Reproducible LC/MS Peptide Separation Using Agilent AdvanceBio Peptide Plus Columns

Transcription

1 Reproducible LC/MS Peptide Separation Using Agilent AdvanceBio Peptide Plus Columns Application Note Biotherapeutics and Biosimilars Author Suresh Babu C.V. Agilent Technologies Inc. Introduction Peptide separation is of paramount importance during the characterization of biopharmaceuticals. Peptide mapping serves as a primary quality control (QC) step in pharmaceutical development. It involves enzymatic digestion to produce peptide fragments, followed by reversed-phase separation and identification by mass spectrometry. Due to the inherent complexity of proteolytic digests of protein mixtures, high-efficiency and high-resolution peptide separation is very important. The quality attributes of peptide mapping such as retention time and peak area/height are critical for reliable peptide maps. Hence, rigorous characterization of peptide fragments requires a robust and reproducible LC/MS method for confident evaluation of the results. This study demonstrates high-efficiency peptide separation using the MS compatible Agilent AdvanceBio Peptide Plus column with the mobile phase containing formic acid modifier. Different sample types were screened to evaluate the column performance for peptide separation. The results showed excellent retention time, peak area, peak height, and full width half maximum (FWHM) reproducibility.

2 Materials and method Samples HSA Peptides Standard (G-8) Peptide Standard (9-8) mab tryptic digest LC system Agilent 9 Infinity II LC MS system Agilent 6XT AdvanceBio LC/Q-TOF Results and Discussion Figures A and B show LC/MS separations for two different peptide standard mixtures. The selected peptide sequences are composed of varying amino acid residues, length, and mass, and, thus, are useful to evaluate column performance. The results show the highly efficient, baseline separation of the peptide standards. The column provided excellent TIC peak shapes with <.6 minutes FWHM. The narrow TIC peak width was obtained using formic acid as a mobile phase modifier. The RSD values demonstrate the robustness of the LC/MS method. Instrument conditions Parameter Column Mobile phase Value Column temperature C Gradient Flow rate Ion mode Agilent AdvanceBio Peptide Plus. mm,.7 µm, Å (679-9) A). % FA in water B). % FA in ACN %B in minutes. ml/min Drying gas temperature C Drying gas flow Positive ion mode, dual AJS ESI L/min Sheath gas temperature 7 C Sheath gas flow Nebulizer Capillary voltage Fragmentor voltage Skimmer voltage Oct RF Vpp L/min psi, V 7 V 6 V 7 V Data analysis Agilent BioConfirm software B.8

3 Counts A Peak ID. Bradykinin frag 7. Bradykinin. Angiotensin II (human). Neurotensin. Angiotensin I (human) 6. Renin substrate porcine 7. [Ace-F-,-H-] Angiotensinogen ( ) 8. Ser/Thr Protein Phosphatase ( ) 9. [F] Ser/Thr Protein Phosphatase ( ). Melittin (honey bee venom) Peak Peak Peak Peak Peak Peak Peak Peak Peak Peak Counts B 6 7 Peak ID. AAFTE[CAM][CAM]QAADK. YLYEIAR. LVNEVTEFAK. KVPQVSTPTLVEVSR. RP[CAM]FSALEVDETYVPK 6. AVMDDFAAFVEK 7. HPYFYAPELLFFAK Peak.... Peak.... Peak Peak...8. Peak.... Peak Peak Figure. Overlaid total ion chromatograms of peptide standard mixture (five replicate injections) using an Agilent AdvanceBio Peptide Plus column. A) Agilent Peptide Standard (p/n 9-8). B) Agilent HSA Peptides Standard (p/n G-8).

4 Figure shows the LC/MS peptide map separation of four different mabs. Various mab preparations were chosen to study the column performance as well as LC/MS method robustness. The result shows excellent peptide separation for different sets of peptide maps with improved peak shapes. There is no observable shift in the chromatographic profile between the replicate injections. The RSD values illustrate that the column and LC/MS method are robust. Counts Counts Counts Counts mab mab mab mab (ADC) Peak.7... Peak Peak Peak Peak Peak Peak Peak Peak Peak.6... Peak Peak Peak Peak Peak Peak Peak Peak Peak..9.. Peak Peak Peak Peak Peak Peak Peak Peak Peak Peak Peak Peak Peak Peak Peak Peak Peak Figure. Overlaid total ion chromatograms of mab tryptic peptide map (five replicate injections) using an Agilent AdvanceBio Peptide Plus column.

5 Conclusions This study demonstrates: High efficiency LC/MS peptide separation using formic acid as a mobile phase modifier with an Agilent AdvanceBio Peptide Plus column Excellent reproducibility of retention time, peak area, peak height, and peak width

6 For More Information These data represent typical results. For more information on our products and services, visit our Web site at For Research Use Only. Not for use in diagnostic procedures. Agilent shall not be liable for errors contained herein or for incidental or consequential damages in connection with the furnishing, performance, or use of this material. Information, descriptions, and specifications in this publication are subject to change without notice. Agilent Technologies, Inc., 7 Printed in the USA May, EN