Accurate Sequence Analysis of a Monoclonal Antibody by Top-Down

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Accurate sequence analysis of a monoclonal antibody by top-down and middle-down Orbitrap mass spectrometry applying multiple ion activation techniques Luca Fornelli, Kristina Srzenti#, Romain Huguet, Christopher Mullen, Seema Sharma, Vlad Zabrouskov, Ryan T. Fellers, Kenneth R Durbin, Philip D. Compton, and Neil L Kelleher Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b00984 • Publication Date (Web): 12 Jun 2018 Downloaded from http://pubs.acs.org on June 12, 2018

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Analytical Chemistry

Accurate sequence analysis of a monoclonal antibody by topdown and middle-down Orbitrap mass spectrometry applying multiple ion activation techniques Luca Fornelli1,#, Kristina Srzentić1,#, Romain Huguet2, Christopher Mullen2, Seema Sharma2, Vlad Zabrouskov2, Ryan T. Fellers1, Kenneth R. Durbin3, Philip D. Compton1 and Neil L. Kelleher1* 1Departments

of Chemistry and Molecular Biosciences, and the Proteomics Center of

Excellence, Northwestern University, 2145 N. Sheridan Road, Evanston, Illinois 60208 2Thermo

Fisher Scientific, 355 River Oaks Parkway, San Jose, California 95134

3Proteinaceous,

#These

Inc., Evanston, IL, 60201

authors contributed equally to this work

*To whom correspondence should be addressed: Departments of Chemistry and Molecular Biosciences, and the Proteomics Center of Excellence, Northwestern University, 2145 N. Sheridan Road, Evanston, IL 60208. Tel.: 847-467-4362; Fax: 847-467-3276; E-mail: [email protected]

Manuscript version: May 30, 2018 Manuscript submitted to: Analytical Chemistry

1

ACS Paragon Plus Environment

Analytical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Keywords: Ultraviolet photodissociation, UVPD; Electron transfer dissociation, ETD; Electron transfer dissociation – higher energy collisional dissociation, EThcD; Top-down, TD; Middle-down, MD; Immunoglobulin G, IgG; Mass spectrometry, MS; Tandem mass spectrometry, MS2; Liquid chromatography, LC; Orbitrap.

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ABSTRACT Targeted top-down (TD) and middle-down (MD) mass spectrometry (MS) offer reduced sample manipulation during protein analysis, limiting the risk of introducing artefactual modifications to better capture sequence information of the proteoforms present. This provides some advantages when characterizing biotherapeutic molecules such as monoclonal antibodies, particularly for the class of biosimilars. Here, we describe the results obtained analyzing a monoclonal IgG1, either in its ~150 kDa intact form or after highly specific digestions yielding ~25 and ~50 kDa subunits, using an Orbitrap mass spectrometer on a liquid chromatography time scale with fragmentation from ion-photon, ion-ion, and ion-neutral interactions. Ultraviolet photodissociation (UVPD) used a new 213 nm solid state laser. Alternatively, we applied high capacity electron transfer dissociation (ETD HD), alone or in combination with higher-energy collisional dissociation (EThcD). Notably, we verify the degree of complementarity of these ion activation methods, with the combination of 213 nm UVPD and ETD HD producing a new record sequence coverage of ~40% for TD MS experiments. The addition of EThcD for the >25 kDa products from MD strategies generated up to 90% of complete sequence information in six LC runs. Importantly, we determined an optimal signal-to-noise threshold for fragment ion deconvolution to suppress false positives yet maximize sequence coverage, and implemented a systematic validation of this process using the new software TDVALIDATOR. This rigorous data analysis should elevate confidence for assignment of dense MS2 spectra and represents a purposeful step toward the application of TD and MD MS for deep sequencing of monoclonal antibodies.

3



INTRODUCTION Within the past five years, monoclonal antibodies (mAbs) have transitioned from being a promising class of biotherapeutics1 to a staple of the pharmaceutical market. In 2015, the Food and Drug Administration (FDA) approved nine new therapeutic antibodies,2 and in the first half of 2016, five of the thirteen newly approved drugs were mAbs.3 Importantly, the current ~50 different therapeutic mAbs already present in the market (with more than 300 in development)4 are likely to be joined by their socalled ‘biosimilar’ versions.5,6 The first biosimilar therapeutic antibody was approved by the FDA at the beginning of 2016.3 Immunoglobulins G (IgGs), which represent the main class of antibodies used for therapeutic purposes, are highly complex molecules composed of four polypeptide chains (two ~25 kDa light and two ~50 kDa heavy) for a total mass of approximately 150 kDa. The tertiary and quaternary structures of an IgG are stabilized by a series of intra- and inter-molecular disulfide bridges, respectively. Importantly, heavy chains are N-glycosylated, with variability of the N-linked glycans that depends on the expression system (e.g., CHO, insect or any other type of cell line).7 Other sources of variation include formation of pyroGlu, Met oxidation, clipping of the C-terminal Lys residue of the heavy chain, and deamidation (i.e., conversion of Gln to Glu). With such complexity, it is apparent that sophisticated analytical tools are required to guarantee high quality IgG is being produced and the quality is maintained throughout storage. Mass spectrometry (MS) is a key analytical technique for molecular quality control (QC) due to its capability to robustly generate information at the single amino acid 4


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residue level. MS can be used to detect and localize different types of biological and artifactual post-translational

modifications

(PTMs) along the

protein.8 Several

approaches are available for the MS analysis of mAbs, the most common of which consists of the tryptic digestion of the intact IgG into short peptides (0.5-2 kDa). This method, called bottom-up (BU),9 is known to introduce artificial PTMs into the sample due to the slightly basic conditions needed for the proteolysis (which can promote deamidation),10 and requires a lengthy and imperfect assembly of peptides to infer whole protein compositional information. An alternative to traditional trypsin-based BU MS is represented by the use of the protease Sap9, which produces peptides in the 3-5 kDa mass range, in a process referred to as extended bottom-up (eBU).11 Sap9 efficiently cleaves IgGs under acidic conditions in about 1 hour, reducing the probability

of

introducing

artifacts

into

the

original

sample.12

However, the analysis of larger subunits or even the whole antibody offers additional information such as the relative order of complementarity determining regions (CDRs) or, in the case of an antibody mixture, the connectivity between light and heavy chains (also known as “chain pairing”) which cannot be inferred from DNA sequencing. The analysis of >10 kDa protein subunits obtained by proteolysis has come to be known as a middle-down (MD) strategy.13 Although several proteases can generate large fragments of IgGs, the IgG degrading enzyme from Streptococcus pyogenes, IdeS, has become popular.14 This protease cleaves the heavy chain below the hinge region in 70% sequence coverage was obtained.17,19 Notably, these results were obtained despite ~510% of ETD and up to 15% of UVPD fragment ions automatically-matched were removed from the final list in each run after manual inspection using TDVALIDATOR. The left panels of Figure 4 show the fragmentation maps generated by combining the 15



results of all the ion activations. The fragmentation maps of the three subunits subjected to a single ion activation are shown in Figures S-13, S-14 and S-15 for ETD, UVPD and EThcD, respectively. Furthermore, we were able to identify fragment ions retaining N-linked glycans (specifically G0F, G1F and G2F) and, although the vast majority of N-terminal fragments for Lc and Fd includes the pyroGlu modification, also a few fragments containing an N-terminal Gln were observed. Careful inspection of these fragmentation maps reveals the highly complementary nature of the activation techniques: as an example, ETD (3 durations) and UVPD (2 different laser irradiations) yielded 59.3 and 71.3% sequence coverage for the Fc/2 subunit, respectively. However, while neither of these fragmentation techniques was able to fragment efficiently the last 5-7 residues of the protein termini, EThcD was able to partially characterize them, presumably due to the vibrational activation of primary reaction products from ETD in the HCD cell promoting the formation of smaller protein fragment ions. Furthermore, the two ion activation techniques that result on average in the higher sequence coverage, ETD and UVPD, do not sequence with equal efficiency all the 25 kDa subunits: for instance, Table 1 indicates that UVPD outperforms ETD in sequencing the Fc/2, but returns a lower sequence coverage than the electron-based fragmentation for the Fd subunit (47.3 vs 38.5% for ETD and UVPD, respectively). The high degree of complementarity between all the three ion activation technologies is summarized for each of the 3 IgG subunits in the Venn diagrams displayed in the right panels of Figure 4. These diagrams show the matched fragment ions that are shared between ETD, EThcD, and UVPD. Unsurprisingly, this analysis showed a larger number of common identified ions between ETD and EThcD, while UVPD adds the highest number of unique matched ions for each of the 16


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subunits. Considering all three ion activation methods, the total number of unique matched ions account for 30-43% of the total. This last observation could be explained considering the high number of a/x-type ions produced by 213 nm UVPD: these ion types cannot be produced by ETD nor EThcD (Figure S-14). Overall, these results underline the importance of applying multiple ion fragmentation methods to obtain the highest achievable sequence coverage. CONCLUSIONS These results show that the use of multiple ion activation techniques coupled with

state-of-the-art

Orbitrap

FTMS

enables

thorough

characterization

of

biotherapeutics such as monoclonal antibodies: the combination of TD and MD MS not only yields an overall sequence coverage (and PTM mapping) in line with what obtainable by BU or eBU MS strategies, but also produces multiple layers of information about the IgG of interest (e.g., CDR pairing, chain pairing, intact mass) not obtainable after extended proteolysis. Owing to the complementarity between ETD, EThcD and UVPD, the overall number of LC-MS/MS experiments required for generating high quality-information is low (a total of only 13 LC runs were considered for the main body of this work). To further ease the transition of TD and MD MS technology from “research grade” to a “quality control-ready” tools, we also introduced more stringent parameters for the standard data analysis workflow (i.e., spectral deconvolution followed by matching of neutral masses) to increase robustness and confidence

of

final

reports.

In

summary,

we

envision

that

the

antibody

characterization protocol based on TD and MD will be useful not only for the analysis of other classes of biotherapeutics (e.g., antibody-drug-conjugates, diverse multivalent 17



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MAb conjugates, and monomeric glycoproteins),41 but also that in the near future, considering the constant development of new dedicated software42 and efficient ion activation technologies,43 TD and MD technologies will serve applications currently performed only by BU MS, such as targeted de novo sequencing.44 ACKNOWLEDGEMENTS This work was supported by the National Institute of General Medical Sciences of the National Institutes of Health under grant number GM067193 (N.L.K.). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. The authors would like to thank Michael Senko for motivating discussion and technical support. Statement of Conflict of Interest Computational tools described in this work are available as either freeware or via commercialization so a financial conflict of interest is declared and actively managed.

SUPPORTING INFORMATION Extended

Experimental

Section

(Sample

digestion

procedures;

Liquid

chromatography details; Top-down MS experiments; Middle-down MS experiments for 50 kDa units; Middle-down MS experiments for 25 kDa units). Variation of sequence coverage and p-score as a function of the fragment mass tolerance. Variation of sequence coverage and p-score as a function of the signal-to-noise ratio used for deconvolution of UVPD spectra. TDVALIDATOR workflow. Tandem mass spectra of 18


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intact Rituximab fragmented by ETD HD and 213 nm UVPD. Graphical fragmentation maps for intact Rituximab (IgG1) resulting from the combination of 5 and 10 ms duration ETD. Graphical fragmentation maps for intact Rituximab obtained with UVPD using 5 laser pulses. Graphical fragmentation maps for intact Rituximab (IgG1) resulting from the combination of matching and validated fragment ions generated by ETD and UVPD. Results of middle-down analysis of Rituximab (IgG1) digested with GingisKHAN (without reduction of disulfide bridges) fragmented by ETD, UVPD and EThcD. Results of the characterization of the IdeS-generated ~25 kDa subunit of Rituximab fragmented by ETD, UVPD and EThcD. Distribution of ion types produced by 213 nm UVPD. Table S-1: mass accuracy of the four most abundant Rituximab glycoforms. Table S-2: list of transients (i.e., microscans) averaged for different TD MS experiments. (pdf)

TABLE LEGEND Table 1. Summary of sequence coverage of Rituximab obtained via top-down or middle-down analysis using multiple ion fragmentation techniques. One single ion activation was used per LC run. Note that EThcD was not applied to the top-down analysis of intact Rituximab. FIGURE LEGEND Figure 1. (A), general workflow for the parallel analysis via Xtract/PROSIGHT LITE and TDVALIDATOR. (B), graphic user interface of TDVALIDATOR showing a confident match 19



between an experimental (black trace) and a theoretical (red triangles) isotopic cluster for the z10610+ fragment ion from MS2 by ETD of a subunit liberated from Rituximab using the IdeS protease (Fc/2). Figure 2. (A), variation of sequence coverage (expressed as a percentage) and –LOG10 (p-score) as a function of fragment ion tolerance used for sequence matching; the set of observed fragment ions returned by the Xtract algorithm was determined with a signal-to-noise threshold (SNR) of 7:1. (B), sequence coverage and –LOG10 (p-score) as a function of signal-to-noise ratio (SNR) threshold selected for Xtract, keeping the fragment ion tolerance fixed at ±5 ppm. For these plots we considered the reduced light chain of Rituximab (obtained via IdeS digestion) fragmented by UVPD using 5 laser pulses. Figure 3. Results of middle-down analysis of Rituximab digested with GingisKHAN (without reduction of disulfide bonds). (A), chromatographic separation of the two ~50 kDa IgG subunits produced by proteolysis, which are present in a stoichiometric ratio of 1:2 for Fc and Fab, respectively. Insets show the isotopic distribution of Fc (left) and Fab (right). (B)-(D): graphical fragmentation maps obtained by combining ETD, UVPD and EThcD data for the three subunits: Fd (B) and Lc forming the Fab subunit (C), and the Fc (D). The presence of the N-linked glycan, mapped using diagnostic fragment ions that include the mass of the sugar chains G0F, G1F and G2F, is indicated by an orange rectangle. Figure 4. Results from LC-MS characterization of the IdeS-generated ~25 kDa subunits of Rituximab. The left side of panels (A), (B) and (C) show the fragmentation maps of Fd, Lc and Fc/2, respectively, obtained by summing data from 3 ETD, 2 20


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UVPD and 1 EThcD, runs (total of 6 LC-MS2 runs). The presence of the N-linked glycan, mapped using diagnostic fragment ions which include the mass of the sugar chains G0F, G1F and G2F, is highlighted by an orange rectangle. For each panel, the right side shows the corresponding Venn diagram of shared/unique matched fragment ions for each of the three ion fragmentation techniques.

Table 1

Species Workflow

Targeted (mass in

Chain

of LC runs

kDa) light Top-downa

Sequence coverage (%)

Number

ETD

UVPD

EThcD

Total

21.2

31.6

x

38.2

25.6

23.4

x

41.2

3b

Intact (150) heavy

21



Fc (50)

heavy - Fc

Page 22 of 29

17.0

21.5

9.9

36.8

11.6

13.2

14.7

29.3

10.4

13.8

11.3

25.0

59.8

61.2

51.7

86.6

Fd (25)

47.3

38.5

38.5

72.0

Lc (25)

53.3

50.9

45.3

79.7

Middle-down 4c

heavy - Fd GingisKHANa

Fab (50) light 6e

Fc/2 (25) Middle-down IdeSd

a,

with all disulfide bridges intact; b, summing 2 ETD and 1 UVPD runs; c, summing 1

ETD, 2 UVPD and 1 EThcD run; d, with reduced S-S bridges; e, summing 3 ETD, 2 UVPD and 1 EThcD run(s)

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Figure 1. (A), general workflow for the parallel analysis via Xtract/PROSIGHT LITE and TDVALIDATOR. (B), graphic user interface of TDVALIDATOR showing a confident match between an experimental (black trace) and a theoretical (red triangles) isotopic cluster for the z10610+ fragment ion from MS2 by ETD of a subunit liberated from Rituximab using the IdeS protease (Fc/2). 325x412mm (300 x 300 DPI)


Sequ

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


30.0

-LOG(p-score) Sequence coverage

28.0 26.0

Page 26 of 29

4

6

8

10

12

Fragment mass tolerance, p

(B)

fragment tolerance = 5 ppm

Sequence coverage, %

38.5 38.0 37.5 37.0 36.5 36.0 35.5 35.0 34.5

3

4


5

SNR

6

126 G P S V F Analytical P LChemistry A P S S K S T S G

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151 K D Y F P E P V T V S W N S G A 176 A V L Q S S G L Y S L S S V V T 201 T Y I C N V N H K P S N T K V D 226 K C

(C) N 26 51 76 101 126 151 176 201

Q S S R T S N S S

I S N V K G A T S

V S L E L T L L P

L V A A E A Q T V

S S S E I S S L T

Q Y G D K V G S K

S I V A R V N K S

P H P A T C S A F

A W V T V L Q D N

I F R Y A L E Y R

L Q F Y A N S E G

S Q S C P N V K E

A K G Q S F T H C

S P S Q V Y E K C

P G G W F P Q V

G S S T I R D Y

T K V S V V Y L F

H D K T S Y P D S

T T F Y N T S S C

C L N R K L D D S

P P C P A M I S R T W Y V D G V V S V L A L P A P P P S R D I A V E W G S F F L ACS Paragon Plus Environment V M H E A

P P V T I E E Y L

E E E V E L S S H

L V V L K T N K N

L T H H T K G L H

G C N Q I N Q T Y

G V A D S Q P V T

P V K W K V E D Q

(D) 26 51 76 101 126 151 176 201


Figure 4. Results from LC-MS characterization of the IdeS-generated ~25 kDa subunits of Rituximab. The left side of panels (A), (B) and (C) show the fragmentation maps of Fd, Lc and Fc/2, respectively, obtained by summing data from 3 ETD, 2 UVPD and 1 EThcD, runs (total of 6 LC-MS2 runs). The presence of the Nlinked glycan, mapped using diagnostic fragment ions which include the mass of the sugar chains G0F, G1F and G2F, is highlighted by an orange rectangle. For each panel, the right side shows the corresponding Venn diagram of shared/unique matched fragment ions for each of the three ion fragmentation techniques. 537x465mm (600 x 600 DPI)


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For TOC only 84x31mm (300 x 300 DPI)


Accurate Sequence Analysis of a Monoclonal Antibody by Top-Down

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