Fast Protein Sequence Verification of Therapeutic Antibodies by Top-Down Mass Spectrometry Top-Down Sequencing can help accelerate your sequence verification projects. In this contribution a simple and straightforward LC-free Top-Down MS method is described that typically delivers the terminal status from light and heavy chains in mAbs after reduction and coverage of CDR1 and CDR2. Keywords: Top-Down Proteomics Consortium Antibody Study, MALDI-TDS, BioPharma Compass 2.0, mAb Authors: Anja Resemann 1 , Detlev Suckau 1 . 1 Bruker Daltonik GmbH, Bremen, Germany. A further in-depth approach utilizes additional steps: FabRICATOR ™ cleavage of the heavy chain, IgGZERO ™ deglycosylation and LC separation of the LC, Fd and Fc/2 fragments followed by Top-Down Sequencing (TDS). Typically > 80 % of the antibody sequence is confirmed by this approach from a single dataset including the CDR3 on the Light Chain. The mAbs NISTmAb, SigmaMAb and Herceptin were used in this study as contribution to the Top-Down Proteomics Con- sortium´s monoclonal antibody study in 2017. YEARS MALDI 25
Transcript
Fast Protein Sequence Verification of Therapeutic Antibodies by Top-Down Mass Spectrometry
Top-Down Sequencing can help accelerate your sequence verification projects. In this contribution a simple and straightforward LC-free Top-Down MS method is described that typically delivers the terminal status from light and heavy chains in mAbs after reduction and coverage of CDR1 and CDR2.
A further in-depth approach utilizes additional steps: FabRICATOR™ cleavage of the heavy chain, IgGZERO™ deglycosylation and LC separation of the LC, Fd and Fc/2 fragments followed by
Top-Down Sequencing (TDS). Typically > 80 % of the antibody sequence is confirmed by this approach from a single dataset including the CDR3 on the Light Chain.
The mAbs NISTmAb, SigmaMAb and Herceptin were used in this study as contribution to the Top-Down Proteomics Con-sortium´s monoclonal antibody study in 2017.
YEARS MALDI25
Introduction
Intact protein sequencing by Top-Down mass spectrometry can elucidate the modification status of the N- and C-termini of a protein. In addition, the sequences of proteins up to approx. 25 kDa can be completely verified in minutes using the approach. The technique can also be applied to much larger molecules such as intact monoclonal antibodies (mAbs) using specific enzymatic cleavage at the hinge region by FabRICATOR and reduction – yielding fragments in the 23-27 kDa range. These fragments can be readily characterized, providing sequence information. Typically, in addition to the N- and C-terminal modification status, the sequences of CDR1, CDR2 and the Light Chain-CDR3 can be confirmed.
Here we describe the Top-Down sequence analysis of the FabRICATOR fragments of Trastuzumab, the NIST antibody and the SigmaMAb, as contribution to the Top-Down Proteomics Consortium Study on mAb analysis in 2017 [1] using both LC-free and LC-MALDI approaches.
Why can Top-Down Sequencing be a good step forward for you ?
Top-Down Sequencing can help accelerate your projects
• In your lab, is the verification of protein (terminal) sequences time-consuming?
• Could detecting sequence errors be more straight-forward?
• Can obtaining CDR sequence information be a bottle-neck for you to move forward in your projects?
Today's Top-Down mass spectrometric techniques can provide straight- forward solutions to such questions.
Figure 1: MALDI-TDS spectrum detail from the NIST mAb analysis. Fragments are isotope-resolved and monoisotopic masses were picked. The fragments matching to the LC (top) and the HC (bottom) are labelled red (N-terminal) and blue (C-terminal). Unassigned picked peaks are labelled in black. Resolution and mass accuracy allow the simultaneous assessment of the N- and C-termini of LC and HC as MALDI-TDS spectra contain only single charged fragment ions.
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Methods
All intact antibody samples were provided by the Top-Down Proteomics Consortium as part of a multi laboratory mAb analysis study [1].LC-free analysis of the mAb employed reduction and direct matrix addition prior to MALDI-TDS analysis. For LC based analysis, mAbs were digested with FabRICATOR™ and IgGZERO™ (both Genovis, Sweden) and reduced with DTT yielding the
LC, Fc/2 and Fd fragments in the 25 kDa mass range, as previously described [2].The digest mixture was separated by cap LC using a phenomenex Jupiter C4 column (250 x 0.3 mm, 5 µm 300 Å) in a 60 min gradient. Samples were collected with a MALDI fraction collector and sDHB matrix added thereafter.All MALDI Top-Down Sequencing spectra were acquired on a rapifleX TOF/TOF instrument.
Reference sequences were stored in BioPharma Compass 2.0 (BPC 2.0), which was used for the entire sequence confirmation work with automated traffic light-style assessment.
Ranked candidate sequences with variable terminal modifications
Fragment ions matching the selected sequence candidate are represented in bricks
Bricks: top and bottom row code for N- and C-term fragments, resp.Red: fragment detected, yellow: residue inferred following gap acceptance rules
Automatic assessment of N/C-terminal sequences were confirmed (green) or rejected (red)
Sequence Coverage (SC) and Validated Sequence (SVP) are given for each sequence candidate
Selection of a protein entry in the top table annotates the fragments ions in the spectrum for review
Figure 2: Fast terminal status assessment: Representation of the NIST mAb terminal sequence assessment spectrum in BPC 2.0. The N-term status pyroglutamylation and C-term Lysine-loss were confirmed in the HC. In addition 62 and 82 residues were detected on the N- and 32 and 37 on the C-terminal of the LC and HC, respectively, excluding the possibility of sequence or modification variants in those regions.
Two approaches for monoclonal antibody sequence analysis were employed.
1. Fast terminal sequence status assessment (Figures 1-3): The mAb is reduced in 30 min and the resulting LC and HC mixture is deposited on the MALDI sample plate without further chromatogra- phic separation. N- and C-terminal sequences from both the Light Chain and the Heavy Chain are obtained and the modification status and possible sequence truncations can be quickly assessed.
2. Sequence verification (Figures 4-6): The mAb is digested by FabRICATOR and IgGZERO and reduced to obtain deglycosylated Fc/2, LC and Fd fragments. These are LC separated and fractions are spotted right on the MALDI sample plate together with matrix. In the LC-MALDI-MS dataset, the fragments of interest are further analyzed by MALDI Top-Down Sequencing providing for valida-tion of the fragment sequences of 80-100%, enabling the detection and localization of sequence errors.
Video: Top-Down Protein Sequencing in a minute
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Sigma
Trastuzumab
Figure 3: Terminal sequence assessment summary for the 3 mAbs. Based on the Rank 1 matches for each Light Chain and Heavy Chain the following assessments were automatically provided in the BPC 2.0 software: NIST: LC: confirmed; HC: pyroQ, Lys-loss, confirmedSigma: LC: pyroQ, confirmed; HC: Lys-loss, confirmedTrastuzumab: LC: confirmed; HC: Lys-loss, confirmed
Figure 4: Sigma mAb Fc/2. The extended sequence coverage (76.9 % SC) yields a verification of 87.7 % (SVP) of the sequence. The candidate sequence without glycosylation (Rank 2) yields no N-term fragments after N60 confirming it is prominent in the sample.
Figure 5: Sigma mAb Fd. The sequence coverage (62.4 % SC) yields a sequence verification of 73.1 % SVP. Concomitantly, the CDR1 and CDR2 sequences can be read through reliably.
Results
Fast terminal sequence status testing (Figures 1-3)
In less than 1 hour and with minimal sample preparation the Heavy and Light Chain terminal sequences were confirmed and the modification status assessed. All mAbs showed C-term Lys truncation of the HC whereas pyroglutamylation was only present on the Sigma mAb LC
and the NIST mAb HC. Typically, the observed Sequence Coverages were 50-60 % and 25-30 % for the correct LC and HC variants, respectively.
Unique for Top-Down Sequencing analyses using MALDI-TDS are:
• Speed through LC-free analysis• Simplicity of spectral interpretation
due to formation of only singly charged fragments, eliminating the need for charge deconvolution
• Specificity of the sequence match due to comparisons with correct and wrong terminal structures providing a quality assessment of the match simultaneously with the result
• Analysis Depth. The terminal modification status of all 4 termini for each antibody was obtained including the CDR1 and CDR2 sequences
Figure 6: Sigma mAb LC. The extended sequence coverage (87.3 % SC) yields a sequence verification of 96.8 % SVP, covering CDR1, CDR2 and CDR3 very well. This sequence, together with the pyroglutamylated N-term, is automatically classified as “confirmed” because the SVP is > 90 %, which is the usual default parameter setting in BPC 2.0 for MALDI-TDS spectra. Rank 2 protein sequence with free N-terminal only shows confirmation of the C-terminal.
Conclusions
For rapid assessment of the terminal modification status and confirmation of the HC and LC sequences, read outs of approximately 30-80 residues can be directly obtained from a reduced antibody. Thus, the direct Top-Down protein sequence analysis with MALDI without any further sample workup is a very efficient approach. Sample preparation is quick (less than 60 min) and data acquisition and analysis is a matter of 1 min per sample.A more detailed analysis includes the cleavage of the HC into Fd and Fc/2, deglycosylation and LC separation, altogether taking approx. 1 h. This yields terminal sequence read outs of up to 110 residues from each terminus and typically provides the direct confirmation of CDRs 1 and 2 in the Fd and CDRs 1-3 in the LC.Top-Down Sequencing using MALDI-TOF is a uniquely fast, easy and automated sequence confirmation approach. The obtained sequence information can be used particularly well hyphenating it with accurate intact mass information from Bruker's maXis II ultrahigh resolution QTOF.
Sequence verification (Figures 4-6)
To further increase the access to larger stretches of the sequence by Top-Down mass spectrometry the following steps were developed [2]:
• FabRICATOR digestion reduces the molecular weight of the HC to the two 25 kDa Fc/2 and Fd fragments, increasing sequence coverage significantly for the HC.
• IgGZERO digestion removes glycosylation heterogeneity of the HC around N300 enabling the sequence to be read through the glycosylation site. GlcNAc-Fuc remains after such digest permitting for unequivocal assessment of the glycosylation site.
• Stringent reduction under denaturing conditions increases the readout throughout the disulfide-bonded sequence stretches.
• Liquid chromatographic separation of the processed antibody purifies the sample and provides a single protein on each spot, further increasing the quality of the results.
LC-MALDI-TDS datasets typically yielded good sequence coverage of 3 CDRs on the LC and 2 CDRs on the Fd domain (see Sigma mAb data in Figures 4-6). Typically the overall sequence coverage after FabRICATOR digest and reduction was in the 70-80 % range. Determination of the intact mass of these fragments as described elsewhere [2,3] is well suited to resolve remaining uncertainties about the sequence stretches not directly covered in the TDS datasets.
Unique for Top-Down Sequencing analyses using LC-MALDI-TDS are:
• Analysis Depth as typically 80-110 residues from each terminus and thus CDRs are covered and usually more than 80 % of the antibody sequence can be validated from a single experiment. The quality is sufficient to detect and character-ize sequence anomalies or errors as previously shown [2,3].
• Simplicity of the analysis as only a single TDS spectrum is acquired for each mAb fragment of interest.
• Speed through the fully automated assessment of the best sequence in the database of possible modifications or sequences.
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References
[1] The Consortium for Top Down Proteomics Launches Pilot Project for Characterization of Monoclonal Antibodies.
[2] Resemann A, Jabs W, Wiechmann A, Wagner E, Colas O, Evers W, Belau E, Vorwerg L, Evans C, Beck A, Suckau D. Full validation of therapeutic antibody sequences by middle-up mass measurements and middle-down protein sequencing. MAbs. 2016;8(2):318-30.
[3] Ayoub D, Jabs W, Resemann A, Evers W, Evans C, Main L, Baessmann C, Wagner-Rousset E, Suckau D, Beck A. Correct primary structure assessment and extensive glyco-profiling of cetuximab by a combination of intact, middle-up, middle-down and bottom-up ESI and MALDI mass spectrometry techniques. MAbs. 2013;5(5):699-710.
