Protein Quantitation using various Modes of High Performance Liquid Chromatography

Transcription

1 Platzhalter für Bild, Bild auf Titelfolie hinter das Logo einsetzen Protein Quantitation using various Modes of High Performance Liquid Chromatography Grotefend, S.; Wroblewitz, S.; Kaminski, L.; Limberger, M.; Watt, S.; El Deeb, S.; Wätzig, H. Innsbruck, Tuesday, September 20 th

2 Quantitative Protein Analysis Gel Electrophoresis Capillary Electrophoresis Protein Quantitation Mass Spectrometry High Performance Liquid Chromatography 20th September 2011 l HPLC for Protein Analysis l Page 2

3 Quantitative Protein Analysis by HPLC Differences, similarities and evaluation SEC SAX RP WCX ProPac WCX-10 (250 x 4 mm, Dionex) Uptisphere-C4 300 Å, 5 µm (150 x 4.6 mm, Interchim) ProSwift SAX-1S (50 x 4.6 mm, Dionex) BioSep S-3000, 5 µm (300 x 7.8 mm, Phenomenex) Proteema 300 Å, 5 μm ( 300 x 8 mm, PSS) 20th September 2011 l HPLC for Protein Analysis l Page 3

4 Ideal Size Exclusion Chromatography (1) Ovalbumin (2) Myoglobin (1) (2) PSS Proteema SEC column Eluent: Phosphate buffer ph 6.6 with 0.3 M NaCl Flow rate: 1 ml/min Detection: 214 nm Injection volume: 60 µl 20th September 2011 l HPLC for Protein Analysis l Page 4

5 Non-Ideal Size Exclusion Chromatography (1) Ovalbumin (2) Myoglobin Concentration each 0.4 mg/ml (1) Repeatability area (RSD% n=20) Day-to-day (2) precision area (RSD% n=60) PSS Proteema SEC column Eluent: 17 mm phosphate buffer ph 6.35 Flow rate: 1 ml/min Detection: 214 nm Injection volume: 20 µl LOQ Ovalbumin < 0.4 µg/ml Myoglobin < 0.4 µg/ml Concept according to: Kopaciewicz, W. and Regnier F.E., Anal. Biochem.;126 (1982) th September 2011 l HPLC for Protein Analysis l Page 5

6 Non-Ideal SEC - Phenomenex (1) Ovalbumin (2) Myoglobin PSS Proteema SEC (2) Phenomenex BioSep 3000 Eluent: 17 mm phosphate buffer ph 6.35 Flow rate: 1 ml/min Detection: 214 nm Injection volume: 40 µl (1) 20th September 2011 l HPLC for Protein Analysis l Page 6

7 SEC of a monoclonal antibody (1) Dimer (2) Monomer (3) Fragment Peak (2) Phenomenex BioSep 3000 Eluent: 100 mm phosphate buffer ph 6.80 Flow rate: 1 ml/min Day-to-day precision t R Detection: Repeatability 214 nm Day-to-day Injection area pooled volume: 10 precision µl area (RSD % n=60) (RSD % n=20) (RSD % n=60) (1) Dimer (2) Monomer (3) Fragment (3) all area (1) Method according to: Phenomenex BioSep Product manual, application: IgG aggregates 20th September 2011 l HPLC for Protein Analysis l Page 7

8 Routine analysis SEC data of mab Phast GmbH Sample all Area A&M Stab Sample Area Monomer Area Dimer RSD % pooled RSD % pooled area RSD % area area per per day between days sample (diff. samples) Phast GmbH (n=2) (n=4) 1.86 (n=4) A&M Stab (n=3/5/6) (n=9/15/18) (n=4) Monomer Dimer th September 2011 l HPLC for Protein Analysis l Page 8

9 Strong Anion Exchange Chromatography (1) Dionex ProSwift SAX-1S RSD % (area) = 2.31%; without Vial 1 = Eluents: 0.784% A) 20 mm Tris-HCl buffer ph 8.6 RSD % (t R ) = 1.03% B) Eluent A + 1 M NaCl Gradient: 98% - 50% A in 5 min Flow rate: 2 ml/min RSD % (area) = 1.90% Detection: 214 RSD nm % (t R ) = 1.98% Injection volume: 60 µl (1) Myoglobin (2) Ovalbumin (3) BSA Area Myoglobin Ovalbumin BSA (2) (3) RSD % (area) = 3.21% RSD % (t R ) = 1.98% Injection-Blank Ovalbumin Injection BSA Blank Myoglobin LOQ < 15 µg/ml Runs Method according to: Dionex Corp., Product manual for ProSwift monolithic columns (2008) 44-45, modified 20th September 2011 l HPLC for Protein Analysis l Page 9

10 RP-HPLC (1) Ovalbumin (2) BSA (3) Myoglobin Repeatability area pooled (RSD% n=20) (2) (3) Day-to-day precision area (RSD% n=60) LOQ BSA Detection: 214 <15 nm µg/ml Ovalbumin Ovalbumin (1) 5.51 Myoglobin Myoglobin Interchim Uptisphere C4 Eluents: A) 5% ACN : 95% water + 0.1% TFA B) 95% ACN : 5% water + 0.1% TFA Gradient: % B in 26 min Flow rate: 0.5 ml/min Injection volume: 50 µl (3) 15 µg/ml (1) ~ 10 µg/ml Modified according to: Bradshaw, T.P., Phenomenex; Introduction to Protein and Peptide HPLC (1998) th September 2011 l HPLC for Protein Analysis l Page 10

11 Routine analysis - external RP data (Phast GmbH) Detection: DAD Sample Area Method/ RSD % pooled Detection area per sample Detection: FLD Sample Area 1 Area RSD % pooled RSD % area area per day between (dif. samples) days DAD 1.69 (n=3) 1.47 (n=9) (n=6) FLD (n=5/2) (n=9) (n=7) Peak Peak th September 2011 l HPLC for Protein Analysis l Page 11

12 Weak Cation Exchange Chromatography (1) (1) Ovalbumin (2) Cytochrome C (3) Lysozyme Repeatability area pooled (RSD% n=20) Dionex ProPack WCX-10 Dionex Eluents: ProPack A) 17 mm WCX-10 phosphate buffer ph 6.0 Eluents: A) B) 17 Eluent mm phosphate A + 1 M NaCl buffer ph 6.0 Gradient: B) Eluent 98-68% A + A 1 in M 14 NaCl min Gradient: Flow rate: % ml/min A in 14 min Flowrate: Detection: ml/min nm Detection: Injection 214 volume: nm 25 LOQ µl Injection volume: 10 µl Day-to-day precision area (RSD% n=60) (3) Ovalbumin (1) <10 µg/ml (1) Ovalbumin Cytochrome C <10 µg/ml (2) (2) Myoglobin Lysozyme <10 µg/ml (2) Method according to: Dionex Corp., Product manual for ProPac IEX columns (2007) th September 2011 l HPLC for Protein Analysis l Page 12

13 Quantitative Protein Analysis by HPLC: Similarities 18 Equipment RSD% Scattered light, electronic noise Typical total noise: - statical: < 5x Detection: 8 - UV DAD - dynamical: < 1x10-3 Sample pretreatment: - weighted sample 2 0 Concentration dependence - filtration - ultrasonic bath 0,0% 0,1% 1,0% 10,0% 100,0% content, log scale Repeatability System precision Udo Schepers, Joachim Ermer, Lutz Preu, Hermann Wätzig J. Chromatogr. B, 810, (2004) 20th September 2011 l HPLC for Protein Analysis l Page 13

14 Quantitative Protein Analysis by HPLC: Differences Separation mechanisms, selectivity Chemical noise S/N ratio Baseline characteristics SAX WCX Peak shapes SEC Myoglobin, Ovalbumin SAX Myoglobin, Ovalbumin, BSA SAX Blank CE separation Ovalbumin Redweik, S. RP Myoglobin, Ovalbumin, BSA RP Blank RP WCX Blank WCX Ovalbumin, Cytochrom C, Lysozym 20th September 2011 l HPLC for Protein Analysis l Page 14

15 Overall Conclusions Peak capacity [peaks/10 min] Selectivity Precision Analysis time LOQ [µg/ml] Sample/ Mobile Repeatability area Day-to-day [min] precision area phase RSD best RSD I % worst (n=20) % (n=60) preparation SEC Oval Myo BSA Oval Myo BSA PSS < Phe SEC ø ø SAX RP (+20) 1.59 < WCX SAX (+18) 1.41 < RP CIEF 16.7 approx approx. +(+) 2.0 ø (+22) 5.63 < 15 ø ++ SEC Oval Cyto Lyso Oval Cyto not yet (+) 12 mab measured Lyso +++ WCX Dimer Monomer Fragment all area Dimer Monomer Fragment all area RSD % area typically < 2% without individual optimization mab CE data: Suratman, A., Wätzig, H.; J. Sep. Sci., 2008, 31, and unpublished 20th September 2011 l HPLC for Protein Analysis l Page 15

16 Further possible improvements Method development for analysis of monoclonal antibodies with WCX, RP-4 and SAX Strategies for individual method optimization Analysis of Variance with data from protein analysis Role of sample pretreatment 20th September 2011 l HPLC for Protein Analysis l Page 16

17 Acknowledgements, Montlucon (Fra), Homburg (Ger) Markus Limberger, Andreas Ziegler, Mainz (Ger) Steven Watt 20th September 2011 l HPLC for Protein Analysis l Page 17

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19 Chromatographic conditions mobile Phase Eluent A SEC non ideal SAX WCX RP phosphate buffer tris-hcl buffer phosphate buffer 5% ACN : 95% water + 0.1% TFA Eluent B Ø Eluent A + 1 M NaCl gradient Ø linear 98% - 50% A Eluent A + 1 M NaCl linear 98%-68% A ionic strength mm 20 mm mm Ø ph-value ~ % ACN : 5% water +0.1% TFA linear 15%-100% B flow rate 1 ml/min 2 ml/min 1 ml/min 0.5 ml/min analysis time 16 min 5 min 17 min 26 min injection volume 40 µl 60 µl 25 µl 50 µl detection wavelength 214 nm 214 nm 214 nm 214 nm 20th September 2011 l HPLC for Protein Analysis l Page 19

20 Chromatographic conditions - Matuzumab mobile Phase Eluent A SEC phosphate buffer RP 5% ACN : 95% water + 0.1% TFA Eluent B Ø 95% ACN : 5% water +0.1% TFA gradient Ø isocratic 50% A : 50% B ionic strength 100 mm Ø ph-value 6.80 ~ 1.9 flow rate 1 ml/min 0.5 ml/min analysis time 15 min 12 min sample content 2.5 mg/ml 3.3 mg/ml injection volume 10 µl 50 µl detection wavelength 214 nm 214 nm 20th September 2011 l HPLC for Protein Analysis l Page 20

21 Protein properties Proteins MW [Da] pi Myoglobin 17, Ovalbumin 45, BSA 66, Cytochrome C 13, Lysozyme 14, Dumetz, A.C. et al, Protein Scie.; 16 (2007) Gochman-Hecht, H., Bianco-Peled, H.; J. Coll. Interf. Scie. 297 (2006) th September 2011 l HPLC for Protein Analysis l Pagee 21

22 HPLC systems SAX Hitachi Merck SEC/RP/WCX Elite LaChrom (VWR) Solvent pump L-6200A L-2130 Autosampler AS-2000A AS 2203 Detector (UV-Vis) L-4250 L-2400 Interface D-6000 organizer Software D7000 HSM (Merck) EZ Chrom Elite Vers th September 2011 l HPLC for Protein Analysis l Page 22

23 Size determination of Matuzumab Phenomenex BioSep Product manual; calibration curve 20th September 2011 l HPLC for Protein Analysis l Page 23

24 Control charts RP peak shapes 20th September 2011 l HPLC for Protein Analysis l Page 24

25 RP-Separation of Matuzumab (EMD 72000) + DTT Interchim Uptisphere C4 Eluents: A) 5% ACN : 95% water + 0.1% TFA B) 95% ACN : 5% water + 0.1% TFA isocratic: 50:50 Flow rate: 0.5 ml/min Detection: 280 nm Injection volume: 50 µl 20th September 2011 l HPLC for Protein Analysis l Page 25

26 SEC of an IgG antibody + DTT (1) MW ~ 740 kda Phenomenex BioSep 3000 Eluent: 100 mm phosphate buffer ph 6.80 Flow rate: 1 ml/min Detection: 214 nm Peak RSD % tinjection R (n=18) volume: RSD 10 µl % area (n=18) (1) (2) (3) (2) (3) MW < 1 kda MW < 5 kda 20th September 2011 l HPLC for Protein Analysis l Page 26