新方式キャピラリー電気泳動　Maurice

Spotlight A Novel Platform for icIEF Fractionation of Antibody Charge Variants While imaged capillary isoelectric focusing Experimental Methods electrophoresis (imaged cIEF or icIEF) has become the The stepwise fractionation workflow on MauriceFlexTM method of choice for monitoring charge variant levels is of biotherapeutics, the in-depth characterization of illustrated below: charge variants has been carried out mostly by mass spectrometry (MS). 1 Setup (~30min) • Load reagents (kit provided) and NIST mAb The MauriceFlexTM system is a new and innovative (1 mg/mL) Maurice platform featuring icIEF-based fractionation for • Insert MauriceFlex cartridge collecting protein charge variants, enabling downstream analysis of these variants with techniques such as MS. The fractionation utilizes a special cartridge to perform 2 Focusing (45min) isoelectric focusing to separate protein charge variants, Voltage and once focused and separated, the charge variants are • 10 min @500 V eluted via chemical mobilization for fraction collection. • 10 min @1000 V Compared to ion-exchange chromatography (IEX)-based • 25 min @1500 V fractionation, MauriceFlexTM fractionation is fast, with pI-based resolution, and overcomes some of the limitations of direct coupled cIEF-MS, such as the need 3 Mobilization (25min) for a dedicated interface and incompatibility of the • Mobilizer: 5 mM NH4Ac background electrolytes with the MS. Moreover, offline • Voltage: 1000V fractionation and potential enrichment of charge variants pooled from multiple fractionations offer more flexibility for mass spectrometry characterization, including intact 4 Elution (20min) mass, reduced mass and peptide mapping. Collect fraction In addition to fraction collection, MauriceFlexTM can • 36 fractions at 25 second/fraction on a 96-well plate perform routine CE-SDS and cIEF analysis using the • Each well contains 30 uL 5 mM NH4Ac relevant Maurice cartridges, and all data is analyzed using the 21CFR Part 11 compliant Compass for iCE software. Voltage • 1000 V (off during transition from well In this spotlight, we demonstrate the fractionation to well) workflow and intact and peptide mapping analysis by liquid chromatography and mass spectrometry 5 Verification (4-5 hours) (LC-MS) on the NIST mAb (RM 8761 from NIST), which is a common reference standard for assessing • Check identify and purity of fractions with analytical Maurice icIEF new analytical technologies for characterizing monoclonal antibodies (mAbs). • 16 fractions checked

For intact mass, the fractions were analyzed directly. A 0.25 mg/mL For peptide mapping, the charge variant fractions from A2 A1 M B1 B2 10 fractionation runs were pooled, lyophilized on a 30,000 SpeedVac, and reconstituted prior to tryptic digestion. Variant Relative The digested samples were lyophilized and reconstituted 25,000 Abundance in 40 µL 5 mM ammonium acetate solution. B2 0.95 20,000 B1 8.5% The LC-MS characterization was performed with a 15,000 M 58.4% Thermo ScientificTM Vanquish UHPLC coupled to a Q 10,000 A1 25.7% ExactiveTM HF Hybrid Quadrupole-Orbitrap™ mass A2 6.5% spectrometer. Reverse phase LC separation was used 5,000 for intact mass analysis and peptide mapping with 0 appropriate gradients of 0.1% formic acid in water and 8.7 8.8 8.9 9 9.1 9.2 9.3 9.4 acetonitrile at 0.3 mL/min. The injection volume was pI 10 µL for both intact and peptide mapping analysis. The data were analyzed using BioPharma Finder 4.1 software. B 1 mg/mL 20,000 A2 A1 M B1 B2 Results The method for fractionation of the NIST mAb sample 15,000 using the MauriceFlex cIEF fractionation cartridge can be developed quickly based on the analytical method 10,000 on a Maurice cIEF cartridge. FIGURE 1 shows profiles of analytical icIEF and fractionation focusing runs of NIST mAb. Five charge variants (B2, B1, M, A1, A2) were 5,000 identified for NIST mAb and their relative abundances were obtained (FIGURE 1A). Note that fractionation 0 separation with cIEF fractionation cartridge is designed 8.6 8.8 9 9.2 9.4 for maximizing the yield of the fraction collection, and for pI this purpose, a higher concentration (1 mg/mL) of NIST FIGURE 1. icIEF separation of NIST mAB charge variants with the Maurice cIEF cartridge (A) and MauriceFlexTM cIEF Fractionation cartridge (B). Both mAb was loaded. While this resulted in apparently lower electropherograms show the same number of charge variants. The relative resolution with overlapped peaks (FIGURE 1B) when abundance (%) of each variant was calculated from the peak areas in the compared to charge separation with the regular Maurice analytical run (A) and listed in the table insert. cIEF cartridge (FIGURE 1A), the same number of charge variants were detected, as seen in FIGURE 1B, and were well separated. As shown in the workflow, the mobilization and elution 40,000 steps take 45 minutes in total when collecting 36 fractions 20,000 Reference Standard 0 on a 96-well plate. The Compass for iCE software has 40,000 a peak prediction feature that provides an estimated 20,000 #10 (65% B2, 35% B1) 0 range of wells that contain the eluted charged variants. 40,000 20,000 #11 (100% B1) Alternatively, a fluorescence plate reader can be used to 0 40,000 select the wells that contain the most abundant charge 20,000 #14 (100% M) variant. For NIST mAb, a total of 16 wells of fractions were 0 40,000 selected, and their identity and purity were verified with 20,000 #17 (100% A1) 0 analytical Maurice icIEF. Among the 16 fractions analyzed, 40,000 20,000 #21 (100% A2) 12 were found to contain the charge variants. 0 8.4 8.6 8.8 9 9.2 9.4 9.6 FIGURE 2 shows icIEF electropherograms of fraction wells pI containing individual charge variants with the highest FIGURE 2. Verification of charge variants of representative fractions for purity. As shown, except for low abundant B2 (0.9%) at 65% identity and purity by Maurice icIEF analytical runs. The numbers assigned represent the fraction numbers. purity in fraction #10, fractions containing 100% purity for the four other charge variants were obtained. Fluorescence Fluorescence Fluorescence

1_25s_NIST_B2 NL: 3.91E5 1_25s_NIST_B2 #6192-6525 RT:5.219-5.500 AV:334 NL: 6.31E4 1_25s_NIST_B2 NL: 2.90E6 5.332 100 3093.8529 100 148455.08 100 3228.3434 148615.67 148293.41 B1 50 50 3374.9532 50 148046.48 148776.81 5.513 3621.8044 148154.77 147832.36 148936.20 2320.1353 4013.4516 4364.7178 0 2_25s_NIST_B1 NL: 8.33E5 0 2_25s_NIST_B1 #6192-6538 RT:5.219-5.500 AV:347 NL: 0 1.49E5 2_25s_NIST_B1 NL: 6.60E6 5.327 3091.1942 148323.80 100 100 100 148162.06 B2 3297.0919 148488.27 50 5.406 50 50 147957.50 3450.4218 5.458 3804.3165 148119.58 148647.80 2179.1503 4125.6578 148812.16 0 NL: 3_25s_NIST_M NL: 1.27E8 0 0 3_25s_NIST_M #6196-6631 RT:5.219-5.500 AV:436 3_25s_NIST_M NL: 1.30E7 5.93E8 5.247 3088.5136 148195.83 100 100 100 148360.19 M 3294.3404 148034.06 147832.55 50 50 3447.4749 50 148521.97 3800.9019 147983.95 148680.13 5.366 2352.1516 4239.9135 0 NL: 2.24E7 0 4_25s_NIST_A1 #6192-6606 RT:5.219-5.500 AV:415 NL: 0 NL: 4_25s_NIST_A1 1.85E6 4_25s_NIST_A1 7.28E7 5.262 100 3091.9231 100 100 148359.86 148520.52 148196.08 3297.8927 50 A1 147985.31 148680.31 50 3451.2775 50 5.309 148037.08 148835.75 3809.3392 148995.39 5.461 2318.6478 4122.2188 149157.33 0 0 5_25s_NIST_A2 #6192-6525 RT:5.219-5.500 AV:334 NL: NL: 5_25s_NIST_A2 NL: 3.43E5 0 5_25s_NIST_A2 4.83E4 2.32E6 3157.5700 100 5.343 148361.48 5.311 100 100 5.372 148199.25 3297.9091 148521.36 A2 147987.39 148683.70 50 50 3455.0008 50 148036.25 148842.23 3805.0788 2355.1556 4239.4999 148994.20 0 0 0 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 2000 2500 3000 3500 4000 4500 147000 147500 148000 148500 149000 149500 150000 RT (min) m/z Mass FIGURE 3. LC-MS intact mass analysis of charge variants showing total ion current (TIC) chromatograms (left), mass spectra of the peaks (middle) and the deconvoluted spectra (right). Peak Deconvoluted Mass (Da) Mass Shift (Da) Modification The LC-MS intact mass analysis on the high purity B2 148455.08 +259.25 2xC-term K charge variant fractions are shown in FIGURE 3 and B1 148323.80 +127.97 C-term K the modifications identified from the deconvoluted mass M 148195.83 0.00 G0F/G1F spectra for each charge variants are summarized in TABLE 1. The results are consistent with the established A1 148359.86 -164.03 Glycation knowledge of PTMs of the charge variants of NIST mAb. A2 148361.48 -165.65 Glycation TABLE 1. Summary of the identified modifications from intact mass analysis. The mass shifts of each variants are relative to the G0F/G1F glycoform of the M peak. NL: 1.32E8 Peptide mapping is an indispensable tool for 22.98 100 90 characterizing the primary structure of biotherapeutics, 80 B1 70 and the capability of pooling charge variants from 60 50 11.56 1.22 multiple fractionation runs on MauriceFlexTM makes it 23.13 40 11.03 15.00 30 9.41 14.74 6.57 22.74 possible to enrich the charge variants for peptide mapping 7.52 20 23.22 4.94 17.80 10 17.38 1.97 4. 19.93 21.30 24.07 analysis. FIGURE 4 demonstrates the peptide mapping 1.05 53 25.12 28.79 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 results from samples pooled from 10 fractionation RT (min) NL: 2.32E8 11.55 runs. As shown, the sequence coverage is above 90% 100 90 A1 22.97 for all variants except for the low abundant B (relative 80 9.41 11.04 70 7.52 14.98 14.72 19.92 abundance 0.9%) and heavy chain (HC) of the A2 peak. 60 6.56 21.27 9.69 17.82 50 40 18.46 8.96 22.18 23.12 30 1.22 20 17.38 23.21 10 4.93 Sequence Coverage % 8. 0 1.97 4.54 23 .68 16.80 24.38 25.17 27.96 29.74 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 Fraction LC HC RT (min) B2 84.0 58.9 FIGURE 4. Representative chromatograms of the peptide mapping of B1 and A1, and sequence coverage of Light Chain (LC) and Heavy Chain (HC) of all B1 94.4 92.4 charge variants. The charge variant samples were pooled from 10 M 95.3 94.0 fractionation runs. A1 95.3 92.2 A2 93.9 73.8 Relative Intensity Relative Intensity Relative Intensity Relative Intensity Relative Intensity

Conclusion By using the NIST mAb sample with the icIEF • A single fractionation run provides sufficient charge fractionation feature on the new MauriceFlexTM system, variant fractions for intact mass analysis we have demonstrated that: • With enrichment from pooling fractions from multiple runs, charge variants can be analyzed with LC-MS • High purity fractionation of charge variants can be peptide mapping obtained in a single day Bio-Techne® | R&D Systems™ Novus Biologicals™ Tocris Bioscience™ ProteinSimple™ ACD™ ExosomeDx™ Asuragen® For research use or manufacturing purposes only. Trademarks and registered trademarks are the property of their respective owners. STRY0297039_BBU_SP_MauriceFlex-Spotlight_KG