CHROMATOGRAPHY MAbPac RP Column High-performance reverse phase chromatography column for monoclonal antibody analysis Product Specifications The Thermo Scientific MAbPac RP is a reverse phase (RP) liquid chromatography column designed for mab characterization, including the separation of mab and variants, light chain () and heavy chain (), Fc and Fab fragments, scfc and F(ab') fragments, using /UV or /MS. The unique column chemistry provides excellent performance under a broad range of ph, temperature, and mobile phase composition. Product Highlights Superior resolution power for monoclonal antibodies and related substances High efficiency with low carry-over Excellent MS compatibility Wide operating ph range: 14 High temperature stability: up to 11 C High throughput Introduction The monoclonal antibody (mab) therapeutics market is growing at a rapid rate owing to increasing demand for targeted treatments. Therapeutic mabs are mostly produced from mammalian cells. These biological products are heterogeneous due to post-translational modifications. Additional modifications such as oxidation can be introduced during the manufacturing process. A comprehensive characterization of mab purity, aggregates, and variants is required for the final biopharmaceutical product approval and subsequent manufacturing processes. There is a growing trend to obtain intact mass information as well as the glycan profile in the QC of mabs using reverse phase chromatography coupled with high resolution mass spectrometry detection. /MS analysis of mab fragments such as light chain (), heavy chain (), Fc, Fab, scfc and F(ab'), can accurately reveal the location, as well as nature, of the modification. Moreover, in most QC environments, /UV analysis of mab fragments has been established as a high throughput assay. Column Technology MAbPac RP is a reverse phase (RP) column specifically designed for separation of intact monoclonal antibodies (mabs) and mab fragments. The stationary phase is fully compatible with mass spectrometry friendly organic solvent such as acetonitrile and isopropanol, as well as low ph eluents containing trifluoroacetic acid or formic acid. The MAbPac RP is based on wide-pore 4 µm polymer particles that are stable at extreme ph ( 14) and high temperature (up to 11 C). The wide-pore size of polymeric particles enables efficient separation of protein molecules with low carry-over.
Applications Fast Separation of Intact Proteins/mAbs The MAbPac RP column is designed for high resolution and fast separation of proteins. High throughput and high-resolution can be achieved by adjusting both gradient slope and flow rate. The wide-pore nature of polymer particles combined with 4 µm particle size provides excellent resolution for intact proteins/mabs with high-throughput and low carry-over. Figure 1 shows the baseline separation of four proteins: ribonuclease A, cytochrome C, lysozyme, and a mab, on a.1 mm MAbPac RP column within min. 14 Format:.1 mm 15 Mobile phase A: H O/TFA (99.9 :.1 v/v) Mobile phase B: MeCN/ H O/TFA (9: 9.9 :.1 v/v/v) 11. 8 88.5.7 75.8 8 4. 8 6 4 Flow Rate:.6 ml/min 8 Temp.: 8 ºC Detection: UV (8 nm) 5 1 1. Ribonuclease A (.5 mg/ml) 1. Cytochrome C (.5 mg/ml). Lysozyme (.5 mg/ml) 4. mab (1 mg/ml) -.. 1. 1.... Separation of mab Fragments Monoclonal antibodies are heterogeneous. Comprehensive analysis of mab posttranslational modifications, such as deamidation, C-terminal lysine truncation, N-terminal pyroglutamation, methionine (Met) oxidation, and glycosylation, requires complete digestion of mabs and sequencing of all the peptides. However, peptide mapping can be time consuming. A simpler and more direct way to analyze mab variants and locate the modifications is to measure mab fragments, which can either be generated by chemical reduction or by enzymatic digestion. Figure shows the analysis of trastuzumab intact molecule and fragments. and (Figure b), Fc and Fab (Figure c), scfc and F(ab') (Figure d) are baseline separated on a. mm MAbPac RP column using a 1 min gradient. Light chain () and heavy chain () are generated by the reduction of mab with DTT, Fc and Fab fragments are generated by papain digestion, and singlechain Fc (scfc) and F(ab') fragments are generated by IdeS digestion. Figure 1: Fast separation of intact proteins/mab 14 (a) mab - 8-1 9-1 (b) (c) (d) -1 1.. 6. 9. 1. 15. Format:. mm Mobile phase A: H O/FA/TFA (99.88 :.1:. v/v/v) Mobile phase B: MeCN/ H O/FA/TFA (9: 9.88 :.1:. v/v/v/v). 8 1. 8 11. 55 45 1. 55 45 14. 8 15. 8 scfc Fc Fab F(ab ) Figure : Separation of mab and mab fragments Flow Rate:.5 ml/min Inj. Volume: 5 µl Temp.: 8 ºC Detection: UV (8 nm) IgG IgG Reduction Papain digestion IdeS DTT F ab F (ab') (a) Trastuzumab (5 mg/ml) (b) Trastuzumab + DTT (4 mg/ml) (c) Trastuzumab + Papain ( mg/ml) (d) Trastuzumab + IdeS ( mg/ml) scfc F ab F c
/MS analysis of Intact mab and mab fragments High resolution mass spectrometers such as the Thermo Scientific Q Exactive Hybrid Quadrupole-Orbitrap Mass Spectrometer provide accurate mass information of large biologic molecules such as mabs. The MAbPac RP column can be directly coupled to the mass spectrometer for MS detection of mab and mab fragments. While trifluoroacetic acid (TFA) as ion-pairing reagent provides excellent separation results, TFA can suppress ionization in the /MS interface, causing a drop in signal. This can be mitigated by reducing the TFA concentration to.% in the presence of.1% formic acid (FA). Figure shows the intact mass detection of trastuzumab. The top trace shows the total ion-current chromatogram. The middle trace shows the MS spectrum of trastuzumab in the mass range of, to 4,. The bottom trace shows a zoom-in spectrum of trastuzumab with + charges. The cluster shows the glycosylation profile of trastuzumab. 9 8 7 6 4 Format:. mm Mobile phase A: H O/FA/TFA (99.88 :.1:. v/v/v) Mobile phase B: MeCN/ H O/FA/TFA (9: 9.88 :.1:. v/v/v/v). 8 1. 8 11. 55 45 1. 55 45 14. 8 15. 8 Flow Rate:.5 ml/min Temp.: 8 ºC MS Detection: Positive-ion mode Mass Spec: Q Exactive Plus Trastuzumab (5 mg/ml) 1 8.6 4.47 5.1 5.4 5.68 1. 1.5 1.74 5.9 6.9 6.74 6.99 7.9 7.64 8.97 9.7 9.7 9.88 5. 5. 5.5 6. 6.5 7. 7.5 8. 8.5 9. 9.5 1. 1.5 11. 11.5 1. 1.5 1. Time (min). 88.9 69.64 MS spectrum 94.8 154.64 9 5.91 8 7 6 4 1 9 8 7 57.49 TIC 745.8 695.91 51.18 647.76 4.8 61.7 471. 94.5 18.1 8.77 at + 851.9 797.55 4 6 8 8.6 965.4 8.44 97.7 965.4 447.98 968.61 5.9 616.14 76. 81.5 91.54 917.6 994.1 4 6 8 4 6 4 96. 971.87 959.4 974.96 1 95.5 9.5 99.7 96.54 99.75 977.49 956.45 98.99 986.8 996..66 9 95 94 945 9 955 96 965 97 975 98 985 99 995 Figure : Intact mab and glycosylation profile by /MS
4 During characterization, mabs are often reduced to and. Mass spectrometry analysis of these fragments can quickly reveal and localize the modifications. Figure 4 shows the separation of trastuzumab and on a. mm MAbPac RP column. Total ion chromatogram (TIC) and UV spectrum show identical retention time of mab fragments. The mass spectrum of shows multiple charge states of a single polypeptide chain while the mass spectrum of the shows multiple glycosylation forms of the heavy chain. Format:. mm O/FA/TFA (99.88 :.1:. v/v/v) O/FA/TFA (9: 9.88 :.1:. v/v/v/v). 8 1. 8 11. 55 45 1. 55 45 14. 8 15. 8 Flow Rate:.5 ml/min Temp.: 8 ºC UV Detection: 8 nm MS Detection: Positive-ion mode Mass Spec: Q Exactive Plus Reduced trastuzumab (4 mg/ml) 9 8 TIC 7.4 8.8 7 6 4 1..6.94 4.4 4.79 5. 5. 5.95 6.1 6.41 6.96 7.95 8.58 8.86 9.11 9.76 1. 1.7 1.77 6 5 UV 8.8 4 4 7.4 9.1 6.5 6.6 7.7 8.85 6.8..5 4. 4.5 5. 5.5 6. 6.5 7. 7.5 8. 8.5 9. 9.5 1. 1.5 11. Time (min) 9 8 7 6 4 1 1954.16 18.88 11.6 1675.14 156.47 44.74 1465.8 179.68 176.96 1. 1967.87 146.6 14.47 1687.1 1819.8 1478.76 1575.6 61.4 19.86 95..1 9 8 7 6 4 1 161.64 168.9 9 165.61 1+ 159. 168.9 8 168.8 16.1 16. 7 164.16 164.91 6 169.8 149.85 165. 164.8 1751.18 4 164.6 1446.47 168.89 1671.7 181.69 1661. 141.9 1646.9 16.16 1666.59 1 188.81 165.79 1615.8 168.6 195.11 16 161 16 16 164 16 166 167 16.67 1.8 115.81 169.96 47.97 118.58.69 18.96 8.14 418. 158.66 5.7 58.1 1 1 14 1 16 17 18 19 4 Figure 4: /MS analysis of mab fragments
9 8 7 6 4 1.1 1.79 11.9 1..85.5.91 4.87 5.5.45 4.1 5.7 5.8 6.8 6.48 7.79 9.8 8.9 9.6 4 5 6 7 8 9 1 11 1 9 8 7 6 4 1 9 8 7 6 4 1 (a) TIC (b) Oxidized scfc (c) scfc 55.6.76 54.8 Oxidizied scfc 7.16 51.98 51.75 9.45 55.6 54.8 Time (min) 59.45 57.98 scfc 46 48 5 54 56 58 6 6 Format:. mm O/FA/TFA (99.88 :.1:. v/v/v) O/FA/TFA (9: 9.88 :.1:. v/v/v/v). 75 5 1. 75 5 11. 6 7 1. 6 7 14. 75 5 15. 75 5 Figure 5: /MS analysis of oxidized scfc 7.7 54.8 545.66 544. 541.99 +1 8.5 557.97 556.9 56.15 566.6 1.48 5.11 56.78 Flow Rate:.5 ml/min Inj. Volume: µl Temp.: 8 ºC MS Detection: positive-ion mode Mass spec: Q Exactive Plus 585.84 Fd 1.4 Oxidized trastuzumab, reduced by DTT and digested by IdeS (1 mg/ml) /MS analysis of mab fragments containing oxidation variants Development of high-throughput release and characterization assays is critical for rapidly growing biologics pipeline for biotherapeutics. Oxidation of mabs is commonly monitored during process optimization, formulation development, and stability studies. The mab can be broken down into scfc,, and Fd' fragments using DTT reduction followed by IdeS enzyme. Figure 5a shows the baseline separation of the scfc,, and Fd' fragments of trastuzumab. This sample was previously treated with H O, resulting in oxidation of a methione in the Fc region (confirmed by peptide mapping, data not shown). The oxidized scfc eluted before the unmodified scfc. Figure 5b shows the +1 charge state of scfc at 55.6 and Figure 5c shows the +1 charge state of unmodified scfc at 54.8. The delta mass between oxidized peak and unmodified peak corresponds to one oxygen mass. This example demonstrates that oxidation occurring in the scfc,, and Fd', regions can be simultaneously monitored by this assay, without going through complete digestion of mab and peptide mapping. This simple reduction/digestion assay coupled with fast separation of the mab fragments using MAbPac RP and high resolution accurate mass detection by Q Exactive Plus Instrument makes it an ideal method for high throughput screening of mab oxidation. 5 Unlabeled Ab ß-1,4- Add in Galactosidase GalT(Y89L) DIBO- UDP-GalNAz 7 C, 5 hr 7 C, ON 5 C, ON Cleave Terminal Gal Azide-Activated Ab (stable for long-term storage) Figure 6: Site-selective antibody-drug conjugates (ADCs) 5.. 15. 1. 5.. DAR DAR1-5. 1. 1..... 4. Figure 7: modified trastuzumab ADC DAR & DAR DAR1 Antibody Drug Conjugate (ADC) Format:.1 mm O/TFA (99.9 :.1 v/v) O/TFA (9: 9.9 :.1 v/v/v). 65 5.5 65 5 4.5 45 55 5. 45 55 5.5 65 5 6. 65 5 Flow Rate:.6 ml/min Inj. Volume: µl Temp.: 8 ºC Detection: UV (8 nm) Trastuzumab- Analysis of Antibody-Drug Conjugate (ADC) Antibody-drug-conjugates are proven to be a highly effective cancer therapy. Due to the heterogeneous nature of the ADC, it is critical to characterize its multiple drug-toantibody ratio (DAR) forms. The MAbPac RP column can be utilized in the separation of mab and its conjugates. In Figure 6, ADCs were prepared by enzymatically activating mab Fc domain glycans with azides using the mutated beta-galactosyltransferase enzyme. The azide-activated antibodies were then conjugated with dibenzocyclooctyne (DIBO)-activated Val-Cit-PAB-Monomethyl Auristatin E () toxin in a copperless click reaction, resulting in a mixture of drugloaded antibody species with to 4 molecules. The unmodified mab and ADCs with DAR values ranging from to 4 are well resolved on the MAbPac RP column (Figure 7).
6 Analysis of PEGylated Protein Protein PEGylation technology has been used successfully to increase the circulating half life and decrease antigenicity of protein drugs. PEGylated protein contains multiple PEGylated forms. Figures 8a and 8b illustrate the separation of a PEGylated protein and its de-pegylated form on a.1 mm MAbPac RP column. At least eight PEGylated forms are resolved based on the degree of PEGylation. Excellent Reproducibility and Chemical Stability Column ruggedness is a critical characteristic for accurate and reproducible results, as well as good column lifetime. MAbPac RP columns are packed using a carefully developed packing protocol to ensure excellent packed bed stability, column efficiency and peak asymmetry. Figure 9 demonstrates that the excellent performance of the MAbPac RP is maintained throughout 1, runs at 8 C providing consistent retention time, peak shape, and peak efficiency. The RSDs of retention time from four protein peaks are tabulated in Figure 9. MAbPac RP chemistry also offers excellent chemical stability in a wide ph range. Figure 1 shows the MAbPac RP column maintains its performance after 6 hours of wash with.8 M NaOH at 8 C. Such chemical stability, especially under alkaline condition provides a great advantage over RP columns based on pure silica or organo-silica hybrid particles. 9. 6. 4.. 1. 5. (a) -1. 14. (b) -. 1... 4. 5. 6. 7. 8. 9. 1. 11. Figure 8: PEGylated protein 1 8 6 4 - -4-6 1-8.. 1. 1.... Figure 9: Excellent reproducibility 14 15 11 88 75 6 8 5 1 After 6 hrs of.8 M NaOH wash at 8 oc Before NaOH wash 4 #111 #91 #81 #71 #651 #1 #41 #1 #1 #11 -. 1. 1..... Figure 1: Superior chemical stability 1 4 Format:. mm O/TFA (99.9 :.1 v/v) O/TFA (9: 9.9 :.1 v/v/v). 55 45 1. 55 45 11. 5 75 1. 5 75 14. 55 45 15. 55 45 Flow Rate:.5 ml/min Temp.: 8 ºC Detection: UV (8 nm) (a) PEGylated protein (11 mg/ml) (b) de-pegylated protein (1.4 mg/ml) Format:.1 mm O/TFA (99.9 :.1 v/v) O/TFA (9: 9.9 :.1 v/v/v). 8.5.7.8 8 4. 8 Flow Rate:.6 ml/min Temp.: 8 ºC Detection: UV (8 nm) 1. Ribonuclease A (.5 mg/ml). Cytochrome C (.5 mg/ml). Lysozyme (.5 mg/ml) 4. mab (1 mg/ml) Peaks 1 4 RSD of Retention Time.55.6.5.1 Format:.1 mm O/TFA (99.9 :.1 v/v) O/TFA (9: 9.9 :.1 v/v/v). 85 15.5.7.8 85 15 5. 85 15 Flow Rate:.6 ml/min Temp.: 8 ºC Detection: UV (8 nm) 1. Ribonuclease A (.5 mg/ml). Cytochrome C (.5 mg/ml). Lysozyme (.5 mg/ml) 4. mab (1 mg/ml)
Consistent Manufacturing Each MAbPac RP column is manufactured according to stringent specifications to ensure column-to-column reproducibility. Each column is shipped with a test chromatogram. Physical Data Chemistry Substrate Phenyl Particle size 4 µm Pore size Column housing Spherical polymer particles 1, Å Stainless steel Product Specifications Operational Specifications Column Column ID (mm) Flow Rate (ml/min) Pressure Limit (psi) Temperature ( C) ph Range MAbPac RP..4 1. 4, < 11 14 MAbPac RP.1..6 4, < 11 14 Ordering Information Description Particle Size Part Number MAbPac RP, Analytical,. mm 4 µm 88644 MAbPac RP, Analytical,. mm 4 µm 88645 MAbPac RP, Guard,. 1 mm 4 µm 88646 MAbPac RP, Analytical,.1 mm 4 µm 88647 MAbPac RP, Analytical,.1 mm 4 µm 88648 MAbPac RP, Guard,.1 1 mm 4 µm 88649 thermofisher.com/biolc 16 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific and its subsidiaries. Specifications, terms and pricing are subject to change. Not all products are available in all countries. Please consult your local sales representative for details. USA and Canada +1 8 1 Australia 1 75 9 (free call domestic) China 8 81 5118 (free call domestic) 4 6 5118 France + ()1 6 9 48 4 Germany +49 () 4 941 or 1 India +91 674 9494 +91 7 1766 5 PS1199-EN 616S Japan 1 75 67 (free call domestic) 1 75 671 (fax) Korea +8 4 86 United Kingdom +44 () 198 54 11 New Zealand 8 9 966 (free call domestic) Singapore +65 689 119 All Other Enquiries +44 () 198 54 Technical Support For advice and support, please visit our website: www.thermoscientific.com/chromexpert