) and antibody–drug conjugates (ADCs). Micro-pillar array columns are
produced by a lithographic etching process creating a perfectly ordered separation bed on a silicon
chip. As a result of the order existing in these columns, peak dispersion is minimized and highly effi-
cient peptide maps are generated, providing enormous structural detail. Using examples from the
author’s laboratory, the performance of these columns is illustrated.
Peptide Mapping of Monoclonal
Antibodies and Antibody–Drug
Conjugates Using Micro-Pillar
Array Columns Combined with
Mass Spectrometry
chromatographyonl ine .com October 2018 Current Trends in Mass Spectrometry 21
Figure 1: Micro-pillar array column. From left to right: (left) Top view of two parallel 315-μm wide separation channels that have been interconnected
with proprietary flow distributor structures, (middle) SEM image showing a transverse section of a separation channel containing 5-μm diameter
cylindrical pillars, (right) HR-SEM image of the 300-nm porous-shell layer incorporated into a 5-μm diameter pillar.
sequence, and higher order structures may coexist, all contributing to the safety and efficacy of the product. Compared to naked mAbs, ADCs f ur t her add to t he complex it y
because the heterogeneity of the initial antibody is superimposed with the variability associated with the conjugation. Conjugation typically takes place on the amino groups of
lysine residues or on the sulphydryl g roups of i ntercha i n c y s te i ne residues. With 80 to 100 lysine and only eight interchain cysteine residues available, lysine conjugation yields a more heterogeneous mixture of species compared to cysteine conjugation.
Peptide mapping is particularly powerful for the detailed structural characterization of these products and has proven to be of enormous value in demonstrating comparability, for example, between mAb originator and biosimi lar. Character ist ics such a s a m i no ac id sequence and modif icat ions l ike N- and O-glycosylation, glycation, N- and C-terminal processing, deamidation (asparagine, glutamine), aspartate isomerization, succinimide, oxidation (methionine, tryptophan), clipping, sequence variants, cysteine variants (S-S bridges, thioether, free cysteine), and drug conjugation sites can readily be extracted out of the generated peptide map data and at low levels.
Look ing back to t he pept ide mapping of the f irst monoclonal antibodies in the late 1980s and early 1990s , one w i l l not ice a substantial leap forward in technical capabilities. Chromatography and mass spectrometry were at that time of modest performance compared to t he current s tate-of-t he-a r t technology. High performance liquid chromatography (HPLC) separations were performed on columns packed with 5–10 μm porous particles and pumps were operated at 400 bar.
Figure 2: LC–MS chromatograms of a Herceptin tryptic digest. (a) Total compound chromatogram.
(b) Compound chromatograms corresponding to the identified peptides presented in Table 1.
chromatographyonl ine .com22 Current Trends in Mass Spectrometry October 2018
Fast atom bombardment (FAB) was used to introduce peptides into low resolution mass spectrometers and peptide identity was further confirmed using Edman degradation following peak collection (16). Today, columns packed w it h sub-2-μ m porous and superficially porous particles operated at system pressures up to 1500 bar and electrospray ionization (ESI) can be used to introduce peptides into high resolution mass spectrometers equipped with a variety of fragmentation modes.
A more re c e nt a d d i t ion to t he ch romatog raphers toolbox
are micro-pi l lar array columns. T he or ig i n of t h is technolog y d ate s bac k to t he l a te 19 9 0 s when Regnier et a l . addressed t he problem of m i n iat u r i z i ng capi l lary electrochromatography (CEC) columns and introduced microfabricated supports as an a lternative for the conventional packed beds (17,18). The theoretical benef it (reduc t ion of t he va n Deemter A-term) of such supports was elucidated only a few years later by Knox (19). In the years to follow, Desmet et a l. conducted severa l quant itat ive studies on K nox’s
argumentation, taking a column f i l led with an array of pil lars as a representative example (20). In 2007, the first micromachined LC columns operated by pressure-driven l iquid f low, later termed micro-
pillar array columns, were reported (21). The inherent high permeability and low “on-column” dispersion obtained by the perfect order of the separation bed makes micro-pillar array-based chromatography unique and offers several benefits for chromatographers. The peak d i s p e r s i o n o r i g i n a t i n g f r o m heterogeneous f low paths in the s e p a r at ion b e d i s e l i m i n at e d (no A-term contr ibut ions) a nd therefore components remain more concentrated (sharp peaks) during separation. The freestanding nature of the pillars also leads to much lower backpressure allowing the use of very long columns. These properties result in excellent chromatographic performance with high resolution and high sensitivity.
This article describes the use of micro-pillar array columns in the characterization of mAbs and ADCs for the first time.
Materials and Methods
Materials
Water and acetonitrile were purchased f rom Biosolve . Tr i f luoroacet ic acid, dithiothreitol (DTT), and 2-iodoacetamide (IAA) were from Sigma-Aldrich. Tris-HCl pH 7.5 was purchased as a 1 M solution (Thermo Fisher Scientific). Porcine sequencing-grade modified trypsin was acquired from Promega and Rapigest from Waters. Herceptin and Kadcyla were obtained from Roche, Remicade from Johnson & Johnson, Adcetris from Seattle Genetics, and the candidate biosimilars from a local biotechnology company.
Sample Preparation
To a volume corresponding to 100 μg of protein, 105 μL of 0.1% Rapigest in 100 mM Tris-HCl pH 7.5 was added followed by the addition of 100 mM Tris-HCl pH 7.5 to a final volume of 192.5 μL. The sample was subsequently reduced
Figure 3: LC–UV 214 nm chromatograms corresponding to the replicate analysis (n = 4) of a
Herceptin tryptic digest.
Figure 4: LC–MS total compound chromatograms of a Herceptin originator and candidate
biosimilar tryptic digest.
chromatographyonl ine .com October 2018 Current Trends in Mass Spectrometry 23
at 60 °C for 30 min by the addition of 5 mM DTT (2.5 μL of 400 mM DTT in 100 mM Tris-HCl) and alkylated at 37 °C for 1 h by adding 10 mM IAA (5 μL of 400 mM IAA in 100 mM Tris-HCl). Digestion proceeded for 16 h at 37 °C
using trypsin as protease added at an enzyme to substrate ratio of 1/25 (w/w). Lyophilized trypsin (20 μg) dissolved in 100 mM Tris-HCl (50 μL) was added in a volume of 10 μL giving rise to a final sample volume of 210 μL.
LC–MS
A n U lt i m ate 3 0 0 0 R SL Cna no system (Thermo Fisher Scientific) was used for LC–ultraviolet (UV)–MS measurements. Tryptic digests were analyzed on a 200 cm C18 μPAC column (PharmaFluidics) at 50 °C. Elution was performed with a linear gradient of (A) 0.1% TFA in H2O and (B) 0.1% TFA in 80:20 (v/v) acetonitrile–water, from 2% B to 70% B in 120 min. The flow rate was 500 nL/min. An injection program allowed the introduction of 100-nL sample in between two plugs of mobile phase A. Loop size was 1 μL and samples were kept at 10 °C in the autosampler tray while waiting for injection. UV detection was performed at 214 nm using a 3 nL detection cell.
High-resolution accurate mass measurements were obtained on a 6530 QTOF mass spectrometer (Agilent Technologies) equipped with a dual nano-ESI source operated in posit ive electrospray ionizat ion mode . T he m icro-pi l la r a r ray column was connected via a 30-μm internal diameter (i.d.), 280-μm outside diameter (o.d.) fused silica capillary to a PicoTip emitter (8-μm tip i.d. [New Objective]) via a true zero dead volume conductive union (UH-634 sta inless steel adapter from Idex). To connect the PicoTip emitter to the union, an F-330 blind f it t ing equipped with an M-125 perfluoroelastomer conductive ferrule was used (Idex). Capillary voltage was set at +2.4 kV, drying gas f low rate and temperature were set at 4 L/min and 300 °C, and fragmentor voltage at 175 V. The TOF was calibrated on a daily basis and subsequently operated at high accuracy (<5 ppm) without using reference masses. Data were col lected in centroid mode at a rate of 1 spectrum/s in the extended dynamic range mode (2 GHz) offering a resolution of 9000 at mass-to-charge ratio (m/z) 622.0296. MS/MS experiments were performed in the data-dependent acquisition (DDA) mode. One survey MS measurement was complemented with three data-dependent MS/MS
Figure 5: Extracted ion chromatograms of selected modified peptides measured in Herceptin
originator and candidate biosimilar. Shown are asparagine deamidation, lysine truncation, and
methionine oxidation, which are elevated in the biosimilar compared to the originator. The
location of the modifications is highlighted in the antibody structure.
chromatographyonl ine .com24 Current Trends in Mass Spectrometry October 2018
measurements. Double, triple, and quadruple charged precursor ions with the Averagine isotope model were selected based on abundance. After being fragmented twice, a particular m/z value was excluded for 30 s. Selecting the same m/z value twice increases the chance of measuring a particular precursor at its maximum intensity while an exclusion time of 30 s allows MS/MS information on chromatographically resolved isomers to be obtained. The quadrupole was operated at medium resolution and the collision energy varied based on the following equation:
(3.6 * m/z)/100 – 4 [1]
A l l-ions MS/MS ex per i ments were per for med at a col l i s ion energ y of 20 eV. LC data were acquired in Chromeleon (Thermo Fisher Scientif ic) and MS data in MassHunter (Agilent Technologies). Data analysis was performed in MassHunter Qualitative Analysis with BioConfirm add-on.
Results and Discussion
Characteristics of
Micro-Pillar Array Columns
The separation beds of micro-pillar array columns are fabricated by carefully etching the interstitial volumes out of a silicon substrate following lithographic definition of an array of pillars. This creates a stationary phase support structure that is organized in a reproducible and ordered pattern. Concatenation of several of these channels allows long column lengths to be fabricated on a small footprint (22). The most important characteristics of the micro-pillar array separation bed design are: pillar diameter, 5 μm; inter pillar distance, 2.5 μm; pillar length or bed depth, 18 μm; external porosity (Vinterstitial/Vtotal), 59%; bed channel width, 315 μm; and bed length, 200 cm. To increase the retentive surface, the pillars are rendered superficially porous with a typical porous shell thickness of 300 nm and pore sizes in the nanometer range. The porous surface has been uniformly modified with octadecyl alkyl
Figure 6: LC–UV 214 nm peptide map of (a) a Remicade originator and (b) a candidate biosimilar
tryptic digest. Unzoomed and zoomed views.
Figure 7: LC–MS total compound chromatograms of (a) a Remicade originator and (b) a candidate
biosimilar tryptic digest. MS/MS spectra associated with peaks 4 and 5 are presented in Figure 8.
chromatographyonl ine .com October 2018 Current Trends in Mass Spectrometry 25
chains to create a hydrophobic stationary phase suited for reversed-phase LC separations. Figure 1 shows some relevant characteristics of the micro-pillar array column.
Because of the high permeability, the 200-cm column used in this study can be operated at moderate LC pump pressures (50 to 300 bar) over a wide range of flow rates (100–1000 nL/min). Van Deemter measurements with heptyl-phenyl ketone demonstrated that a total of 400,000 theoretical plates could be generated at the optimal li nea r solvent veloc it y, corresponding to a f low rate of 200–250 nL/min and generat ing a column backpressure of on ly 70 bar. By increasing the f low rate
up to 750 nL/min, eff iciencies up to 200,000 theoretical plates could be ach ie ved w it h i n 30 m i n at operating pressures well below 250 bar (mobile phase 50% acetonitrile with 0.1% [v/v] FA).
Peptide Mapping of
Herceptin Originator and
a Candidate Biosimilar
Herceptin (scientific international non-proprietary names [INN] name tras-tuzumab) is a humanized IgG1 mAb recombinantly produced in Chinese hamster ovary (CHO) cells and used in the treatment of human epidermal growth factor receptor 2 (HER2) posi-tive breast cancer. Herceptin is open to the European market and evolves out of
patent in the US in 2019 (5). Given its market potential (global sales of $6.6 bil-lion in 2015), dozens of companies are actively developing a Herceptin biosim-ilar. In developing these products, simi-larity to the originator has to be demon-strated and for that, an enormous weight is placed on analytics; both the biosimilar and originator need to be characterized and compared in great detail. Peptide mapping is a powerful method to charac-terize and compare mAbs in great detail.
When digesting Herceptin with the enzyme trypsin, which cleaves the protein next to arginine and lysine residues, 62 identity peptides are formed. Taking into account post-translational modifications and incomplete and aspecific cleavages tak ing place, over 100 peptides w it h va r y i ng phy sicochem ic a l properties present in a wide dynamic concent rat ion ra nge a re to be expected. This is quite a complex sample demanding the best in terms of chromatographic resolution.
Figure 2(a) shows the LC–MS tota l compound chromatogra m of a Herceptin tryptic digest on a m ic ro -pi l l a r a r r ay c o lu m n . The compound chromatograms corresponding to the identi f ied peptides presented in Table 1 are shown in Figure 2(b). Approximately 95% of the sequence is covered by this peptide map and post-translational modifications such as glycosylation, asparagine deamidation, aspartate i s o m e r i z a t i o n , m e t h i o n i n e oxidation, N-terminal cyclization (pyroglutamate), and C-terminal lysine truncation, amongst others, are revealed. Peptides that are not detected are typically small and their signal might be suppressed in the column f low through. The UV 214 nm chromatograms corresponding to replicate analyses (n = 4) of the Herceptin tryptic digest are demonstrated in Figure 3. These measurements are highly precise making this technology attractive to compare different mAb production batches a nd to compa re m Ab originator products to biosimilars.
T h e L C – M S p e p t i d e m a p s of a Hercept in or ig inator a nd
Figure 8: LC–MS/MS spectra of peak 4 and 5 (Figure 7) acquired on the fly in data-dependent
MS/MS mode confirming the threonine to serine substitution in the variable part of the heavy
chain.
chromatographyonl ine .com26 Current Trends in Mass Spectrometry October 2018
Table I: Identified peptides in the Herceptin tryptic digest analyzed by LC–MS
RT (min) Mass Sequence Seq Loc. Theor. Mass ∆m (ppm) Modifications
61.29 1880.99676 EVQLVESGGGLVQPGGSLR Hc(001-019) 1880.9956 0.62
66.83 1862.98605 EVQLVESGGGLVQPGGSLR Hc(001-019) 1862.98503 0.55 Pyroglutamate
51.04 1166.57794 LSCAASGFNIK Hc(020-030) 1166.5754 2.18 Cys-Alkylation
71.21 2237.10621 LSCAASGFNIKDTYIHWVR Hc(020-038) 2237.10516 0.47 Cys-Alkylation
55.35 1088.54237 DTYIHWVR Hc(031-038) 1088.54033 1.87
52.54 829.44557 GLEWVAR Hc(044-050) 829.44464 1.12
39.97 1084.52052 IYPTNGYTR Hc(051-059) 1084.51893 1.47 Deamidation
40.39 1083.53637 IYPTNGYTR Hc(051-059) 1083.53491 1.35
41.46 1084.52241 IYPTNGYTR Hc(051-059) 1084.51893 3.21 Deamidation
28.81 681.33403 YADSVK Hc(060-065) 681.33336 0.99
44.14 968.48309 FTISADTSK Hc(068-076) 968.48148 1.66
43.48 1366.66793 NTAYLQMNSLR Hc(077-087) 1366.66634 1.17 Met-Alkylation
53.78 1310.63608 NTAYLQMNSLR Hc(077-087) 1310.62889 5.49 Deamidation
54.96 1309.6462 NTAYLQMNSLR Hc(077-087) 1309.64487 1.02
40.75 1333.56246 AEDTAVYYCSR Hc(088-098) 1333.56087 1.19 Cys-Alkylation
70.51 2840.27436WGGDGFYAMDYWGQ
GTLVTVSSASTKHc(099-124) 2840.2752 -0.3 Met-Alkylation
79.37 2783.25248WGGDGFYAMDYWGQ
GTLVTVSSASTKHc(099-124) 2783.25374 -0.45
58.45 1185.64103 GPSVFPLAPSSK Hc(125-136) 1185.63938 1.4
54.84 1320.67286 STSGGTAALGCLVK Hc(137-150) 1320.67075 1.59 Cys-Alkylation
92.81 6712.27146
DYFPEPVTVSWNSGALTSG
VHTFPAVLQSSGLYSLSSVVTVP
SSSLGTQTYICNVNHKPSNTK
Hc(151-213) 6712.3072 -5.32 Cys-Alkylation
24.21 599.36222 KVEPK Hc(217-221) 599.36426 -3.41
23.25 471.26917 VEPK Hc(218-221) 471.2693 -0.27
21.56 508.19936 SCDK Hc(222-225) 508.19515 8.28 Cys-Alkylation
82.24 2618.30392 THTCPPCPAPELLGGPSVFLFPPK Hc(226-249) 2618.30254 0.53 Cys-Alkylation
80.29 2843.4514 THTCPPCPAPELLGGPSVFLFPPKPK Hc(226-251) 2843.45027 0.4 Cys-Alkylation
41.18 891.44858 DTLMISR Hc(252-258) 891.44841 0.19 Met-Alkylation
41.64 850.42241 DTLMISR Hc(252-258) 850.42186 0.65 Oxidation
45.43 834.42764 DTLMISR Hc(252-258) 834.42694 0.84
64.07 2138.02125 TPEVTCVVVDVSHEDPEVK Hc(259-277) 2138.02016 0.51 Cys-Alkylation
61.55 1676.79346 FNWYVDGVEVHNAK Hc(278-291) 1676.79471 -0.75 Isomerization
62.47 1676.7955 FNWYVDGVEVHNAK Hc(278-291) 1676.79471 0.47
32.86 2957.13923 EEQYNSTYR Hc(296-304) 2957.14427 -1.7 G2F
32.92 2795.08868 EEQYNSTYR Hc(296-304) 2795.09144 -0.99 G1F
33.05 2633.03635 EEQYNSTYR Hc(296-304) 2633.03862 -0.86 G0F
81.52 1807.98677 VVSVLTVLHQDWLNGK Hc(305-320) 1807.98324 1.95 Deamidation
82.11 1807.0017 VVSVLTVLHQDWLNGK Hc(305-320) 1806.99922 1.37
chromatographyonl ine .com October 2018 Current Trends in Mass Spectrometry 27
Table I: (Continued) Identified peptides in the Herceptin tryptic digest analyzed by LC–MS
82.92 1807.98553 VVSVLTVLHQDWLNGK Hc(305-320) 1807.98324 1.27 Deamidation
84.33 1789.97519 VVSVLTVLHQDWLNGK Hc(305-320) 1789.97267 1.4 Succinimide
46.50 837.49753 ALPAPIEK Hc(330-337) 837.49601 1.82
23.71 447.26932 TISK Hc(338-341) 447.2693 0.04
50.08 1285.66847 EPQVYTLPPSR Hc(348-358) 1285.66665 1.41
24.31 636.2778 EEMTK Hc(359-363) 636.27888 -1.69
58.87 1160.62392 NQVSLTCLVK Hc(364-373) 1160.62235 1.36 Cys-Alkylation
68.04 2544.11037 GFYPSDIAVEWESNGQPENNYK Hc(374-395) 2544.10812 0.88 Deamidation
68.51 2543.12268 GFYPSDIAVEWESNGQPENNYK Hc(374-395) 2543.12411 -0.56
69.70 2526.09392 GFYPSDIAVEWESNGQPENNYK Hc(374-395) 2526.09756 -1.44 Succinimide
70.26 1872.90824 TTPPVLDSDGSFFLYSK Hc(396-412) 1872.91455 -3.37 Isomerization
71.48 1872.91517 TTPPVLDSDGSFFLYSK Hc(396-412) 1872.91455 0.33
32.14 574.33213 LTVDK Hc(413-417) 574.33263 -0.86
66.07 2800.25791 WQQGNVFSCSVMHEALHNHYTQK Hc(420-442) 2800.25984 -0.69 Cys-Alkylation
44.81 659.34888 SLSLSPG Hc(443-449) 659.34901 -0.19 Lys-Truncation
43.20 787.44611 SLSLSPGK Hc(443-450) 787.44397 2.72
46.55 1934.90169 DIQMTQSPSSLSASVGDR Lc(001-018) 1934.90038 0.68 Met-Alkylation
54.09 1877.87963 DIQMTQSPSSLSASVGDR Lc(001-018) 1877.87891 0.38
35.39 748.39074 VTITCR Lc(019-024) 748.39016 0.78 Cys-Alkylation
53.62 1707.82255 ASQDVNTAVAWYQQK Lc(025-039) 1707.82165 0.53
54.73 1708.80311 ASQDVNTAVAWYQQK Lc(025-039) 1708.80567 -1.5 Deamidation
51.50 1990.97492 ASQDVNTAVAWYQQKPGK Lc(025-042) 1990.97486 0.03 Deamidation
53.37 1989.99336 ASQDVNTAVAWYQQKPGK Lc(025-042) 1989.99084 1.27
54.43 1990.97614 ASQDVNTAVAWYQQKPGK Lc(025-042) 1990.97486 0.64 Deamidation
83.35 4040.11375ASQDVNTAVAWYQQKPG
KAPKLLIYSASFLYSGVPSRLc(025-061) 4040.116 -0.55
76.25 1771.95123 LLIYSASFLYSGVPSR Lc(046-061) 1771.95088 0.2
81.29 4186.90432SGTDFTLTISSLQPEDFAT
YYCQQHYTTPPTFGQGTKLc(067-103) 4186.91062 -1.5 Cys-Alkylation
30.68 487.3006 VEIK Lc(104-107) 487.3006 -0.01
72.96 2101.12277 RTVAAPSVFIFPPSDEQLK Lc(108-126) 2101.1208 0.94
74.60 1945.02035 TVAAPSVFIFPPSDEQLK Lc(109-126) 1945.01968 0.34
80.21 1796.88935 SGTASVVCLLNNFYPR Lc(127-142) 1796.88796 0.77 Cys-Alkylation
21.45 346.18747 EAK Lc(143-145) 346.18524 6.46
34.98 559.31157 VQWK Lc(146-149) 559.31183 -0.48
39.99 2134.96295 VDNALQSGNSQESVTEQDSK Lc(150-169) 2134.96146 0.7
59.63 1501.75181 DSTYSLSSTLTLSK Lc(170-183) 1501.75118 0.42
24.33 624.27451 ADYEK Lc(184-188) 624.27551 -1.6
50.86 1874.92219 VYACEVTHQGLSSPVTK Lc(191-207) 1874.91965 1.35 Cys-Alkylation
26.74 522.25451 SFNR Lc(208-211) 522.25505 -1.03
chromatographyonl ine .com28 Current Trends in Mass Spectrometry October 2018
candidate biosimilar are shown in Figure 4. While both peptide maps are highly comparable, differences in post-translational modifications are detected. This is illustrated in the extracted ion chromatograms presented in Figure 5.
Peptide Mapping of
Remicade Originator
and Candidate Biosimilar
Remicade (scient i f ic INN name inf liximab) is a chimeric IgG1 mAb on the market since 1998 targeting t u mou r ne c ro s i s f a c tor a lph a (TNF-α) and consequent ly used in the treatment of autoimmune diseases. Remicade reached global sales of $8.4 billion in 2015. Several Remicade biosimilars have already been approved bot h in Europe and the US and many more are in development (8).
Figures 6 and 7 show the UV and MS total compound chromatograms of a Remicade orig inator and a candidate biosimilar tryptic digest, respectively. Chromatograms are ver y simi lar but some st r i k ing differences are noted (peaks 1–5), wh ich c a n be ex pla i ned upon consulting the corresponding MS data. Peaks 1 and 2 in the originator chromatogram, corresponding to C-terminal heav y chain peptides SLSLSPGK and SLSLSPG respectively, are replaced by peak 3, corresponding to SLSLSPGI in the biosimilar. The origin of the two peaks SLSLSPG and SLSLSPGK in the originator mAb can be explained by the knowledge that heavy chains are historically cloned with a C-termina l lysine but during cell culture production, host cel l carbox y pept idases act on the antibody result ing in the par t ia l remova l of t hese lysine residues . A m issense mutat ion (Ly s" I l e) i n t he C HO c lone producing the candidate biosimilar e x pl a i n s p e a k 3 (SL SL SP GI). Peaks 4 and 5 corresponding to, respectively, heavy chain peptides NYYGSSYDY WGQGTTLTVSSASTK in the candidate biosimilar and NYYGSTYDY WGQGTTLTVSSASTK i n t he or ig i nator aga i n resu lt
Figure 9: LC–UV 214 nm peptide map of Herceptin and Kadcyla.
Figure 10: LC–MS peptide map of Herceptin and Kadcyla.
Figure 11: All-ions MS/MS chromatograms of the Herceptin and Kadcyla tryptic digest. The ion at
m/z 547.2206 was extracted at high mass accuracy.
chromatographyonl ine .com October 2018 Current Trends in Mass Spectrometry 29
from a point mutation (Thr" Ser) in the biosimilar CHO clone. The corresponding MS/MS spectra are shown in Figure 8.
According to US and European regulatory authorit ies , identica l primary sequence is primordial to similarity thereby ruling out this candidate biosimilar from further development.
Peptide Mapping
of the Antibody–Drug
Conjugate Kadcyla
The antibody–drug conjugate Kadcyla (ado-trastuzumab emtansine) has been used in the treatment of HER2 positive breast cancer since 2013. It combines the anti-HER2 antibody trastuzumab (Hercept in) with the cy totox ic microtubule-inhibiting maytansine
derivative DM1 conjugated to lysine residues via a non-reducible thioether linker. With a drug distribution of 0 to 8, a drug-to-antibody ratio (DAR) of 3.5, and various lysine residues available for conjugation, thousands of species can be generated. To obtain insight in the drug conjugation sites, peptide mapping is the gold standard technology.
Figures 9 and 10 show, respectively, the LC–UV and MS total compound chromatograms of a Herceptin and Kadcyla tryptic digest. Since Herceptin and Kadcyla have the same amino acid sequence, the majority of the peptide map is identical. Differentiating peaks, corresponding to the DM1 conjugated peptides, are nevertheless observed and are located in the late eluting part of the chromatogram. Indeed, the conjugation of DM1 makes the peptides more hydrophobic explaining the elution behavior. Upon collision-induced dissociation (CID), DM1 conjugated peptides give rise to specific fragments originating from the cytotoxic drug, for example, at m/z 547.2206. This ion can be used to selectively recognize DM1 conjugated peptides in the LC–MS chromatogram. For that, one can operate the QTOF-MS system in the a l l-ions MS/MS mode, which means that the quadrupole is operated in the RF only mode, thereby transferring all peptides to the collision cell where CID takes place. As illustrated in Figure 11, when extracting from the data the specific fragment ion at m/z 547.2206 at high mass accuracy, all conjugated peptides are revealed in the chromatogram of Kadcyla compared to Herceptin. The latter chromatogram is virtually empty, il lustrating the selectivity that is offered by this all-ions MS/MS functionality.
Figure 12 shows the extracted ion chromatograms associated with a selection of identified conjugated peptides. A striking observation is the appearance of isomeric DM1 conjugated peptides, which can be explained by the existence of two stereochemical configurations (diastereomers) of the antibody–drug linkage through maleimide.
Figure 12: Extracted ion chromatograms of selected peptides showing the appearance of isomeric
conjugated peptides. The location of the conjugation site in the mAb structure is shown as well.
Figure 13: LC–MS peptide map of Adcetris. (a) Total compound chromatogram and (b) all-ions
MS/MS chromatogram. The ion at m/z 718.5113 was extracted at high mass accuracy. The location
of the conjugation site in the mAb structure is shown as well.
chromatographyonl ine .com30 Current Trends in Mass Spectrometry October 2018
Peptide Mapping of the
Antibody–Drug Conjugate Adcetris
A simi lar st rateg y was appl ied to revea l t he conjugat ion sites o n A d c e t r i s ( b r e n t u x i m a b -vedotin). Approved in 2011, this ADC is directed to CD30, a major ma rker of Hodg k in ly mphoma a nd s y s tem ic a napla s t ic l a rge cel l lymphoma (ALCL). Adcetris combines the antibody brentuximab w i t h t h e a n t i m i t o t i c d r u g monomethylauristatin E (MMAE) conjugated to interchain cysteine residues via a cathepsin cleavable valine-citrulline linker. With only eight residues (four on each half antibody) available for conjugation, Adcetris is much simpler compared to Kadcyla.
Figure 13 shows the LC–MS total compound chromatogram and the a l l-ions MS/MS chromatog ra m of a n Adcet r i s t r y pt ic d iges t , respectively. In analogy to DM1 c o n j u g a t e d p e p t i d e s , M M A E conjugated peptides a lso contain specific fragment ions originating f rom t he c y totox ic mole c u le , t hat is , at m/z 718.5113. W hen ex t rac t i ng t he lat ter ion f rom t he a l l - ions MS/MS d at a , t he conjugated peptides are revealed. Three intense peaks explaining the full conjugation of MMAE at the four interchain cysteine residues in light chain peptide GEC (peak 2), heav y chain pept ides SCDK ( p e a k 1) , a n d T H T C P P C PA PE L L G G P S V F L F PPK PK (p e a k 3) a re obser ved . Of pa r t icu la r interest is the detection of isomeric p a r t i a l l y c o n j u g a t e d h e a v y c h a i n p e p t i d e T H T C PP C PA P ELLGGPSVFLFPPK PK (pea ks a and b), in which only one of the two cysteine residues is conjugated.
ConclusionsIn t he 21st centur y, numerous novel developments were made in LC column technolog y and the best known are columns packed with sub-2-μm porous part icles or sub-3-μm superficially particles. Micro-pi l lar array columns are another novel development offering
highly ef f icient separat ions. In this article, the use of the micro-pillar array column in mAb and ADC peptide mapping has been i l lustrated. In combination with high resolution mass spectrometry, high sequence coverage is obtained and post-translational modifications such as glycosylation, deamidation, isomerization, oxidation, N-terminal cyclization, and C-terminal lysine truncation can be elucidated. Its use in assessing comparability between an originator and biosimilar mAb has furthermore been demonstrated. The resolving power offered by a micro-pillar array column allows an in-depth study of ADC conjugation s i t e s a n d a c c o m m o d a t e s t h e separation of isomeric conjugated peptides. In combination with all-ions MS/MS, conjugated peptides can selectively be recognized to assist data interpretation.
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Koen Sandra is the editor of “Biophar-
maceutical Perspectives”. He is the Scien-
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chromatographyonl ine .com October 2018 Current Trends in Mass Spectrometry 31
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Bioscience titled “Size-Exclusion
Chromatography/ Mass Spectrometry
(SEC/MS) Analysis of a Bispecific
Antibody” reportedly describes a
bispecific T cell engager (BiTE) consisting
of two linked single-chain variable
fragments that was analyzed by SEC–MS
using a TSKgel UP-SW3000, 2-μm column.
Tosoh Bioscience LLC, King of Prussia, PA.www.tosohbioscience.com
32 PHARMACEUTICAL/DRUG DISCOVERY ADVERTISEMENT
Ineffi ciently designed fl ow splitter technology causes signifi cant
problems that are often overlooked, underestimated, or tolerated simply
due to a lack of product options.
Mott Corporation has developed a breakthrough fl ow splitter tech-
nology for use in HPLC and UHPLC analysis. The new PerfectPeak®
Flow Splitters utilize proprietary porous metal fl ow restrictor technology
as a means of creating a differential fl ow while achieving the appropriate
post-column split ratio. These fl ow splitters are designed for +/-10%
split accuracy with only ~5 μL post-column dwell volumes, signifi cantly
minimizing peak broadening. Additionally, PerfectPeak Flow Splitters
are easier to install, and more convenient to use with their interchange-
able cartridges and fi nger-tight fi ttings.
We subjected new PerfectPeak Flow Splitters to a repeat injection test
to determine the variability between injections.
Repeat Injection Test
The PerfectPeak Flow Splitter was tested for injection-to-injection
reproducibility with a small-molecule analysis method. The method
used was modifi ed from an Agilent application note on the United
States Pharmacopeia (USP) method for the evaluation of caffeine and
acetaminophen (https://www.agilent.com/cs/library/applications/5991-
5920EN.pdf) in which 1 mg/mL of caffeine and acetaminophen was
prepared in deionized water. The method was run under the conditions
shown in Table I.
Results
Peak retention was monitored upon successive injections for fl ow
stability. It was found that upon four consecutive injections, the caffeine
and acetaminophen peaks had a 0.11% relative standard deviation
(RSD). The USP requirements of RSD for replicated injections is no
more than 2.0% variation. The results of the testing can be found in
Figure 2 where it can be seen that peak
resolution was 10.3 on average. Figure
3 highlights the reduction in peak
broadening due to the lower internal
volume of the Mott PerfectPeak Flow
Splitter compared to generic “T” style
New Post-Column Flow Splitters Improve Accuracy and RepeatabilityChristopher J. Martino, James Steele, and Vincent Palumbo, Mott Corporation
Mott Corporation
84 Spring Lane, Farmington, CT 06032
tel. (860) 747-6333
Website: www.mottcorp.comFigure 1: Schematic diagram of the low-pressure gradient experimental test system.
Pump G1312B
Auto Injector G1329B
Column Comp. G1316A
Detector VWD G1314B
Figure 2: Data overlay of the caffeine and acetaminophen analysis from the UV detector at 275 nm showing 0.11% relative standard deviation an average peak resolution of 10.3 after four consecutive injections.
Figure 3: Reductions in peak broadening are observed by converting a method setup from a generic “T” with cut tubing to the Mott PerfectPeak Flow Splitter.
splitting that uses cut
tubing.
Mott’s new Perfect-
Peak Flow Splitters offer
convenience and high
accuracy for 1:1, 2:1,
3:1, 5:1, and 10:1 split
ratios with the use of
interchangeable car-
tridges and fi nger-tight
fi ttings. Each fl ow
splitter is packaged in a small, lightweight tee body that will not
kink instrument lines when connected. The footprint of the device
is under 2 in. in any dimension and rated for up to 15,000 PSIG.
More case studies on improving
peak dispersion and time savings
on system setup can be found at
www.mottcorp.com/splitter.
Table I: Repeat injection method
configuration
ColumnZorbax Eclipse Plus C18
4.6 × 100 mm, 3.5 μm
Mobile PhaseWater:Methanol:Acetic
Acid (69:28:3)
Temperature 45 °C
Flow Rate 1.0 mL/min
Injection Volume 5 μL
Detection UV, 275 nm
Medical/Biological 33ADVERTISEMENT
The analysis of synthetic cannabinoids and their metabolites can be a
diffi cult task, one that is further complicated by the ever-growing list of
synthetic cannabinoids produced by illicit drugmakers. As shown here,
the retention and selectivity of the Raptor Biphenyl column allows new
drugs to be added to an existing method, providing labs with an important
vehicle for improving effi ciency and productivity.
When developing methods for synthetic cannabinoids and their
metabolites, optimization of analysis time, resolution of isobaric
compounds, method robustness, and the ability to add emerging
compounds are of ultimate importance as they infl uence method
effectiveness and longevity. Because the Raptor Biphenyl column
combines the speed of superfi cially porous particles (SPP) with the
resolution of highly selective USLC technology, we used it to develop
a simple, dilute-and-shoot method for synthetic cannabinoids and
their metabolites in urine. Our original work produced a fast method
that separated all target compounds in less than 7 min.
Here, we expand on our original method and show proof of
concept for how new drugs can be added, while still maintaining
complete resolution of JWH-018 and JWH-073 isobars, as well as
good separation of all target analytes from matrix interferences in
urine. In the ever-changing landscape of illegal drugs, the ability
to add emerging drugs to existing methods allows for much more
effective use of laboratory resources as analysts and instruments
can focus on routine sample analysis rather than new method
development.
Experimental Conditions
For the existing method, samples were prepared at 5 ng/mL in
human urine and diluted 3x in a 0.2 μm PVDF Thomson SINGLE
StEP fi lter vial with water:methanol (50:50) prior to analysis. The
additional compounds were prepared at 50 ng/mL in urine and also
were diluted 3x and fi ltered using the same technique. Analyses
were performed on a Waters ACQUITY UPLC I-Class equipped
with a Xevo TQ-S and a Shimadzu Nexera UHPLC equipped with a
SCIEX API 4500 MS/MS. Instrument conditions were as follows, and
analyte transitions for the original analytes and the new drugs are
provided in Tables I and II, respectively.
Adding New Synthetic Cannabinoids to an Existing LC–MS/MS Method
Sharon Lupo and Frances Carroll, Restek Corporation
Time (min)0.00
m/z 328�155
m/z 344�155
m/z 358�155
0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50
Time (min)
2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50
2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50
2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50
Figure 1: Original method developed for the combined analysis of synthetic cannabinoids and metabolites in diluted human urine
34 Medical/Biological ADVERTISEMENT
Table I: Figure 1 analyte identification, retention times, and ion transitions
Figure 1 Peak Identifications tR (min) Precursor Ion Quantifier Product Ion Qualifier Product Ion
1. Pravadoline 2.15 379.29 135.04 114.16
2. AM2233 2.44 459.25 112.2 98.15
3. JWH-200-d5 2.47 390.34 155.07 NA
4. JWH-200 2.48 385.28 155.07 114.16
5. WIN 55, 212 3.34 427.29 155.07 127.14
6. JWH-073 N-butanoic acid 3.39 358.27 155.08 127.11
7. JWH-073 4-hydroxybutyl 3.4 344.24 155.09 127.09
8. JWH-018 N-pentanoic acid 3.49 372.18 155.08 127.14
9. JWH-018 5-hydroxypentyl-d5 3.54 363.5 155.08 NA
10. JWH-018 5-hydroxypentyl 3.55 358.27 155.08 127.11
11. JWH-073 6-hydroxyindole 3.77 344.24 155.09 127.09
12. JWH-073 5-hydroxyindole-d7 3.81 351.21 155.07 NA
13. JWH-073 5-hydroxyindole 3.83 344.24 155.09 127.09
14. JWH-073 7-hydroxyindole 3.92 344.24 155.09 127.09
15. JWH-018 6-hydroxyindole 3.94 358.27 155.08 127.11
16. JWH-018 5-hydroxyindole 3.99 358.27 155.08 127.11
17. JWH-018 7-hydroxyindole 4.08 358.27 155.08 127.11
18. RCS-4 4.15 322.27 135.12 77.09
19. XLR-11 4.21 330.25 232.17 125.1
20. JWH-015-d7 4.27 335.28 155.07 NA
21. JWH-250 4.27 336.28 121.12 91.07
22. JWH-015 4.29 328.26 155.07 127.13
23. AM2201 4.30 360.26 155.07 127.14
24. JWH-203 4.39 340.23 188.18 125.09
25. JWH-073 4.42 328.26 155.07 127.13
26. UR-144 4.44 312.32 214.17 125.1
27. JWH-073 4-hydroxyindole 4.53 344.24 155.09 127.09
28. JWH-018-d9 4.55 351.34 155.07 NA
29. JWH-018 4.57 342.27 155.08 127.11
30. JWH-081 4.64 372.28 185.12 157.09
31. JWH-018 4-hydroxyindole 4.66 358.27 155.08 127.11
32. JWH-122 4.69 356.29 169.12 141.11
33. JWH-019 4.70 356.29 155.07 127.1
34. JWH-210 4.84 370.31 183.12 153.26
Table II: Figure 2 analyte identification, retention times, and ion transitions
Figure 2 Peak Identifications tR (min) Precursor Ion Quantifier Product Ion Qualifier Product Ion
1. AB-FUBINACA 3.15 369.0 253.0 109.1
2. AB-PINACA 3.23 331.2 215.0 286.1
3. Salvinorin A 3.39 433.1 373.0 91.1
4. 5F-PB-22 4.04 377.2 232.1 143.9
5. PB-22 4.30 359.2 214.0 144.0
6. APINACA (AKB-48) 4.76 366.3 135.1 93.1
Medical/Biological 35ADVERTISEMENT
Analytical column: Raptor Biphenyl (2.7 μm, 50 mm x 3.0 mm;
cat.# 9309A5E)
Guard column: Raptor Biphenyl EXP guard column (2.7 μm,
5 mm x 3.0 mm; cat.# 9309A0253)
Mobile phase A: 0.1% Formic acid in water
Mobile phase B: 0.1% Formic acid in acetonitrile
Gradient: Time (min) %B
0.00 25
1.00 25
5.00 95
5.50 95
5.51 25
7.00 25
Flow rate: 0.6 mL/min
Injection volume: 2 μL
Column temp.: 30 °C
Ion mode: Positive ESI
Results
Chromatographic results for the original method are presented in
Figure 1. Complete resolution of all isobaric compounds and good
separation from major matrix interferences were achieved with a
cycle time of 5 min and a total analysis time of 7 min because of
the unique selectivity and retention of the Raptor Biphenyl column.
To determine whether new drugs could be successfully added
to the original method, fi ve additional synthetic cannabinoids
and salvinorin A (a psychotropic terpenoid) were analyzed under
conditions identical to the established method. As shown in Figure
2, which is an overlay of the new compounds (numbered peaks)
and the existing separation from Figure 1 (unnumbered peaks), all
compounds were separated from early-eluting matrix interferences
as well as from each other without the need for adjustments to the
mobile phase, gradient, or analytical column.
Conclusions
Labs analyzing synthetic cannabinoids are under increasing
pressure to add new compounds to their analytical testing services.
While this can be done through new method development, adding
new compounds to an existing method can save time and resources.
The method shown here demonstrates the advantages of the Raptor
Biphenyl SPP column for this analysis: due to the column’s highly
retentive, selective characteristics, 35 drugs (including isomers and
emerging compounds) can be analyzed quickly and effectively.
Time (min)
0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50
1
2
3
4
5
6
Figure 2: Overlay of new drugs (numbered peaks) and existing method chromatogram from Figure 1.
Restek Corporation
110 Benner Circle, Bellefonte, PA 16823
tel. (814) 353-1300
Website: www.restek.com
www.gerstel.com
Dynamic Headspace
(DHS), Headspace
Automated Pyrolysis
(PYRO)
Extraction, Clean-Up,
Evaporation,
Addition of Standards
Olfactory Detection Port
(ODP)
MAESTRO PrepAhead
productivity
SPME, SBSE,
Thermal desorption
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