Supporting Information To

Supporting Information to: Glycoforms of immunoglobulin G based biopharmaceuticals are differentially digested by the trypsin due to the glycoform influence on higher order structure

David Falcka,#, Bas C. Jansena, Rosina Plompa, Paul J. Hensbergena, Dietmar Reuschb, Markus Habergerb, and Manfred Wuhrera,c a Center for Proteomics and Metabolomics, Leiden University Medical Center, Albinusdreef 2, 2333 ZA Leiden, The Netherlands b Pharma Biotech Development Penzberg, Roche Diagnostics GmbH, 82377 Penzberg, Germany c Division of BioAnalytical Chemistry, VU University Amsterdam, De Boelelaan 1083, 1081 HV Amsterdam, The Netherlands

#to whom correspondence should be addressed Table S1: Calibration list Identity Exact m/z FeO-propionic acid cluster 621.9729 IgG1_G0F_[M+3H]3+ 878.6820 IgG1_G1F_[M+3H]3+ 932.6996 IgG1_G2F_[M+3H]3+ 986.7172 IgG1_G2SF_[M+3H]3+ 1083.7490 IgG1_G0F_[M+2H]2+ 1317.5229 IgG1_G1F_[M+2H]2+ 1398.5494 IgG1_G2F_[M+2H]2+ 1479.5758 IgG1_G2SF_[M+2H]2+ 1625.1235 Table S2: Time course of the tryptic cleavage of IgG1 mAb without denaturation step. Relative abundances and their standard deviations are given. Time point 1h 2h 3h 4h 6h 8h 22h 26h

Glycan composition H5N2 5.0 9.3 8.7 9.9 11.1 9.5 3.9 3.1 (Man5) ±2.1% ±0.5% ±0.8% ±0.3% ±0.2% ±0.6% ±0.1% ±0.3% H6N2 3.4 2.4 3.3 2.6 1.4 1.1 0.50 0.39 (Man6) ±0.2% ±<0.1% ±0.2% ±0.2% ±<0.1% ±0.1% ±0.06% ±0.06% H7N2 5.5 3.8 4.0 2.9 1.1 0.90 0.45 0.33 (Man7) ±3.6% ±0.5% ±0.9% ±0.7% ±<0.1% ±0.14% ±0.06% ±0.04% H8N2 3.6 2.3 3.2 2.2 1.2 1.1 0.77 0.66 (Man8) ±0.2% ±0.2% ±0.5% ±0.3% ±<0.1% ±<0.1% ±0.08% ±0.08% H4N3 2.0 2.2 1.9 2.3 ±<0.1 1.7 1.5 0.80 0.66 ±<0.1% ±0.2% ±0.4% % ±<0.1% ±<0.1% ±0.02% ±0.05% H4N3F1 4.3 3.4 4.0 3.2 3.3 2.8 1.4 1.2 ±0.3% ±0.1% ±0.1% ±0.2% ±0.1% ±<0.1% ±<0.1% ±<0.1% H3N3 5.3 4.4 4.7 4.2 3.5 3.0 1.4 1.1 ±<0.1% ±0.3% ±0.4% ±0.3% ±0.2% ±0.3% ±<0.1% ±<0.1% H3N3F1 3.2 3.4 4.1 3.7 3.5 3.0 1.5 1.3 ±0.2% ±0.1% ±0.7% ±0.2% ±<0.1% ±0.1% ±<0.1% ±<0.1% H5N3 3.0 1.7 2.3 1.8 1.0 0.83 0.36 0.28 ±0.3% ±0.1% ±0.4% ±0.3% ±<0.1% ±0.05% ±0.03% ±0.04% H5N3F1 3.4 1.8 2.5 1.7 0.84 0.84 0.32 0.27 ±0.5% ±0.1% ±0.5% ±0.2% ±0.03% ±0.03% ±0.04% ±0.03% H6N3 4.2 2.9 3.3 2.3 1.38 1.2 0.51 0.40 ±0.5% ±0.6% ±0.5% ±0.3% ±0.04% ±0.1% ±0.05% ±0.05% H6N3F1 3.9 1.6 2.8 1.8 0.91 0.82 0.46 0.37 ±1.9% ±0.1% ±0.5% ±0.2% ±0.08% ±0.09% ±0.07% ±0.05% H5N4S2F1 3.2 3.9 4.1 3.3 3.3 3.2 1.3 1.1 ±0.5% ±0.2% ±0.5% ±0.3% ±0.3% ±0.3% ±0.1% ±<0.1% H5N4S1F1 4.9 5.4 4.9 4.4 4.5 4.4 2.4 2.0 ±0.1% ±0.4% ±0.3% ±0.4% ±0.4% ±0.3% ±0.1% ±0.1% H4N4S1F1 4.0 3.6 3.9 3.5 3.0 3.1 2.2 2.1 ±0.3% ±0.3% ±0.4% ±0.2% ±0.1% ±0.2% ±<0.1% ±0.1% H5N4 4.5 2.7 3.9 2.7 1.3 1.2 0.88 0.78 ±0.9% ±0.2% ±0.4% ±0.4% ±0.2% ±0.1% ±0.06% ±0.06% H5N4F1 7.5 5.1 5.0 5.0 5.8 6.6 8.0 8.3 ±3.4% ±0.5% ±0.5% ±0.2% ±0.3% ±0.3% ±0.2% ±0.2% H4N4 2.9 3.0 2.8 2.9 ±<0.1 2.6 2.5 3.0 3.1 ±0.2% ±0.2% ±<0.1% % ±<0.1% ±<0.1% ±<0.1% ±<0.1% H4N4F1 11.6 19.0 14.3 19.2 25.0 28.3 36.7 38.0 ±4.4% ±0.3% ±2.0% ±1.9% ±0.2% ±1.1% ±0.2% ±0.7% H3N4 3.0 2.8 3.1 3.3 2.9 2.9 4.5 4.7 ±0.5% ±0.4% ±0.2% ±0.1% ±0.2% ±0.1% ±0.1% ±<0.1% H3N4F1 11.2 15.9 12.9 17.5 20.7 21.3 28.7 30.0 ±4.1% ±0.6% ±1.6% ±1.7% ±0.5% ±1.1% ±0.1% ±0.5% Values in gray contain at least one value under the LLOQ (S/B<3; S/B=3 approximately corresponds to S/N=9). Figure S1: Absolute abundances of glycopeptides in the tryptic cleavage of IgG1 mAb without denaturation.

G Table S3: Comparison of the relative abundances of IgG1 mAb glycoforms obtained with different sample preparation methods. P-values are given throughout, for analyses performed after 3 h and 26 h incubation. DRA: denaturation, reduction and alkylation; FA: acidic denaturation (with formic acid); SDS: detergent-assisted denaturation (using sodium dodecyl sulphate); n: no denaturation.

3 h vs 26 h 3 h 26 h n DR FA SDS n/DRA n/FA DRA/ n/SDS DRA/SDS n/DR n/FA DRA/FA n/SDS DRA/SDS A FA A H3N4F1 / G0F 0.004 n.s. n.s. 0.002 0.002 0.002 n.s. 0.015 0.00003 n.s. n.s. n.s. n.s. n.s. H4N4F1 / G1F 0.003 n.s. n.s. 0.003 0.001 0.003 n.s. 0.007 0.00005 n.s. n.s. n.s. n.s. n.s. H5N4F1 / G2F 0.016 n.s. n.s. n.s. 0.006 0.007 n.s. 0.034 0.001 n.s. n.s. n.s. n.s. n.s. H3N4 / G0 0.006 n.s. n.s. 0.002 0.0002 0.001 n.s. n.s. 0.002 0.018 0.002 0.007 n.s. n.s. H4N4 / G1 0.013 n.s. n.s. 0.024 0.027 0.00006 n.s. 0.018 0.004 n.s. 0.0004 n.s. 0.002 n.s. H5N4 / G2 0.006 n.s. n.s. 0.011 0.006 0.006 n.s. 0.010 0.002 n.s. n.s. 0.046 0.006 0.006 H4N4F1S1 / G1FS 0.028 n.s. n.s. 0.021 0.016 0.021 0.016 0.043 0.007 0.035 n.s. 0.027 n.s. 0.008 H5N4F1S1 / G2FS 0.010 n.s. n.s. 0.023 0.004 0.004 0.002 0.026 0.012 0.006 0.007 n.s. n.s. 0.012 H5N4F1S2 / G2FS2 0.014 n.s. n.s. n.s. 0.009 0.010 0.005 0.035 0.030 0.007 0.010 n.s. n.s. 0.0004 H5N2 / Man 5 0.011 n.s. 0.040 0.00006 0.005 0.006 0.047 0.024 0.0001 0.021 0.021 n.s. n.s. 0.012 n.s. = not statistically significant Table S4: Comparison of the relative abundances of IVIG (Flebogamma) glycoforms obtained with different sample preparation methods. P- values are given throughout, for analyses performed after 3 h and 26 h incubation. FA: acidic denaturation (with formic acid); SDS: detergent- assisted denaturation (using sodium dodecyl sulphate); n: no denaturation.

IgG1 IgG2/3 3 h vs 26 h 3 h 3 h 26 h n FA S n/FA n/SDS S n/FA n/SDS SDS/FA n FA SDS n/FA n/SDS SDS/FA n/FA n/SDS SDS/F D D A S S / F A H3N4F 0.012 n.s. 0 0.019 0.002 0 n.s. 0.016 0.008 0 n.s. 0.026 n.s. 0.001 n.s. n.s. n.s. n.s. 1 / . . . G0F 0 0 0 2 0 3 3 1 2 H4N4F n.s. n.s. 0 n.s. 0.013 0 0.008 n.s. 0.005 n n.s. 0.042 0.014 0.008 0.002 0.009 0.033 0.006 1 / . . . G1F 0 0 s 1 0 . 9 6 H5N4F 0.022 n.s. n 0.002 0.027 0 0.026 n.s. 0.021 n n.s. n.s. 0.000 0.026 0.007 0.044 n.s. 0.046 1 / . . . 2 G2F s 0 s . 1 . 3 H3N4 / n.s. n.s. n n.s. n.s. 0 0.021 n.s. n.s. ------G0 . . s 0 . 2 1 H4N4 / n.s. n.s. 0 n.s. n.s. 0 0.027 n.s. n.s. ------G1 . . 0 0 1 0 7 2 H5N4 / 0.046 n.s. n 0.001 0.024 0 0.026 n.s. n.s. ------G2 . . s 0 . 2 8 H4N4F n.s. n.s. n n.s. n.s. n n.s. 0.003 n.s. n n.s. n.s. n.s. n.s. n.s. n.s. 0.044 n.s. 1S1 / . . . G1FS s s s . . . H5N4F 0.003 n.s. n 0.005 0.002 n n.s. n.s. n.s. 0 n.s. n.s. n.s. 0.002 0.013 n.s. 0.030 n.s. 1S1 / . . . G2FS s s 0 . . 2 2 H3N5F n.s. n.s. n 0.015 n.s. 0 0.010 n.s. 0.003 n n.s. 0.003 0.008 0.006 0.0004 0.007 0.039 0.010 1 / . . . G0FN s 0 s . 0 . 1 H4N5F n.s. n.s. 0 0.0001 0.007 0 0.002 n.s. 0.001 0 n.s. 0.013 0.008 n.s. 0.023 0.002 n.s. 0.002 1 / . . . G1FN 0 0 0 0 0 4 0 1 4 0 2 H5N5F n.s. n.s. n n.s. 0.010 0 0.007 0.026 0.0001 0 n.s. n.s. 0.023 0.004 0.005 0.026 n.s. 0.013 1 / . . . G2FN s 0 0 . 0 3 3 3 H5N4S n.s. n.s. n 0.035 0.036 n 0.021 0.036 n.s. ------1 / . . G2S s s . . n.s. = not statistically significant A Man5 12 ] % [

e 9 c n a d n

u 6 b a

e v i t 3 a l e R 0 0 10 20 30 Incubation time [h]

Figure S2: Comparison of cleavage time curves of IgG1 mAb including detergent-assisted denaturation. The time curves obtained for detergent-assisted denaturation are compared to the non-denatured incubation on the one hand and to the incubations with prior acidic denaturation on the other hand. A) The high mannose glycoform Man5 is shown as an example of the high mannose and hybrid classes of glycans. B) G1F represents the fucosylated complex glycans. C) G2FS exemplifies the sialylated complex glycans. Considering no denaturation and acidic denaturation as extremes of no and full denaturation, respectively, the detergent-assisted denaturation shows a clear bias which is however less pronounced as without denaturation. Therefore, only partial efficiency of this denaturation method can be deduced. Figure S3: SDS-PAGE analysis of the IgG mAb1 cleavage mixture after 6 h cleavage with prior SDS denaturation. The band at 25 kDa was excluded as it very likely (partially) corresponded to the light chain. The three 6 h samples of the detergent-denatured digest (see Figure S2) were pooled, the protein fragments were separated from the fully digested glycopeptides by SDS-PAGE, the four bands were excised and an in-gel cleavage was performed. Although complete in-gel cleavage cannot be proven, the thorough denaturation and reduction afforded by the SDS-PAGE analysis should prevent major biases. Four bands were cut and named H1 to H4 in declining order of size (see Supporting Information Figure S3). From H4 no glycopeptide profile could be extracted. H1 was a very faint band and not surprisingly resulted in a very low abundance of glycopeptides. The profile, though it has to be considered cautiously, was quite similar to those of H2 and H3. Profiles of these two bands were, in terms of abundance, fair and good, respectively (see Table S6). Overall, the in-gel profiles were much closer to the final profiles than to the glycopeptide profiles of the 6 h detergent-denatured sample. However, there were two important differences between the in- gel profiles and the final profiles. These were highly statistically significant as the in-gel values were far outside of the 99% confidence interval (99% CI) of the final profiles. Man5, which had a relative abundance of 9.81% (±0.19%) in the initial glycopeptide analysis and of 1.77% (±0.01%; 99% CI 1.74 to 1.80) in the final profiles, was barely detectable in the H2 and H3 with 0.54% and 0.27%, respectively. G1FS and G2FS2 (1.76±0.02% and 0.66±0.01%, respectively, in the final profile) were not detected at all in H2 or H3 while they exhibit abundances of 2.71±0.07% and 2.71±0.08%, respectively, in the initial glycopeptide analysis. While the relative abundance of G2FS was comparable in H2, it was also reduced from 1.47±0.02% (final profile; 99% CI 1.41 to 1.52) to 0.94% in H3 and increased to 3.97±0.08% in the initial glycopeptide analysis. Table S5: Comparison of glycan profiles between fully digested glycopeptides and partially digested fragments at 6 h from the SDS denaturation time curves (see Figure S2) Sample Final DRA Glycopeptides H1 H2 H3 profile from the (Mean+SEM) 6 h sample Glycan (Mean+SEM) composition H5N2 (Man5) 1.77±0.01% 9.81±0.19% 0.5% 0.3% H6N2 (Man6) 0.97±0.03% H7N2 (Man7) 0.65±0.05% H8N2 (Man8) 0.97±0.05% H4N3 1.61±0.06% H4N3F1 2.72±0.07% 0.3% H3N3 3.10±0.05% H3N3F1 2.93±0.07% 0.8% 0.5% H5N3 0.78±0.03% H5N3F1 0.67±0.03% 0.3% H6N3 1.06±0.03% 0.3% H6N3F1 0.72±0.02% H5N4F1S2 0.66±0.01% (G2FS2) 2.71±0.08% H5N4F1S1 (G2FS) 1.47±0.02% 3.97±0.08% 1.5% 0.9% H4N4F1S1 (G1FS) 1.76±0.02% 2.71±0.07% H5N4 (G2) 0.81±<0.01% 1.26±0.08% 2.1% 1.7% H5N4F1 G2F) 9.10±0.10% 6.44±0.09% 11.0% 10.2% 9.4% H4N4 (G1) 3.37±0.03% 2.64±0.03% 4.0% 3.8% H4N4F1 (G1F) 41.89±0.18% 28.4±0.3% 45.0% 41.3% 43.9% H3N4 (G0) 5.44±0.06% 3.18±0.11% 6.5% 5.9% 5.8% H3N4F1 (G0F) 33.74±0.25% 22.7±0.3% 37.6% 33.8% 33.0% Values in gray contain at least one value under the LLOQ (S/B<3; S/B=3 approximately S/N=9). H3 and H2 have good intensity and should give reliable glycan profiles. H1 is weak and rather an estimation. Table S6: Time course of the tryptic cleavage of IVIG without denaturation step. Relative abundances and their standard deviations are given. Time point 1h 2h 3h 4h 6h 9.5h 22h 26h

Glycan composition IgG1 H3N4F1 22.2 21.5 21.6 21.2 16.7 16.7 17.0 16.9 (G0F) ±0.4% ±0.5% ±0.7% ±0.1% ±0.4% ±0.2% ±0.1% ±0.1% H4N4F1 29.9 30.0 29.6 29.2 26.8 27.9 28.4 28.8 (G1F) ±0.7% ±0.7% ±1.0% ±0.9% ±0.2% ±0.4% ±0.3% ±0.3% H5N4F1 11.9 (G2F) ±<0.1 12.5 12.2 12.8 13.4 13.4 14.4 14.6 % ±0.4% ±0.3% ±0.5% ±0.2% ±0.5% ±<0.1% ±0.3% H3N5F1 7.3 7.1 7.5 7.6 7.9 7.6 6.6 6.5 (G0FN) ±0.1% ±0.4% ±0.5% ±0.4% ±0.4% ±0.6% ±<0.1% ±0.2% H4N5F1 12.0 12.4 12.6 12.4 14.7 14.8 12.1 11.9 (G1FN) ±0.2% ±0.5% ±0.2% ±0.7% ±0.1% ±0.8% ±0.3% ±0.6% H5N5F1 1.9 1.9 2.2 1.8 2.7 2.8 2.4 2.3 (G2FN) ±0.2% ±0.4% ±<0.1% ±0.2% ±<0.1% ±<0.1% ±<0.1% ±<0.1% H4N4F1S1 2.0 2.0 1.9 1.8 2.2 2.0 2.2 2.1 (G1FS) ±0.5% ±0.3% ±0.3% ±0.1% ±0.2% ±0.1% ±0.05% ±<0.1% H5N4F1S1 8.4 8.8 8.6 9.1 11.1 9.7 11.7 11.9 (G2FS) ±0.4% ±0.2% ±0.4% ±0.6% ±0.9% ±0.5% ±0.4% ±0.2% H3N4 1.5 0.92 1.0 1.4 1.1 1.2 1.1 0.87 (G0) ±0.4% ±0.49% ±<0.1% ±0.1% ±0.2% ±0.2% ±<0.1% ±0.10% H4N4 1.5 0.93 1.4 1.3 1.6 1.8 2.0 2.0 (G1) ±0.3% ±0.57% ±0.4% ±0.4% ±0.1% ±0.1% ±<0.1% ±0.1% H5N4 1.1 (G2) ±<0.1 1.6 1.1 1.1 1.2 1.3 1.4 1.3 % ±0.8% ±<0.1% ±0.1% ±0.2% ±<0.1% ±<0.1% ±<0.1% H5N4S1 0.4 0.3 0.3 0.4 0.8 0.80 0.87 0.84 (G2S) ±0.2% ±0.3% ±0.2% ±0.2% ±0.2% ±0.13% ±0.03% ±0.07% IgG2/3 H3N4F1 31.3 30.0 30.4 30.5 26.7 27.6 28.4 27.0 (G0F) ±0.9% ±0.4% ±0.5% ±1.9% ±0.4% ±0.8% ±0.7% ±0.4% H4N4F1 30.3 29.6 29.3 29.8 28.1 28.9 29.6 30.0 (G1F) ±0.3% ±0.4% ±0.4% ±0.8% ±0.4% ±0.2% ±0.3% ±0.4% H5N4F1 10.7 11.3 10.9 11.0 11.7 11.4 11.6 12.3 (G2F) ±0.5% ±0.3% ±0.1% ±0.9% ±0.2% ±0.5% ±0.6% ±0.4% H3N5F1 8.0 (G0FN) ±<0.1 8.3 8.2 8.3 8.8 8.8 8.2 7.5 % ±0.4% ±0.4% ±0.4% ±0.2% ±0.2% ±0.2% ±0.2% H4N5F1 4.5 4.6 5.3 4.9 5.5 5.4 5.2 5.1 (G1FN) ±0.3% ±0.3% ±0.2% ±0.4% ±0.3% ±0.2% ±0.1% ±0.1% H5N5F1 1.5 1.7 (G2FN) ±<0.1 1.8 1.6 ±<0.1 2.0 2.1 1.7 1.8 % ±0.2% ±<0.1% % ±0.1% ±<0.1% ±<0.1% ±<0.1% H4N4F1S1 5.9 (G1FS) ±<0.1 6.0 6.0 6.0 6.3 6.2 6.2 6.5 % ±0.2% ±0.4% ±0.2% ±0.1% ±0.3% ±0.2% ±0.1% H5N4F1S1 7.8 8.4 8.3 7.9 10.9 9.6 9.2 9.8 (G2FS) ±0.3% ±0.6% ±<0.1% ±0.8% ±0.7% ±0.3% ±0.5% ±0.3% Values in gray contain at least one value under the LLOQ (S/B<3; S/B=3 approximately S/N=9).