Supporting Information

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1 Supporting Information Online Hydrophobic Interaction Chromatography-Mass Spectrometry for the Analysis of Intact Monoclonal Antibodies Bifan Chen a, Ziqing Lin b,c, Andrew J. Alpert d, Cexiong Fu e, Qunying Zhang e, Wayne A. Pritts e, and Ying Ge a,b,c,* a Department of Chemistry, University of Wisconsin-Madison, Madison, Wisconsin, USA b Department of Cell and Regenerative Biology, University of Wisconsin-Madison, Madison, Wisconsin, USA c Human Proteomics Program, School of Medicine and Public Health, University of Wisconsin- Madison, Madison, Wisconsin, USA d PolyLC Inc., Columbia, Maryland, USA e Process Analytical, AbbVie Inc., North Chicago, Illinois, USA *To whom correspondence may be addressed: Dr. Ying Ge, 8551 WIMR-II., 1111 Highland Ave., Madison, Wisconsin 53705, USA. ge2@wisc.edu; Phone: ; Fax:

2 Part I. Experimental Procedures Materials and Reagents. HPLC grade water, acetonitrile (ACN), and ammonium acetate (NH 4 OAc) were purchased from Fisher Scientific (Fair Lawn, NJ, USA). Sample preparation. mab1 is a model antibody AB004 in 7 mg/ml (Abbvie), and mab2 is IgG1κ purchased from NIST (Reference material 8671) in 10 mg/ml. A mixture was prepared containing equal amount of mab1 and mab2. Subsequent dilution using 1 M NH 4 OAc (at least 3:1 volume ratio) brought the final concentration to mg/ml for a single mab as well as for the mixture unless noted otherwise. Deglycosylation was performed using IgGZERO (EndoS) (GENOVIS). 1 unit of enzymes and 1 µg of mabs were incubated in 15 mm Tris buffer for 30 min incubation time at 37 C. HIC-MS and -MS/MS. 100 mm 0.2 mm i.d. PolyPENTYL A capillaries (PolyLC, Columbia, MD, USA) with 1.9 µm particle were used on a Waters ACQUITY UPLC M-class system (Milford, MA, USA). Mobile phase A (MPA) contained 1M NH 4 OAc and mobile phase B (MPB) contained 20 mm NH 4 OAc (overall) in 50:50 water/acn without any adjustment of the ph. For Q-TOF MS experiments, a total of 45 min run consisting of a 30 min linear gradient (0 min 99% MPA; 5 min 99% MPA; 35 min 1% MPA; 40 min 1% MPA; 42 min 99% MPA; 45 min 99% MPA) was used at a flow rate of 2.6 µl/min. Later on for FT-ICR MS experiments, a faster 30 min run consisting of a 20 min linear gradient (0 min 99% MPA; 2 min 99% MPA; 22 min 1% MPA; 27 min 1% MPA; 28 min 99% MPA; 30 min 99% MPA) was used at a flow rate of 2.6 µl/min. Samples eluted from HIC columns were electrosprayed into a maxis II ETD Q-TOF or a 12 T solarix FT-ICR mass spectrometer (Bruker Daltonics, Bremen, Germany). For maxis II

3 ETD, the end plate offset and capillary voltage were optimized at 500 V and 3800 V, respectively. The nebulizer was set to 1.5 bar, and the dry gas flow rate was 5.0 L/min at 150 C. In-source CID was set to ev, collision cell RF to 4000 V pp, transfer time to 180 µs, and pre pulse storage to 60 µs to improve desolvation and transmission of large ions. The quadruple low mass was set to 1600 m/z. For solarix, dry gas flow rate was set to 3 L/min at 106 C. Skimmer 1 was set to 120 V and 25 V collision energy was set for the quadrupole to improve desolvation. Lowest frequency values for octopole (2 MHz), quadrupole (1.4 MHz), and transfer hexapole (1 MHz) were used to improve ion transition. Time of flight was set to 2 ms. Ions were accumulated for 1.3 s and 1 M words of data were collected. For ECD parameters, pulse length, ECD bias, and ECD lens were set to 50 ms, 0.6 V, and 15 V, respectively. Mass range was set to 200 or ,000 m/z calibrated by 10 mm cesium iodide salt clusters. Data Analysis. All data were processed and analyzed by Compass DataAnalysis 4.3, 4.4, and MASH Suite Pro. Chromatograms and spectra were smoothed. Maximum Entropy was used for spectrum deconvolution with resolution was set to 10,000. SNAP algorithm were used for peak picking, in which quality threshold was set to 0.3. All fragment ions were manually validated. The crystal structure of the Fab fragment of mab2 (5K8A) was obtained and modified using Protein Workshop from RCSB protein data bank.

4 Part II. Supplementary Data Compound Retention Time Area Under Curve (AUC) Normalized AUC mab mab Table S1. Retention time and area under curve (AUC) of mab1 and mab2 eluted from HIC-MS on a maxis II Q-TOF mass spectrometer. Glycoproteoforms Experimental Mass (Da) Theoretical Mass (Da) Mass Error (ppm) G0F/G0F G0F/G1F G1F/G1F or G0F/G2F G1F/G2F G2F/G2F Table S2. Experimental vs. theoretical masses of mab2 glycoproteoforms 1 detected from HIC- MS on a maxis II Q-TOF mass spectrometer.

5 Area under curve (10 6 ) (a) (b) 0.05 µg 0.25 µg 0.50 µg 1.00 µg Time (min) y = x R² = Injection amount (µg) Figure S1. Quantitative HIC-MS analysis of mab2 on a maxis II QTOF mass spectrometer. (a) Overlaid chromatograms of mab2 in different injection amounts; (b) working curve of peak area versus the amount of antibody from a single injection in (a) showing an R 2 of

6 Monomer G0F/G0F G0F/G1F G1F/G1F or G0F/G2F Dimer G1F/G2F Mass (Da) Deconv m/z Figure S2. Average mass spectrum of mab1 showing the detection of monomer and dimer from a maxis II Q-TOF mass spectrometer. The call-out shows the deconvoluted mass spectrum of mab1 monomer with multiple glycosylated proteoforms.

7 (a) Glycosylated mab2 Deglycosylated mab Time (min) (b) 27+ (c) Deconv Deconv Ο Ο +Lysine (128 Da) +Hexose (162 Da) m/z Da Figure S3. Comparison of HIC-MS analysis of glycosylated and deglycosylated mab2 on a maxis II QTOF mass spectrometer. (a) Overlaid total ion chromatograms of glycosylated (solid) and deglycosylated (dash) mab2; (b) mass spectra of glycosylated (top) and deglycosylated (bottom) mab2; (c) Deconvoluted spectra of glycosylated (top) and deglycosylated (bottom) from b. Solid triangle represents a +Lysine (+128) modification and hollow circle represents a + Hexose (+162 Da) modification.

8 Relative intensity Relative intensity (a) BPC (ii) (i) (b) 100% Time (min) (i) No collisional energy 0% 100% (ii) 25 V collisional energy 0% m/z Figure S4. Improvement of mab2 signals in the 12T solarix XR FT-ICR mass spectrometer by applying collisional energy. (a) Overlaid HIC-MS base peak chromatograms of deglycosylated mab2 in a shorter gradient with no (i) or 25 V (ii) collisional energy applied; (b) Average mass spectra of deglycosylated mab2 when applying no (top) and 25 V (bottom) collisional energy with normalized intensity.

9 (a) ECD 0.6 ev 50 ms (b) ECD 0.8 ev 50 ms (c) ECD 1.2 ev 50 ms m/z Figure S5. Online broadband ECD fragmentation with different electron energies: (a) 0.6 ev (b) 0.8 ev (c) 1.2 ev and 50 ms irradiation time using a 12T solarix FT-ICR mass spectrometer. The intensities of all the spectra are normalized to the most abundant ion (9.4E6) in (a).

10 Light Chain 1 D I Q M T Q S P S T L S A S V G D R V T I T C S A S S R V G Y M H W Y Q Q K P G K A P K L L I Y D T S K L A S G V P S R F S G S G S G T E F T L T I S S L Q P D D F A T Y Y C F Q G S G Y P F T F G G G T K V E I K R T V A A P S V F I F P P S D E Q L K S G T A S V V C L L N N F Y P R E A K V Q W K V D N A L Q S G N S Q E S V T E Q D S K D S T Y S L S S T L T L S K A D Y E K H K V Y A C E V T H Q G L S S P V T K S F N R G E C 1 Light Chain 1 D I Q M T Q S P S T L S A S V G D R V T I T C S A S S R V G Y M H W Y Q Q K P G K A P K L L I Y D T S K L A S G V P S R F S G S G S G T E F T L T I S S L Q P D D F A T Y Y C F Q G S G Y P F T F G G G T K V E I K R T V A A P S V F I F P P S D E Q L K S G T A S V V C L L N N F Y P R E A K V Q W K V D N A L Q S G N S Q E S V T E Q D S K D S T Y S L S S T L T L S K A D Y E K H K V Y A C E V T H Q G L S S P V T K S F N R G E C 1 1 pq V T L R E S G P A L V K P T Q T L T L T C T F S 26 G F S L S T A G M S V G W I R Q P P G K A L E W L 51 A D I W W D D K K H Y N P S L K D R L T I S K D T 76 S K N Q V V L K V T N M D P A D T A T Y Y C A R D 101 M I F N F Y F D V W G Q G T T V T V S S A S T K G 126 P S V F P L A P S S K S T S G G T A A L G C L V K 151 D Y F P E P V T V S W N S G A L T S G V H T F P A 176 V L Q S S G L Y S L S S V V T V P S S S L G T Q T 201 Y I C N V N H K P S N T K V D K R V E P K S C D K 226 T H T C P P C P A P E L L G G P S V F L F P P K P K D T L M I S R T P E V T C V V V D V S H E D P E V K F N W Y V D G V E V H N A K T K P R E E Q Y N S T Y R V V S V L T V L H Q D W L N G K E Y K C K V S N K A L P A P I E K T I S K A K G Q P R E P Q V Y T L P P S R E E M T K N Q V S L T C L V K G F Y P S D I A V E W E S N G Q P E N N Y K T T P P V L D S D G S F F L Y S K L T V D K S R W Q Q G N V F S C S V M H E A L H N H Y T Q K S L S L S P G pq V T L R E S G P A L V K P T Q T L T L T C T F S 26 G F S L S T A G M S V G W I R Q P P G K A L E W L 51 A D I W W D D K K H Y N P S L K D R L T I S K D T 76 S K N Q V V L K V T N M D P A D T A T Y Y C A R D 101 M I F N F Y F D V W G Q G T T V T V S S A S T K G 126 P S V F P L A P S S K S T S G G T A A L G C L V K 151 D Y F P E P V T V S W N S G A L T S G V H T F P A 176 V L Q S S G L Y S L S S V V T V P S S S L G T Q T 201 Y I C N V N H K P S N T K V D K R V E P K S C D K 226 T H T C P P C P A P E L L G G P S V F L F P P K P K D T L M I S R T P E V T C V V V D V S H E D P E V K F N W Y V D G V E V H N A K T K P R E E Q Y N S T Y R V V S V L T V L H Q D W L N G K E Y K C K V S N K A L P A P I E K T I S K A K G Q P R E P Q V Y T L P P S R E E M T K N Q V S L T C L V K G F Y P S D I A V E W E S N G Q P E N N Y K T T P P V L D S D G S F F L Y S K L T V D K S R W Q Q G N V F S C S V M H E A L H N H Y T Q K S L S L S P G Heavy Chain c ----CO--NH--CHR-- ECD z. Heavy Chain Figure S6. Broadband ECD (0.6 ev and 50 ms) fragmentation map of intact deglycosylated mab2 from the online HIC-MS experiment on the 12T solarix FT-ICR mass spectrometer. The solid purple lines represent known disulfide linkages. For heavy chains, pq indicates pyroglutamic acid, and the highlighted N300 has Da after deglycosylation by IgGZERO. Complementarity-determining regions are underlined in red.

11 Reference (1) Formolo, T.; Ly, M.; Levy, M.; Kilpatrick, L.; Lute, S.; Phinney, K.; Marzilli, L.; Brorson, K.; Boyne, M.; Davis, D.; Schiel, J. State-of-the-Art and Emerging Technologies for Therapeutic Monoclonal Antibody Characterization, Vol 2: Biopharmaceutical Characterization: The Nistmab Case Study 2015, 1201, 1-62.