Supplementary Information for:

2D 1HN, 15N Correlated NMR Methods at Natural Abundance for Obtaining Structural Maps and Statistical Comparability of Monoclonal Antibodies

Luke W. Arbogast1; Robert G. Brinson1; Trina Formolo2; J. Todd Hoopes1; John P. Marino*,1

1Institute for Bioscience and Biotechnology Research, National Institute of Standards and Technology and the University of Maryland, 9600 Gudelsky Drive, Rockville, MD 20850.

2National Institute of Standards and Technology, 100 Bureau Drive, Gaithersburg, MD 20899.

*Corresponding Author:

Institute for Bioscience and Biotechnology Research

National Institute of Standards and Technology

9600 Gudelsky Drive

Rockville, MD 20850

(o) 240-314-6160

(f) 240-314-6225

Supplementary Methods for Mass Spectrometry

Enzyme digestion. The NISTmAb or PolyAb were exchanged into buffer comprising 6M guanidine HCl in 1 mM ethylenediaminetetraacetic acid (EDTA) and 0.25 M Tris(hydroxymethyl)aminomethane/ Tris(hydroxymethyl)aminomethane HCl (Tris), pH 7.5. Dithiothreitol (DTT) was added to a final concentration of 10 mM and the Abs reduced at 37°C for 30 min. Iodoacetamide (IAM) was added to a final concentration of 20 mM and the sample alkylated at room temperature for 30 min in the dark. Alkylation was quenched by the addition of DTT to a total, final concentration of 20 mM. The sample was then buffer exchanged with solution comprising freshly made 2 M urea in 0.1 M Tris, pH 7.8. Mass spectrometry grade trypsin or sequencing grade chymotrypsin (Promega, Madision, WI) was added at a 1:35 (enzyme:IgG) mass ratio. Samples were either digested overnight at room temperature (trypsin) or by utilizing a microwave hydrolysis system (Discover Proteomics model 908005, CEM Corp, Matthews, NC), in SPS mode for 30 min at 50 °C (chymotrypsin). Digests were brought to a concentration of 0.5 µg/µL with 0.1 % trifluoroacetic acid (TFA) for mass spectrometry analysis.

Mass spectrometry analysis. LC-MS/MS was performed using the Dionex UltiMate 3000 UHPLC system coupled to the OrbiTrap Elite mass spectrometer with heated electrospray ionization source (HESI II) (Thermo Scientific, Waltham, MA). Peptides (10 µg) were loaded onto a Supelco Analytical Discovery BIO Wide Pore C18 column (3 µm, 15 cm x 2.1 mm; Sigma-Aldrich, St. Louis, MO) or XSelect CSH C18 Column (2.5 µm, 15 cm x 2.1 mm; Waters Corp, Milford, MA) via autosampler and washed for 10 min using 97 % mobile phase A (volume fraction of 0.1 % FA in water) and 3 % mobile phase B (volume fraction of 0.1 % FA in acetonitrile) at a flow rate of 0.200 mL/min. Peptide elution was achieved over a 110 min linear gradient increasing from 3 % to 45 % mobile phase B. Mass spectrometry data were collected using data dependent mode with one cycle of experiments consisting of one full MS scan followed by MS/MS of the ten most intense peaks, with dynamic exclusion enabled. CID fragmentation was performed using a normalized collision energy of 35, activation Q of 0.250 and activation time of 10 ms. MS/MS fragment ions were analyzed in the ion trap.

Peptide identification. Mass spectrometry data were interrogated using the Byonic software package (Protein Metrics, Inc., San Carlos, CA). Data were searched for peptides with a static modification of cysteine carbamidomethylation (+57.021464 Da) and variable modification of protein C-terminal loss of lysine (-128.094963 Da). Chymotrypsin cleavage sites were set at low specificity (Cterminal to Phe, Trp, Tyr, Met and Leu residues). Mass tolerance was set at 10ppm for parent ions and 0.8 Da for fragment ions. A high confidence level of |Log Prob| > 3 was used for acceptance of peptide identifications obtained through Byonic searches. Glycopeptides were identified using manual inspection of MS/MS spectra.

S14

Table S1. Comparison of Fc region peptides identified by LC-MS/MS analysis after (A) tryptic or (B) chymotryptic digestion. Peptides are named according to the N-terminal and C-terminal amino acid residue positions they cover, with the N-terminal of the full heavy chain considered as residue 1 and “H” indicating heavy chain. Peptide identification begins at residue H(222) which begins two residues N-terminal to the hinge region. The H(296-304) peptide containing an N-glycosylation site was found occupied with multiple glycan compositions for both Abs. While the peptide sequence was confirmed as identical, some differences in glycan populations were observed between the two Abs. One representative, highly abundant glycopeptide found to have the same glycan composition for both IgGs is listed. Note that the NISTmAb sequence continues to H(450), although the majority of NISTmAb species (≈90 %) terminates at residue H(449). No peptide with the C-terminal H(450) residue was detected for the PolyAb. Peptide identification covers the entire Fc region of both Abs between the two enzyme digests. Peptide analysis begins at residue H(222) which just above the hinge region. nd = not detected. See Supplementary Methods.

A) Tryptic Peptides

/ Retention Time (min) / Mass Error (ppm) / |log prob| /
Peptide / Start / End / Theoretical
mass (M+H) (Da) / PolyAb / NISTmAb / PolyAb / NISTmAb / PolyAb / NISTmAb /
H(222-251) / 222 / 251 / 3334.6420 / 55.98 / 56.16 / 1.5 / -9.2 / 3.05 / 5.41
H(226-251) / 226 / 251 / 2844.4575 / 58.89 / 58.94 / -7.6 / -6.7 / 18.71 / 18.67
H(252-258) / 252 / 258 / 835.4342 / 31.57 / 31.62 / -7.5 / -7.2 / 7.96 / 8.94
H(259-277) / 259 / 277 / 2139.0274 / 48.71 / 48.60 / -7.2 / -0.9 / 15.84 / 15.71
H(278-291) / 278 / 291 / 1677.8020 / 45.75 / 45.71 / -6.9 / -6.0 / 11.05 / 11.87
H(292-295) / 292 / 295 / 501.3144 / 1.28 / 1.28 / -9.4 / -6.7 / 5.61 / 4.38
H(296-304) + G0F glycan / 296 / 304 / 2634.0459 / 19.3 / 19.39 / -7.6 / -7.3 / manual
H(305-323) / 305 / 323 / 2228.2073 / 59.56 / 59.55 / 2.2 / -7.4 / 9.97 / 16.51
H(324-329) / 324 / 329 / 735.3818 / 3.13 / 3.18 / -9.7 / -4.9 / 5.52 / 7.07
H(330-337) / 330 / 337 / 838.5033 / 28.45 / 28.44 / -7.3 / -7.2 / 4.40 / 5.89
H(338-341) / 338 / 341 / 448.2766 / 4.24 / 3.85 / -9.3 / -4.4 / 5.17 / 3.56
H(342-347) / 342 / 347 / 656.3838 / 3.09 / 3.12 / -9.2 / -4.8 / 3.03 / 4.64
H(344-358) / 344 / 358 / 1724.9078 / nd / 32.24 / nd / -7.9 / nd / 6.95
H(344-363) / 344 / 363 / 2343.1761 / nd / 32.96 / nd / -7.4 / nd / 11.90
H(348-358) / 348 / 358 / 1286.6739 / 35.82 / 35.80 / -7.3 / -7.7 / 10.59 / 10.12
H(348-363) / 348 / 363 / 1904.9422 / 36.24 / 36.04 / -7.3 / -5.0 / 10.34 / 13.84
H(364-373) / 364 / 373 / 1161.6296 / 42.17 / 42.08 / -7.5 / -7.2 / 9.84 / 10.07
H(374-395) / 374 / 395 / 2544.1313 / 59.88 / 59.88 / -7.4 / -6.9 / 14.06 / 13.29
H(396-412) / 396 / 412 / 1873.9218 / 61.73 / 61.73 / -4.8 / -4.8 / 6.49 / 8.14
H(413-417) / 413 / 417 / 575.3399 / 4.37 / 4.41 / 1.5 / -8.8 / 3.04 / 4.58
H(420-442) / 420 / 442 / 2801.2671 / 43.33 / 43.19 / -7.5 / -6.7 / 3.28 / 4.49
H(443-449) / 443 / 449 / 660.3563 / 35.29 / 35.29 / 2.8 / 10.0 / 5.65 / 4.13
H(443-450) / 443 / 450 / 788.4513 / nd / 24.36 / nd / nd / -8.00 / 8.86
B) Chymotryptic Peptides
Retention Time (min) / Mass Error (ppm) / |log prob|
Peptide / Start / End / Theoretical
mass (M+H) (Da) / PolyAb / NISTmAb / PolyAb / NISTmAb / PolyAb / NISTmAb
H(239-244) / 239 / 244 / 563.282 / 43.99 / 43.81 / -3.6 / -2.5 / 4.62 / 6.31
H(245-254) / 245 / 254 / 1155.677 / 48.23 / 48.57 / -2.9 / -2.0 / 6.24 / 5.17
H(245-255) / 245 / 255 / 1286.718 / 53.09 / 53.49 / -1.2 / -1.9 / 7.21 / 9.39
H(246-255) / 246 / 255 / 1173.634 / 53.04 / 53.37 / -6.7 / -3.6 / ms1 / 7.11
H(247-255) / 247 / 255 / 1026.565 / 53.2 / 53.37 / -3.2 / -2.6 / 8.52 / 7.12
H(255-278) / 255 / 278 / 2773.354 / 69.22 / 69.4 / -2.1 / -2.6 / 4.98 / 11.04
H(256-278) / 256 / 278 / 2642.313 / 67.31 / 67.63 / -3.3 / 1.1 / 9.24 / 5.70
H(304-309) / 304 / 309 / 672.440 / 44.85 / 44.73 / -3.5 / -2.0 / 3.59 / 3.43
H(304-322) / 304 / 322 / 2256.214 / 76.59 / nd / -8.5 / nd / 5.00 / nd
H(310-316) / 310 / 316 / 898.442 / 44.27 / 44.18 / -2.7 / -2.0 / 6.63 / 7.41
H(310-317) / 310 / 317 / 1011.526 / 59.15 / 59.36 / -2.9 / -2.6 / 6.78 / 5.18
H(310-322) / 310 / 322 / 1602.791 / 53.93 / 54.24 / 1.0 / -2.8 / 7.40 / 12.04
H(313-316) / 313 / 316 / 585.242 / 23.59 / 23.00 / -6.0 / -3.1 / ms1 / 3.57
H(313-322) / 313 / 322 / 1289.591 / 43.92 / 43.81 / -3.3 / -2.2 / 8.46 / 10.20
H(317-322) / 317 / 322 / 723.367 / 21.44 / 20.38 / -3.7 / -2.5 / 4.90 / 5.40
H(318-322) / 318 / 322 / 610.283 / 21.34 / 20.38 / -3.9 / -1.1 / 4.19 / 3.37
H(332-352) / 332 / 352 / 2337.293 / 53.33 / 53.79 / -2.8 / -1.9 / 3.76 / ms1
H(353-368) / 353 / 368 / 1829.943 / 47.44 / 47.54 / -2.9 / -1.9 / 4.66 / 5.91
H(362-368) / 362 / 368 / 789.447 / 33.54 / 33.25 / -3.2 / -2.8 / 5.22 / 3.90
H(369-375) / 369 / 375 / 824.434 / 49.67 / 49.72 / 8.5 / -2.7 / ms1 / 5.14
H(369-384) / 369 / 384 / 1884.920 / 78.74 / 78.96 / -3.8 / 0.0 / 6.96 / 6.98
H(372-384) / 372 / 384 / 1510.758 / 73.45 / 73.65 / -2.8 / -1.9 / 7.74 / 9.00
H(376-384) / 376 / 384 / 1079.504 / 64.94 / 65.31 / -3.4 / -2.8 / 3.76 / 4.87
H(385-394) / 385 / 394 / 1151.460 / 22.72 / 22.05 / -2.9 / -2.5 / 5.32 / 6.23
H(385-408) / 385 / 408 / 2643.185 / 68.54 / nd / -7.1 / nd / 3.28 / nd
H(395-407) / 395 / 407 / 1363.674 / 51.82 / 52.11 / -3.4 / -2.7 / 9.03 / 11.71
H(395-408) / 395 / 408 / 1510.742 / 66.04 / 66.54 / -3.4 / -1.8 / 4.34 / 6.80
H(395-410) / 395 / 410 / 1786.890 / 75.80 / nd / -8.9 / nd / 7.71 / nd
H(409-426) / 409 / 426 / 2169.145 / 56.75 / nd / -9.2 / nd / 4.21 / nd
H(411-420) / 411 / 420 / 1219.679 / 37.69 / nd / -3.6 / nd / 9.30 / nd
H(411-426) / 411 / 426 / 1892.998 / 50.88 / 51.25 / -4.2 / -3.5 / 5.91 / 7.43
H(414-420) / 414 / 420 / 891.468 / 26.69 / 26.05 / -3.2 / -1.7 / 5.89 / 4.28
H(414-426) / 414 / 426 / 1564.787 / 48.01 / 48.10 / -3.3 / -2.3 / 7.00 / 5.11
H(421-426) / 421 / 426 / 692.336 / 35.82 / 35.66 / -3.9 / -2.1 / 5.54 / 4.06
H(427-431) / 427 / 431 / 583.221 / 24.83 / 24.13 / -4.7 / -2.5 / 5.62 / 3.83
H(427-435) / 427 / 435 / 1033.444 / 43.34 / 43.33 / -3.7 / -2.1 / 8.21 / 9.38
H(427-439) / 427 / 439 / 1584.668 / 38.51 / 38.60 / -3.3 / -2.1 / 8.87 / 11.21
H(427-444) / 427 / 444 / 2141.986 / 42.72 / nd / -9.2 / nd / 9.90 / nd
H(432-435) / 432 / 435 / 469.241 / 15.58 / 14.56 / -4.2 / -3.7 / 4.13 / 5.13
H(432-439) / 432 / 439 / 1020.465 / 16.53 / 15.13 / -3.8 / -2.2 / 8.31 / 5.89
H(440-444) / 440 / 444 / 576.335 / 11.18 / 9.92 / -3.5 / -1.7 / 6.10 / 3.68
H(445-449) / 445 / 449 / 460.240 / 18.78 / 18.17 / -3.7 / -3.2 / 3.82 / 3.26
H(445-450) / 445 / 450 / 588.335 / nd / 11.82 / nd / nd / -1.90 / 3.97

nd = not determined

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Table S2. Correlation Coefficients from Comparison of NISTmAb and PolyAb 1HN-15N Correlation Spectra. The Pearson’s correlation coefficients were calculated as described in the Experimental Section of the main text.

Spectral Correlation / MHz / 1D coefficient / 2D coefficient / Calc. 2d coefficientⱡ
NISTmAb full length
PolyAb full length / 900 / 0.878 / 0.776 / 0.911
NISTmAb digest
PolyAb digest / 900 / 0.819 / 0.590 / 0.9110
NISTmAb Fc
PolyAb Fc / 900 / 0.943 / 0.930 / 0.936
NISTmAb Fc
PolyAb Fc / 600 / 0.900 / 0.911 / 0.899
NISTmAb Fab
PolyAb Fab / 900 / 0.795 / 0.524 / 0.928
NISTmAb Fab
PolyAb Fab / 600 / 0.746 / 0.516 / 0.890
NISTmAb Fc
NIST Fc-deglycosylated / 900 / 0.832 / 0.768 / 0.910
NISTmAb Fc
duplicate / 900 / 0.976 / 0.935 / 0.932
NISTmAb Fc
metG-CSF / 900 / 0.621 / 0.207 / 0.945
PolyAb Fc
600-900 MHz / 600/900 / 0.930 / 0.919 / 0.908
PolyAb Fab
600-900 MHz / 600/900 / 0.913 / 0.897 / 0.908
PolyAb digest
duplicate / 900 / 0.923 / 0.911 / 0.922
NISTmAb Fc US
NIST Fc 50% NUS / 900 / N.D. / 0.871 / 0.878
NISTmAb digest US
NISTmAb digest 50% NUS / 900 / N.D. / 0.924 / 0.926
15N metG-CSF
gHSQC duplicate / 900 / 0.997 / 0.9978 / N.A.
metG-CSF SOFAST HMQC 4k duplicate / 900 / 0.9842 / 0.9943 / N.A.
metG-CSF SOFAST
HMQC 2k duplicate / 900 / 0.9790 / 0.9849 / N.A.
metG-CSF SOFAST
HMQC 1k duplicate / 900 / 0.9321 / 0.9670 / N.A.

ⱡ Predicted form experimental S/N from eq. (2). Values have an RMSD of ± 0.006. N.D. = not determined

Table S3. Correlation Coefficients from Comparison of NISTmAb and PolyAb 1H-13C Correlation Spectra. The Pearson correlation coefficients were calculated as described in the Experimental Section of the main text. Typical 1H-13C spectra were analyzed over the bounds of 11.0 ppm to 30.5 ppm in F1 and -0.7 ppm to 2.35 ppm in F2. The RMS noise amplitude of the spectrum was analyzed over the bounds of 30.0 ppm to 37.0 ppm in F1 and -1.5 ppm to -0.5ppm in F2.

Spectral Correlation / MHz / 1D coefficient / 2D coefficient
PolyAb Fc
600-900 MHz / 600/900 / 0.950 / 0.920
PolyAb Fab
600-900 MHz / 600/900 / 0.960 / 0.890
NISTmAb Fc
NISTmAb Fc-deglycosylated / 900 / 0.853 / 0.708
NISTmAb Full length
PolyAb Full length / 900 / 0.922 / 0.770
NISTmAb Full length
PolyAb Full length / 600 / 0.935 / 0.825
NISTmAb Fc
PolyAb Fc / 900 / 0.967 / 0.932
NISTmAb Fc
PolyAb Fc / 600 / 0.949 / 0.923
NISTmAb Fab
PolyAb Fab / 900 / 0.872 / 0.655
NISTmAb Fab
PolyAb Fab / 600 / 0.916 / 0.689
NISTmAb digest
PolyAb digest / 900 / 0.885 / 0.638
NISTmAb Fab
duplicate / 900 / 0.990 / 0.988

Figure S1. 1HN-15N SOFAST HMQC natural abundance correlation spectra. (A) Overlay of purified NISTmAb Fab (red) and Fc (blue) domains; (B) overlay of NISTmAb Fab/Fc (2:1) digest (blue) with the purified NISTmAb Fab and Fc domains (red). In panel B, both spectra of the Fab and Fc domains were shown in red so that a visual evaluation can be made of their combined spectral correlation to the Fab/Fc (2:1) digest spectra. Contours levels in panel B were chosen to best match signal intensities between the different spectra. The extraneous blue peaks are from high amplitude noise. All experiments were recorded at 900 MHz and 50 °C. Negative contours are in gray and represent a t1 ridge from residual l-histidine-d3 buffer.