Agilent Protein analysis by aqueous size exclusion chromatography
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1. ment between molecular weight Mp 149000 calculated by SEC and the reference value M 157000 from the Agilent 2100 bio analyzer The difference of 5 is mainly due to the fact that SEC separates under native conditions while the Agilent 2100 bioanalyzer uses the denaturing sodiumdode cylsulfate SDS Antibody High MW Impurity 0 2 Area 347 719 Time min Figure 5 Sensitive fluorescence detection of an human antibody and an impurity Columns TSK SW 3000xl in series with TSK SW 4000xI Eluent A 0 1 M Na sulfate 0 1 mM Na phosphate pH 6 7 Flow rate 0 8 ml min Column compartment temperature 22 C Injection volume 100 pl Proteins were dissolved in the mobile phase concentration 0 1 Sample preparation 2 Impurity testing and molecu lar weight characterization of Erythropoietin EPO Erythropoietin EPO is the main factor responsible for regulating red blood cell production during steady state conditions and accel erating recovery of red blood cell mass following hemorrhage EPO synthesis in adult organisms takes place in the kidney It is a 165 amino acid glycoprotein with a molecular weight of approximate ly 30000 to 35000 Dalton EPO can nowadays be produced by a recombinant process and is used to treat anemia associated Absorbance mAU 1400 ei M 55400 ref SEC 55000 1000 D 1 007 800 with chronic renal failure for patients on dialysis It has become wide2. Calibration SEC calibration of the chromato graphic system is performed by injecting macromolecule stan dards with known molecular weight and then connecting the data points The graph shows the retention time elution volume on the x axis and the logarithm of standard s molecular weight on the y axis Commercially avail able proteins with the molecular weights as specified by the suppli er were used Depending on pro tein type of column buffer and buffer concentration the hydrody namic volume changes and there fore a mixed mode separation mechanism for example SEC mixed with IEC ion exchange chromatography occurs To ensure a widely SEC based sepa ration it is important to carefully determine correct conditions for each standard and sample protein The proteins listed in table 1 ful filled the criterion of a widely size based separation in the various mobile phases The peptides Angiotensin II molecular weight MW 1046 Da and Leucin Enke phalin MW 555 Da could not be used because they eluted with all three eluents with or even behind the amino acid tyrosine MW 180 This means they did not elute at all in the SEC mechanism To determine the best calibration curve it is extremely useful that the Agilent ChemStation GPC SEC data analysis software offers a wide variety of curve fits such as linear 2nd to 7th fits and three special ones PSS 3 PSS 5 and PSS 7 The best curve fit for the data poin
3. Technologies GmbH Waldbronn Germany The author would like to thank Dr Rudolf Grimm Hexal Biotech Research GmbH Munich Germany for providing the EPO sample and for helpful discussions Caliper LabChip and the LabChip logo are US registered trademarks of Caliper Technologies Corp www agilent com chem Copyright 2001 Agilent Technologies All Rights Reserved Reproduction adaptation or translation without prior written permission is prohibited except as allowed under the copyright laws Published September 1 2001 Publication Number 5988 4266EN 7 Agilent Technologies
4. of a human plasma sample using a DAD and FLD within one run Elu ent A and one TSK SW3000x col umn were in series with one TSK SW 4000 column Due to the many proteins which are present in plasma a full separation of all compounds was not expected Despite the limited resolution the largest peak could be easily identi Absorbance mAU 300 250 200 Immumoglobuline Mp 717000 fied as human albumin by molecu lar weight comparison The molec ular weight of 65500 Dalton deter mined by SEC agrees very well with the reference value of 66000 Dalton The first peak eluting is obviously an immunoglobuline with a molecular weight of 717000 Dalton To obtain a better separa tion especially in the area between the immunoglobuline and the albumin peak a further col umn for example a TSK SW 2000x1 column should be installed __ Albumin Mp 65500 100 FLD 50 DAD 0 0 5 10 15 20 25 30 35 Time min Figure 9 Protein identification in human plasma by molecular weight Protein Reference MW SEC MW Mp Delta Myoglobin 17000 PAGE 17100 Eluent A 0 5 Ovalbumin 43000 PAGE 42700 Eluent A 0 7 Ovalbumin dimer 86000 PAGE 84400 Eluent A 0 7 Human albumin in plasma 66000 PAGE 65000 Eluent A 1 5 Antibody 157000 2100 bioanalyzer 149000 Eluent B 5 0 Ferritin 460000 PAGE 459600 Eluent A 0 1 EPO approx 55000 SEC 54000 Eluent B 1 8 approx 30000 MALDI 80 Table 2 Comparison of molecul
5. pure commercial beta lac toglobulin including the identity confirmation by the molecular weight Peak purity was confirmed by e Visual inspection of the peak shape e A polydispersity D of 1 007 which is significantly smaller than 1 01 e The overlay of the peak spectra acquired at inflection points and apex The molecular weight Mp 35600 is in very good agreement with the reference value M 35000 from gel elec trophoresis Columns TSK SW 2000xI in series with TSK SW 3000xl Eluent C 0 15 M NaCl 20mM Na phosphate 0 01 Tween 20 pH 6 8 Flow rate 0 8 ml min Column compartment temperature 42 C Injection volume 100 ul Proteins were dissolved in the mobile phase concentration 0 1 Sample preparation Detector response 400 300 Ovalbumin 200 1007 Dimer of 0 Ovalbumin T T 7 T T i T 0 5 10 15 20 25 30 Elution volume ml dad1B dad1B Mn 4 2810e4 8 7127e4 g mol Mw 4 3249e4 8 9261e4 g mol Mz 4 3712e4 9 1589e4 g mol Mv 0 000000 0 000000 g mo D 1 0103e0 1 0245e0 n 0 000000 0 000000 ml g Vp 1 9340e1 1 7761e1 ml Mp 4 272664 8 4358e4 g mol Mp 42726 OVA and A 2 5864e2 25751e1 mi V 84358 dimer ae Aozet BOSTA fal neterence values h 0925e 0457e g mol x 50 4 2849e4 8 6867e4 g mol M 43000 OVA and 86000 dimer 70 4 4925e4 9 4617e4 g mol 90 4 8602e4 1 0943e5 g mo Figure 3 SEC report for ovalbumin and ovalbumin dimer Absorb
6. Protein analysis by aqueous size exclusion chromatography Application Heinz Goetz Abstract Aqueous size exclusion chromatography SEC often referred to as gel filtration chromatography GFC is a entropically controlled separa tion technique in which molecules are separated based on hydrody namic volume or size In protein analysis the technique is used for dif ferent applications such as e impurity testing e following progress of a reaction e separation of reaction components and products antibodies fragments and conjugates folding studies e purification and e desalting and exchange of sample buffer In all these application areas except for the last one the protein mole cular mass is typically determined in the same analysis To obtain this important parameter a proper column calibration with well character ized macromolecules or molecular mass sensitive detectors such as mass spectrometry or light scattering are used This Application Note describes results in protein impurity testing and simultaneous molecu lar mass characterization with the Agilent 1100 Series GPC SEC analy sis system RE Agilent Technologies Introduction Using recombinant DNA tech niques to obtain proteins through high level expression in bacterial cells rather than extraction and purification techniques from other biological resources has become very popular lately and has brought forth a growing number of protei
7. ance mAU 24 389 pure beta lactoglobulin 804 Molecular weight data Mn 33493 60 Mw 33727 Polydisp 1 007 Mp 35600 ref 35000 0 5 10 15 20 25 30 35 Time min Figure 4 Protein impurity testing and identity confirmation of a beta lactoglobulin sample Protein analysis of research samples 1 Impurity testing and molecu lar weight characterization of a human antibody UV detection at 214 or 278 nm is most commonly used for protein analysis Fluorescence detection is also of interest due to improved selectivity and sensitivity Protein fluorescence derives from the aro matic amino acids tryptophan and tyrosine which are present in almost every protein The UV detector used should be a diode array detector DAD because it acquires several wavelengths and spectra at the same time Figure 5 shows the SEC analysis of a human antibody sample with fluo rescence detection at an excita tion wavelength of 220 nm and an Luminescence LU 300 280 260 240 4 220 200 4 1804 0 5 10 emission wavelength of 350 nm The large antibody peak and a small impurity of only 0 2 can be seen The impurity could not be detected with the DAD at 214 nm due to the small concentration A typical excitation and emission fluorescence protein spectrum obtained with the Agilent 1100 Series fluorescence detector FLD without stopping the flow is shown in figure 8 In addition there was good agree
8. ar weight data obtained by aqueous SEC and reference methods Table 2 shows a comparison of the molecular weight results obtained by aqueous SEC and the reference methods The difference between the methods is typically smaller than 5 in most cases even smaller than 2 The larger differ ence between the MALDI MS and the SEC value for EPO is due to the 40 glycolization of the sam ple which makes it larger and very hydrophilic In SEC this results in smaller elution volumes but does not effect the MALDI experiment There was a good agreement only 1 8 difference between the EPO molecular weight determined in our lab and the reference value determined in the supplier s lab using SEC Columns TSK SW 3000xl in series with TSK SW 4000x Eluent A 0 1 M Na sulfatel 0 1 mM Na phosphate pH 6 7 Flow rate 0 8 ml min Column compartment temperature 22 C Injection volume 100 pl Proteins were dissolved in the mobile phase concentration 0 1 Sample preparation Conclusions Aqueous SEC is an important technique for protein analysis In a single run quantitative infor mation on the degree of dimeriza tion oligomerization lower molec ular weight impurities and the molecular weight is obtained Molecular weight accuracy and precision are typically better than 2 Information on purity is obtained not only by the chromatographic signals of the UV diode array detector and the fluorescence detector but also b
9. denatured during the analysis and cannot be collected Excellent mass accuracy of 0 1 is obtained by mass spectroscopy for example by MALDI MS Matrix Assisted Laser Desorption Ionization MS however it cannot distinguish between the monomer and oligomers and is limited to molecular masses up to approxi mately 120000 Dalton This tech nique also requires a significant financial investment Experimental Chromatography was performed on an Agilent 1100 Series GPC SEC analysis system equipped with a micro vacuum degasser an isocratic pump with seal wash option a thermostatted autosam pler a thermostatted column com partment a diode array detector and a fluorescence detector The data was transferred to an HPLC 3D ChemStation with GPC SEC data analysis software The columns were purchased from Agilent Technologies GmbH Germany These included TSK SW 2000x1 7 8 x 300 mm 5 pm Agi lent part number 79912S2 597 TSK SW 3000xl 7 8 x 300 mm 5 pm Agilent part number 79912S3 597 and TSK 4000 SW 7 5 x 300 mm 10 pm Agilent part number 79912S4 197 All com mercially available proteins of purest analytical grade were pur chased from Sigma Munich Ger many The following mobile phas es were prepared Eluent A 0 1 M Na Sulfate 0 1 M Na phosphate pH 6 7 Eluent B 0 40 M NaCl 10 mM Na phosphate pH 7 4 Eluent C 0 15 M NaCl 20 mM Na phosphate 0 01 Tween 20 pH 6 8 Results and Discussion
10. ection volume 100 pl Molar Mass D Proteins were dissolved in the mobile phase concentration 0 1 Sample preparation T 13 5 14 0 14 5 150 15 5 160 1 17 0 175 18 0 18 5 19 0 19 5 Elution Volume ml Volume Molar Mass Stat Weight 13 5227 432000 00 15 1653 66000 00 1 00 BSA dimer 1 00 BSA Slope Deviation 0 2440 0 1094 01380 0 4901 1 00 OVA 1 00 Cytochronec bration curve aorst 0 6707 Bees Deviation between cali bration point and curve Figure 1 Typical calibration curve and table obtained with commercially available proteins Impurity testing and molecular weight characterization of commercially available pro teins Several commercially available proteins were analyzed by aqueous SEC for impurities and molecular weight The advantage of aqueous SEC is that both types of informa tion are obtained with good preci sion and accuracy from a single fully automated analysis Automa tion here includes the complete process from sample injection to chromatographic analysis impuri ty identification and quantification protein molecular weight calcula tion and even printing of the user specified reports Figure 2 shows the SEC chromatogram of an oval bumin sample with the software calculated area data The impuri ty is the ovalbumin dimer as iden tified easily by the molecular weight Figure 3 shows the user Abso
11. ly known in the context of misuse by athletes for blood dop ing in endurance sports Figure 6 shows the overlay of a simultaneous diode array and fluo rescence detector analysis of an EPO sample For medical use it is mandatory that the sample is high ly pure that it does not form aggregates and that the molecular weight is correct Therefore by using two different detectors additional confidence is gained Tyr ISTD 0 5 10 20 25 30 Time min Figure 6 Impurity testing of erythropoietin EPO with UV diode array and fluorescence detection Columns Eluent B Flow rate Column compartment temperature Injection volume Sample preparation TSK SW 2000xl in series with TSK SW 3000x 0 40 M NaCl 10 mM Na phosphate pH 7 4 0 8 ml min 42 C 100 pl Proteins were dissolved in the mobile phase concentration 0 1 Besides looking at the chromato graphic signals purity was also demonstrated by the overlay of the UV spectra acquired at inflec tion points and apex of the EPO peak figure 7 and polydispersity D 1 007 Figure 8 shows the excitation and emission spectra Norm 150 100 220 230 240 250 260 acquired with the Agilent 1100 Series FLD The molecular weight Mp 55400 agreed very well with the reference value M of 55000 which was determined in the sup plier s laboratory also using SEC It should be noted that 55000 is not the true molecular weight of 278 n
12. m 270 280 290 300 310 Wavelength nm Figure 7 Overlay of UV spectra acquired at inflection points and apex of EPO peak which represents a fur ther indication of peak purity Luminescence LU 400 200 Excitation spectrum T T 240 260 T T 280 300 340 nm Emissiom spectrum T T T 300 350 400 T T T 450 500 550 Wavelength nm Figure 8 Excitation and emission spectra of EPO this EPO sample Since it is gly cosilated by 40 and therefore larger and very hydrophilic it elutes earlier than an EPO without glycolization With MALDI MS a molecular weight of 30000 Dalton was determined The disadvantage of MS based techniques in protein analysis is that aggregates cannot be distinguished from the monomeric protein Columns TSK SW 2000xl in series with TSK SW 3000x Eluent B 0 40 M NaCl 10 mM Na phosphate pH 7 4 Flow rate 0 8 ml min Column compartment temperature 42 C Injection volume 100 pl Proteins were dissolved in the mobile phase concentration 0 1 Sample preparation Columns TSK SW 2000xl in series with TSK SW 3000x Eluent B 0 40 M NaCl 10 mM Na phosphate pH 7 4 Flow rate 0 8 ml min Column compartment temperature 42 C Injection volume 100 pl Proteins were dissolved in the mobile phase concentration 0 1 Sample preparation 3 Protein identification and molecular weight characteri zation of proteins in human plasma Figure 9 shows the analysis
13. ns of interest SEC is a mainstay technique for the analysis of proteins due to its excellent properties for impurity testing quantification molecular weight characterization and automation The technique can handle a very wide range of mole cular weights from several hun dred to about 10 million Dalton A 50 100 difference in the molec ular weight is required With the Agilent 1100 Series GPC SEC analysis system the area amount report for example on impurities is obtained at the same time as the molecular weight report Molecu lar weight accuracy and precision are typically better than 2 Another advantage is that the pro tein is not denatured and can easi ly be collected for further investi gations Although protein analysis tech nologies are developing fast many still rely on traditional methods such as sodium dodecylsulfate polyacrylamide gel electrophore sis SDS PAGE which include a number of laborious time con suming manual steps It requires only a 10 difference in molecu lar weight but offers limited repeatability in quantification Another technique is offered by the Agilent 2100 bioanalyzer with the Protein 200 LabChip which is a compact electrophoresis based system for rapid and auto mated analysis of proteins on a chip Here also only a 10 differ ence in molecular weight is neces sary The molecular weights can range from approximately 14000 to 200000 Dalton The protein is
14. rbance mAU 400 300 200 100 8 9 dimer Ng configured molecular weight report for the sample The molecu lar weight data of ovalbumin monomer and dimer match agree well with the reference molecular weights figure 3 and table 1 Besides the molecular weight Mp derived from the peak apex the software also calculates the poly dispersity D and other molecular weight data which are derived from synthetic polymer characteri zation The polydispersity value D is also of interest in protein analy sis A D value smaller than 1 01 typically indicates peak purity Of course this requires a minimum separation of the compounds often not visible from the peak shape In the example here the D value for the monomer is 1 0145 for the dimer it is 1 02 Polydisper sity D is calculated by dividing the weight average molecular weight Mw by the number average molec ular weight Mp 24 178 91 1 Ovalbumin 22 201 Figure 2 20 25 30 35 Time min SEC chromatogram of an ovalbumin sample with the software calculated area values for oval bumin and an impurity ovalbumin dimer Columns Eluent A Flow rate Column compartment temperature Injection volume Sample preparation TSK SW 3000xI in series with TSK SW 4000xI 0 1 M sulfate 0 1 mM Na phosphate pH 6 7 0 8 ml min 22 C 100 pl Proteins were dissolved in the mobile phase concentration 0 1 Figure 4 shows the analysis of a 100
15. ts was determined by the following criteria meaningful theory Protein amino acid Molecular weight Da Remark Dimer of bovine serum albumin 132000 Present in serum albumin Dimer of ovalbumin 86000 Present in ovalbumin Bovine serum albumin 66000 Ovalbumin 43000 Carbonic anhydrase 29000 Cytochrome C 12400 Strongly tailing with eluent C Lysozyme 14300 Tyrosine 180 Table 1 Compounds used to calibrate the Agilent 1100 Series GPC SEC analysis system e Small deviation between each measured data point and curve Deciding on the the curve fit simply based on an average parameter such as the chi square value or the coeffcient of regression is not recom mended Such a parameter is only an average value for all data points and does not pro Figure 1 shows the calibration curve and table used for charac terizing the erythropoietin All important information such as calibration file name selected curve fit and calibration point data including their deviation from the curve itself is shown Such clearly presented information makes the correct decision on curve fit easy e Curve shape must be physically vide detailed information at a and fast certain elution volume e Curve slope must be negative for all points as predicted in wa aoa Ce File name Columns ee AA te wi x Eluent B 0 40 M NaCl 10mM Na phosphate pH 7 4 Flow rate 0 8 ml min Column compartment temperature 42 C Inj
16. y the polydis persity value calculated by the software and by overlaying spec tra Low concentration impurities can be quantitatively determined in the presence of the main com pound The molecular weight cali bration of the Agilent 1100 Series GPC SEC analysis system is per formed with commercially avail able well characterized proteins Since proteins are used for cali bration absolute detection such as cumbersome light scattering or viscosimetry is not needed The technique requires a minimum molecular weight difference of about 50 100 The molecular weight range is very large ranging from several hundert to about 10 million Dalton SEC is also very flexible The column and the mobile phases can be optimized to ensure a size based separation for a wide range of proteins The purified proteins mostly separated under native conditions can be easily fraction ated for further investigations for example for sequencing References 1 Polymer analysis by GPC SEC Agilent Technologies Technical Note 5988 0110 2000 2 Mikes O High performance liq uid chromatography of biopoly mers and biooligomers Part B Elsevier Science Publisher B V Amsterdam Netherlands 1988 3 Kopaciewicic W Regnier FE Nonideal size exclusion chro matography of proteins effects of pH at low ionic strength Anal Biochem 126 8 16 1982 Heinz Goetz is an application chemist at Agilent
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Agilent Protein analysis by aqueous size exclusion chromatography