Structure Characterization and Differentiation of Biosimilar and Reference Products Using Unique Combination of Complementary Fragmentation Mechanisms

Structure Characterization and Differentiation of Biosimilar and Reference Products Using Unique Combination of Complementary Fragmentation Mechanisms Zhiqi Hao, 1 Chen Li, 2 Shiaw-Lin Wu, 2,3 David M. Horn 1 and Jonathan Josephs 1 1 Thermo Fisher Scientific, San Jose, CA; 2 BioAnalytix Inc, Cambridge, MA; 3 Barnett Institute, Northeastern University Boston, MA

c1 178.1 c2 279.1 c3 c4 494.2 2 6 274.1 (S) 292.1 2 Overview Purpose: To analyze difference of protein structure in biosimilar and reference products using Orbitrap LC-MS/MS Methods: A unique data-dependent instrument method that utilizes two different fragmentation mechanisms was applied for peptide sequence and PTM identification and quantification using a Thermo Scientific TM Orbitrap Fusion TM Tribrid TM mass spectrometer. While generating HCD MS/MS spectra on peptides in a data-dependent experiment, the method identifies glycopeptides on-the-fly using the diagnostic ions from glycan fragmentation. A subsequent ETD fragmentation is then triggered on the same peptide to produce information of amino acid sequence and site of glycosylation. The new Thermo Scientific TM PepFinder TM 1. software for peptide imaging, was used for data analysis. Results: A LC-MS/MS workflow was developed for differentiating minor difference of protein structure in biosimilar and reference products using an Orbitrap Fusion LC-MS/MS and PepFinder 1. software. This new approach offers efficient, confident and comprehensive analysis, not only for biosimilari il comparability study but also for lot-to-lot t l tcomparison of a same compound. Introduction Biosimilars are subsequent versions of innovator biopharmaceutical products created after the expiration of the patent on the innovator product. The approval of a biosimilar product by a regulatory agency requires thorough characterization that demonstrates comparability with a reference product in quality, safety and efficacy. High resolution mass spectrometry provides accurate characterization of various protein properties including primary structure, type and location of post-translationaltranslational modifications (PTMs), and low abundant sequence variants or impurities. In this study, we developed a robust approach for comparability study of biosimilar and reference product. Minor differences in products including glycosylation were systematically compared using high resolution LC/MS/MS with complementary fragmentation methods and a new peptide mapping software package. Methods Samples An original drug, a recombinant variant and its biosimilar product, TPA, I-TNK and G-TNK, were digested using trypsin after reduction and alkylation. Tenectelplase (TNK) is a recombinant TPA with the following minor sequence changes: T13->N (Becomes N-glycosylation site) N117->Q (Removes N-glycosylation site) KHRR (296-299) -> AAAA Liquid chromatography Peptides were separated using with a Thermo Scientific TM EasySpray TM source setup containing -cm C 18 column (2 µ particle size) and a high-pressure easy nanolc (U- HPLC). The LC solvents were.1% formic acid in H 2 O (Solvent A) and.1% formic acid in acetonitrile (Solvent B). Flow rate was 2 µl/min. A min gradient was used to elute peptides from the column. Mass spectrometry Samples were analyzed using an Fusion mass spectrometer with a Thermo Scientific TM EASY-ETD ion source. An instrument method designed for glycopeptide analysis was used for this study. This method primarily acquires HCD MS/MS spectra on peptides in a data-dependent top-ten experiment. However, if diagnostic sugar oxonium ions from glycan fragmentation are detected in the HCD MS/MS spectrum, a subsequent ETD fragmentation is then triggered on the same precursor peptide to produce amino acid backbone sequence information to identify the site of glycosylation. Therefore, for each glycopeptide, this HCD product-dependent d d t ETD method (HCDpdETD) generatesapair of HCD and ETD spectra, producing information for the peptide sequence and the site of glycosylation as well as confirming glycan structure. Orbitrap MS spectra were acquired at 12, resolution (at 2) with an AGC target of 4x1 5. MS/MS spectra were acquired at, resolution (at 2) with an AGC target of 5x1 4. Capillary temperature was set to 275 C and the S-lens level was set at. The priority for precursor selection for data-dependentdependent MS/MS was for the highest charge state followed by the lowest. HCD collision energy was and ETD activation time was charge dependent based on the standard calibration. Data analysis Data was analyzed using PepFinder 1. software. This software provides automated analyses of liquid chromatography/tandem mass spectrometry data for large-scale identification and quantification of known and unknown modifications. Peptide identification is achieved by comparing the experimental fragmentation spectrum to the predicted spectrum of each native or modified peptide. Peak areas of related peptide ions under their 2 selected-ion chromatograms (SIC) are used for relative quantification of modified peptides. A mass tolerance of 5 ppm was used to ensure accurate identification. Table 1. Identified glycosyla number of glycoforms iden Site of glycosylation N 13 N 13 N117 N 184 N 448 N 448 N 448 Figure 2. Characterization o C441-R449 with glycosylati showing peptide backbon preserved) and fragmentatio C441-R449, N448 g 2 Structure Characterization and Differentiation of Biosimilar and Reference Products Using Unique Combination of Complementary Fragmentation Mechanisms

c1 178.1 c3 M[4+] 798.3 2 1 12 1 1 1 1 c2 279.1 274.1 (S) 292.1 c4 494.2 631.3 Y1-F++ 666.3 (SGGn) Y2-F++ 767.9 M1++ 848.9 Y2++ M2++ 929.9 M1F++ M2F++ 2.9 z6[3+] 966.7 M[3+] 164.1 z 9[3+] z9[3+] a 9[3+] M3F++ M3++ 184.5 -SGGn A1G1M2++ 1186. -SGGn++ A1G1++ 1267. A1GM2++ A1GM2F++ 2 1 12 1 1 1 1 1 1 A2G1++ -GGn 1331.6 1214. 1267.5 z 4++ z4++ -G-F++ Y1 1477.7 z 5++ z5++ z'5++ ++ Y2-F 1534.7 1413.1 z6++ z'6++ y 6++ ++ z'7++ c8++ c 8++ z 8++ z8++ b 8++ M1 Y2 1696.8 1574.7 1596.2 z 9++ a 9++ z9++ M2 1858.8 M1F 159.1 (G) z 15++ 838.9 z 3 z'15++ 3.2 z 4 466.2 z 2 z 1++ z 11++ z 12++ z 14++ 2 579.3 4++ 2 5++ Results 1. Peptide identification and protein sequence coverage The data was analyzed and the results were compared. Peptide mapping results indicated % sequence coverage for all of the data files. The relative abundance of each modified peptide forms was calculated and compared between files. A five order magnitude dynamic range for identified peptide abundance was achieved, which allowed identification of modified peptides with less than.1% in abundance of the unmodified versions (data not shown). Figure 1 shows an example of the sequence coverage view for one of the data files. Figure 1. % sequence coverage of I-TNK s), and low abundant sequence t approach for comparability study products including glycosylation LC/MS/MS with complementary re package. product, TPA, I-TNK and G-TNK, ation. Tenectelplase (TNK) is a es: ific TM EasySpray TM source setup high-pressure easy nanolc (Uolvent A) and.1% formic acid in min gradient was used to elute Table 1. Identified glycosylation sites, percentage of glycosylation and the number of glycoforms identified with high confidence Site of glycosylation Sample # glycoforms % glycosylation N 13 I-TNK 18 >99 N 13 G-TNK 11 >99 N117 TPA 14 >99 N 184 I-TNK 12 19 N 448 TPA 44 >99 N 448 I-TNK 36 >99 N 448 G-TNK 47 >99 ++ (Bn-1)-GGnM 3++ Table 2. Comparison of N448 gly relative abundance higher than 1 The five major glycoforms are structure: Antenna A, core fucos N-acetyl neuraminic acid (NANA) meter with a Thermo Scientific TM ed for glycopeptide analysis was MS/MS spectra on peptides in a ic sugar oxonium ions from glycan a subsequent ETD fragmentation e amino acid backbone sequence, for each glycopeptide, this HCD apair of HCD and ETD spectra, site of glycosylation as well as cquired at 12, resolution (at ere acquired at, resolution rature was set to 275 C and the ction for data-dependentdependent MS/MS /z. HCD collision energy was the standard calibration. is software provides automated ectrometry data for large-scale odifications. Peptide identification tion spectrum to the predicted of related peptide ions under their antification of modified peptides. A ntification. Figure 2. Characterization of glycopeptides using HCDpdETD. G-TNK peptide C441-R449 with glycosylation on N448. Top left is fragment ion coverage showing peptide backbone fragmentation from ETD (black, with glycan preserved) and fragmentation of peptide and glycan from HCD (red). C441-R449, N448 glycosylation, Relative abundance =.52% c5 845. 913.1 (Bn-1)-SGGnM Y1++ ETD Spectrum z 6++ 14.1 z7++ z 7++ ++ a 8++ N448 Glycoform N448+A2G2F N448+A2S1G N448+A2S1GF N448+A2S1G1F N448+A2S2F N448+A3G3F N448+A2Sg1S1F N448+A3S1G2F N448+A3S2G N448+A3S2G1F N448+A4S2G2F N448+A4S1G3F N448+A3S3F N448+A4S3G1F N448+A4S4F Thermo Scientific Poster Note PN-64145-ASMS-EN-614S 3

[3+] 64.1 1596.2 1574.7 1413.1 c8++ z6++ z 9++ [3+] 3+] z'6++ c 8++ z 8++ +] 1267.5 z 5++ z5++ y 6++ ++ z8++ 1214. z 4++ z'5++ ++ z'7++ a 9++ b 8++ z9++ z4++ 1 12 1 1 1 1 -GGn 1331.6 Y1 1477.7 n 86. -SGGn++ M2 1G1++ 1267. Y2-F M1 1858.8 1534.7 Y2 1696.8 M1F A2G1++ -G-F++ 12 1 1 1 1 1 1 159.1 (G) 963.6 z 15++ a 28[3+] 838.9 c13++ z28[3+] c27[3+] c 13++ 1484. c 27[3+] c25[3+] z 3 z'15++ z 16++ 1113.2 c9++ c1++ 3.2 z 18++ c11++ c 25[3+] z 27[3+] 889.4 1339.1374.5 c12++ z27[3+] z 4 134. 466.2 z'16++ 924.5 c 9++ 1439.1 c24[3+] z'27[3+] z 15 z 19++ z19++ z 2++ z2++ c 7++ c 1++ c 12++ c 8++ c8++ c 24[3+] 1677.8 z 2 z 1++ z 11++ z 12++ z 14++ z 25[3+] z'18++ c 3++ c3++ z'2++ z 21++ 1266.5 c 11++ c7++ c 26[3+] c26[3+] z21++ y 21++ a 25[3+] a27[3+] a28[3+] a 27[3+] c14++ c16++ c17++ z'16 c 18++ c18++ 2 1 12 1 1 1 1 1 1 1 579.3 4++ 5++ y8 6++ 6.5 7++ 8++ 116.5 978. Y1-F[3+] 9.8142.5 6[3+] 9++ 2 1 12 1 1 1 1 1 1 1 1++ Y2-F[3+] M[4+] 124.5 2++ 1214.6 5++ 4++ 1344.1 1.6 6++ 1437.2 1522.7 1564.2 M[3+] 16. z 28[3+] 7[3+] y 27[3+] Y1-F++ 1624.2 Y1++ 1697.3 Y2-F++ M1++ 16.8 M2++ 133. 145.1 2.1 b4 511.3 b5 446.2 571.3 b6-h2o++ ++ 326.2 b6++ y8++ b1++ ++b1 2 2.1 397.2 b4 494.3 2 511.3 57 2. Glycosylation of TPA, I-TNK and G-TNK A total of four glycosylation sites were identified, three of which are over 99% glycosylated. N448 was glycosylated in all three samples, while N13 was detected in I-TNK and G-TNK and N117 only in TPA. The forth glycosylation site, N184, was identified only in I-TNK and only 19% of this site is glycosylated (Table 1). I-TNK has an additional glycosylation site (N184) even though it shares the same amino acid sequence as G-TNK, suggesting a different manufacturing process. Examples of two identified glycopeptides are shown in Figure 2 and Figure 3. Figure 3. Characterization of glycopeptides using HCDpdETD. I-TNK peptide G12-R129 with glycosylation on N13. The fragment ion coverage at the top of this figure shows peptide backbone fragmentation from ETD (black, with glycan preserved) and fragmentation of peptide and glycan from HCD (red). G12 R129, N13 glycosylation, Relative abundance = 13.85% ETD Spectrum samples. Deamidation on N1 TPA. Other sites of N-deamid Figure 5 shows examples of and deamidated on two differe ntage of glycosylation and the Figure 4. Identification of lo onfidence localization of double oxida # glycoforms % glycosylation 18 >99 11 >99 14 >99 12 19 44 >99 36 >99 47 >99 using HCDpdETD. G-TNK peptide op left is fragment ion coverage from ETD (black, with glycan glycan from HCD (red). abundance =.52% ETD Spectrum z 6++ 14.1 z7++ z 7++ ++ a 8++ ++ (Bn-1)-GGnM 3++ Table 2. Comparison of N448 glycoforms in the three samples. Only those with relative abundance higher than 1% in at least one of the samples are included. The five major glycoforms are highlighted in bold. Abbreviations for glycan structure: Antenna A, core fucose (Fuc) F, mannose (Man) M, galactose (Gal) G, N-acetyl neuraminic acid (NANA) S, N-glycolyl neuraminc acid (NGNA) Sg ++ 11. N448 Glycoform TPA I-TNK G-TNK N448+A2G2F 6.41% 5.% 3.23% N448+A2S1G 5.18% 2.57% <1% N448+A2S1GF <1% <1% 1.79% N448+A2S1G1F 23.11% 16.86% 14.43% N448+A2S2F 37.96% 35.34% 37.59% N448+A3G3F <1% 1.29% <1% N448+A2Sg1S1F 1.32% <1% <1% N448+A3S1G2F 1.59% 2.48% <1% N448+A3S2G 1.43% <1% <1% N448+A3S2G1F 5.19% 7.% 5.4% N448+A4S2G2F <1% <1% 2.2% N448+A4S1G3F <1% 1.16% <1% N448+A3S3F 9.33% 11.61% 16.% N448+A4S3G1F 1.17% 6.55% 2.62% N448+A4S4F 1.67% 7.2% 6.51% Relative Abundance 15.25 72 477.1969 25.38 731.821 25.17 546.2712 8.81 364.2164 2 1.35 368.11 1 TVCLPPADLQLPDW.45..35..25.2.15.1.5. Peak area = 1.88 928.135 5 1 15 2 25 Time (min) 2 1 928.135 5 1 15 2 25 W6 doub 4 Structure Characterization and Differentiation of Biosimilar and Reference Products Using Unique Combination of Complementary Fragmentation Mechanisms

y 27[3+] a 28[3+] c13++ z28[3+] c27[3+] c 13++ 1484. c 27[3+] c25[3+] c9++ c1++ c11++ c 25[3+] z 27[3+] 1339.1374.5 c12++ z27[3+] c 9++ 1439.1 c24[3+] z'27[3+] z 15 c 1++ c 12++ c 8++ c8++ c 24[3+] 1677.8 5 c 11++ c 26[3+] c26[3+] a 25[3+] a 27[3+] a27[3+] a28[3+] c14++ c16++ c17++ z'16 c 18++ c18++ 1 1 1 1 1 1 1 Y1-F++ 1624.2 1522.7 Y1++ 1697.3 5++ + 1344.1.6 6++ Y2-F++ M1++ 1437.2 1564.2 16.8 M2++ 1 1 1 1 1 1 622.3 735.4 3++ 771.3 854.4 b4 511.3 b1 195.6 4++ y8 827.9 913.4 142.5 b11 2.1 128.6 b9 1 967.5 133. 1329.6 b5 654.3 446.2 947.5 8.5 1444.6 4 571.3 1654.8 b6-h2o++ ++ 1++ b16++ 6++ 326.2 b6++ y8++ b1++ ++b11++ b13++ b14++ 9++ 1++ 1++ 5 1 12 1 1 1 1 1 1 1 3++ 787.3 622.3 854.4 735.4 b1 195.6 4++ b4 511.3 b11 128.6 y8 913.4 b9 142.5 4 967.5 2.1 1686.7 494.3 b11-h2o b1-h2o 11.6 9.5 177.5 1 895.4 1476.6 661.2 1361.6 397.2 571.3 5 p 2 1 12 1 1 1 1 1 1 ++ 1139. ++ 1155. 3 1541.7 3 1573.7 1 2 2 3 4 three samples. Only those with e of the samples are included. old. Abbreviations for glycan ose (Man) M, galactose (Gal) G, euraminc acid (NGNA) Sg The type and relative abundance of gllycoforms were compared across the three samples and the following was observed: 1. The relative abundance and identity of the various glycoforms gy on N448 were consistent among all three samples (Table 2). Most of glycans on this site contain sialic acid. 2. The identity of the glycoforms on N13 are similar between I-TNK and G-TNK, but the relative abundance profiles are markedly different. Although the most abundant form, A2S1G1F, is the same in the two samples, the second and the third most abundant forms are not. For the top five most abundant forms, only two of them were common in the two samples (data not shown). 3. The glycoforms on N117 are primarily high mannose, which is very different from the glycans identified on any of the other sites (data not shown). 4. Glycosylation on N184 was only detected for the I-TNK sample (data not shown), with all of the glycans containing sialic acid. 3. Other identified and quantified modifications Besides glycosylation, other covalent modifications that were indentified in these three samples included cysteine alkylation, deamidation, overalkyation, Cys+DTT, oxidation, formylation, and glycation. Figure 4 shows confident identification and localization of a low abundant double oxidation on W6. The relative abundance of the oxidized form is less than.1%. A total of 12 N-deamidation sites were indentified with high confidence in the three samples. Deamidation on N1 was only identified in I-TNK and G-TNK, but not in TPA. Other sites of N-deamidation were consistent across all three samples (Table 3). Figure 5 shows examples of a peptide that were identified in 3 different forms: native and deamidated on two different Asp residues, respectively. Figure 4. Identification of low abundant double oxidized d peptide T393-K416 and localization of double oxidation to W6..45..35..25.2.15.1.5. Relative Abundance 2 1 1.35 368.11 8.81 364.2164 15.25 477.1969 25.17 546.2712 28.59 727.3284 25.38 731.8215 31.41 918.4182 8 34.29 754.3414 43.45 5.3152 TVCLPPADLQLPDWTECELSGYGK Peak area = 1.88e+5 48.88 114.9824 49.32 199.4645 55.72 8.4586 59.23 193.7893 66.23 371.123 51.49 917.4414 78.7171 376.2612 49. 928.1 47.52 928.136 47.8 42.43 928.141 928.135 928.149 5 1 15 2 25 35 45 51.15 Time (min) 2 917.4415 1 42.43 49.9 52.55 59.83 64.41 74.2 85.23 928.135 928.149 928.136 917.49 917.48 917.44 917.41 917.47 5 1 15 2 25 35 45 55 65 75 85 Time (min) 89.77 371.13 TVCLPPADLQLPDWTECELSGYGK Peak area = 1.98e+8 Table 3. Identified N-deamidati Location of N-deamidation N524 I-TNK G-TNK 5.% 3.23% Conclusion 2.57% <1% <1% 1.79% 16.86% 14.43% 35.34% 37.59% 1.29% <1% <1% <1% 2.48% <1% 2 <1% <1% 7.% 5.4% <1% 2.2% 1.16% <1% 11.61% 16.% 6.55% 2.62% 7.2% 6.51% 145.1 W6 double oxidation, Relative abundance =.9% N1 N142 N25 N218 N234 N37 N3 N454 N469 N486 N516 A LC-MS/MS workflow was develop biosimilar and reference products us software, PepFinder 1.. This workflo product comparison. 1. % sequence coverage was o 2. The identified covalent modifi alkylation, deamidation, overa glycosylation. The relative abu between datasets. Confident ide achieved. 3. Glycosylated peptides were char information of peptide sequence, glycosylation sites as well as the are significant differences in glyc All other trademarks are the property of Therm This information is not intended to encourage intellectual property p rights of others. Thermo Scientific Poster Note PN-64145-ASMS-EN-614S 5

1 1329.6 3 1541.7 5 1 1 1 1 1 1 1 1 1361.6 1444.6 1476.6 3 1573.7 4 1654.8 4 1686.7 5 p 1 1 1 1 1 1 4.7 ++ 517.2 y8++ 545.8 ++ ++ 612.3 175.1 375.2 92.4 749.3 ++ -H2O++ 498.2 335.2 432.2 452.2 -H2O++ y8-h2o++ 537.2 8.7 143.1 863.4 223.1 284.2 443.2 325.2 b4 b5 564.3 1 2 2 3 4 5 6 7 8 9 498.2 ++ 546.2 2.3 593.8 M-H2O++ 2.8 594.3 M-H2O++ ++ y8++ 461.2 517.7 7.3 921.4 145.1 ++ b6 864.3 175.1 335.2 432.7 1 2 2 3 4 5 2.8 6 7 8 9 1 461.2 ++ 517.7 y8++ 546.2 ++ 593.8 M-H2O++ ++ 375.2 ++ -H2O++ 498.2 432.7 537.2 -H2O++ y8-h2o++ 145.1 452.2 8.7 171.1 y8 335.2 b6 134.5 -H2O 284.2 b5 528.7 585.3 3.3 228.1 31.1 b4 732.3 b9 1 2 2 3 4 5 6 7 8 9 1 613.2 612.3 749.3 864.3 921.4 191.5 Figure 5. Identification and localization of two deamidation sites, N1 and N142, on peptide L136-R145. High resolution HCD spectrum of this peptide in native form (top), with deamidation either on N142 (middle) or on N1 (bottom). YCR NYCR NHNYCR for the I-TNK sample (data not acid. s that were indentified in these idation, overalkyation, Cys+DTT, ows confident identification and W6. The relative abundance of with high confidence in the three 1 d in I-TNK and G-TNK, but not in across all three samples (Table 3). entified in 3 different forms: native ectively. oxidized d peptide T393-K416 and YCR YCR NYCR NYCR N142 deamidation. Relative abundance = 1.99% NHNYCR N1 deamidation. Relative abundance = 5.93% NHNYCR Table 3. Identified N-deamidation sites and relative abundance of deamidation Location of N-deamidation TPA I-TNK G-TNK N1 ND 12.24% 1.21% 59.23 193.7893 66.23 371.123 N142 3.68% 3.82% 2.% N25 2.8% 1.61% <.5% N218.63% <.5% <.5% N234 <.5% <.5% <.5% N37 29.83% 22.83% 19.64% 78.7171 N3 8.24% 13.56% <.5% 376.2612 89.77 371.13 CLPPADLQLPDWTECELSGYGK Peak area = 1.98e+8 N454 3.62% 2.71% 2.27% N469 3.71% 2.5% 1.24% N486 11.2% 1.% 7.64% N516 3.68% 2.87% 2.2% 59.83 64.41 74.2 85.23 17.48 917.44 917.41 917.47 65 75 85 nce =.9% Conclusion N524 1.32% <.5% 1.% A LC-MS/MS workflow was developed to differentiate minor differences in protein structure for biosimilar and reference products using an Orbitrap Fusion instrument and new peptide mapping software, PepFinder 1.. This workflow provides qualitative and quantitative biosimilar to reference product comparison. 1. % sequence coverage was obtained for all the nine data files analyzed. 2. The identified covalent modifications, both expected and un-expected, include cysteine alkylation, deamidation, overalkyation, Cys+DTT, oxidation, formylation, glycation and glycosylation. The relative abundance of the modified forms was calculated and compared between datasets. Confident identification and precise localization of low abundant PTMs was achieved. 3. Glycosylated peptides were characterized using the unique HCDpdETD method which generates information of peptide sequence, site of glycosylation as well as glycan structure. Comparison of glycosylation sites as well as the type and relative abundance of glycoforms indicated that there are significant differences in glycosylation between the three samples. All other trademarks are the property of Thermo Fisher Scientific and its subsidiaries. This information is not intended to encourage use of these products in any manners that might infringe the intellectual property p rights of others. PO64145-EN 614S 6 Structure Characterization and Differentiation of Biosimilar and Reference Products Using Unique Combination of Complementary Fragmentation Mechanisms

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