1. Heinz Goetz World Wide Product Manager Liquid Chromatography
Protein Analysis by Aqueous Size Exclusion Chromatography (SEC) with the 1100 SEC System

2. SEC: What is it?
HPLC technique for analysis/characterization of macromolecules
SEC: - w/ organic eluents (Gel Permeation Chromatogr.- GPC) w/aqueous eluents (Gel Filtration Chromatogr. - GFC)
GPC: - typical eluents: tetrahydrofuran, toluene, dimethylacetamide … for synthetic macromolecules(plastics)
GFC: - eluent: water, buffered water biomacromolecules (proteins, poly saccharides, DNA, RNA, water-soluble synthetic polymers
Tasks: - MW determination, mono-,di, purity analysis
Selfexplanatory
Title of Presentation
1 March, 2001
Agilent Restricted

3. SEC: What is it? GPC (plastics analysis, organic eluents) SEC
GFC (biomolecule analysis,
aqueous eluents)
Size Exclusion Chromatography (SEC) is the modern name. As a modern company we should use this term. To differentiate SEC with aqueous eluents from synthetic polymer SEC we call it aqueous SEC.
Title of Presentation
1 March, 2001
Agilent Restricted

4. SEC Separation mechanism
Mobile
phase
Column
packing A B C A
Inject B B
Pores
For those of you not as familiar with the technique, SEC is based on size exclusion of the analytes (the polymer molecules) in a column filled with a porous packing. Based on this separation principle the larger polymer molecules are eluted first and the smaller ones take a long ride through the pores. Therefore they arrive later at the column end. This is a major difference compared to standard liquid chromatography. In order to calculate SEC results there is always the need to first of all do a calibration of your SEC column set (e.g. 1-6 columns) with reference standards of known molecular mass. The results will not depend on the peak heights or areas, but on retention times and peak shapes. C
Elution volume C
Polymer
Analytes
Title of Presentation
1 March, 2001
Agilent Restricted

5. Mechanism of SEC Separation
FLOW
The mechanism
Molecules that come right out RED(exclusion)
Molecules that are delayed to different extents Green(separation)
Molecules that are fully retained and not separated Yellow (void, total inclusion)
From the figure, it can be seen that a plot of logM vs. retention volume is linear for components
that selectively permeate the column packing pores.
From a calibration plot and the retention volume of the sample, its molecular weight or
molecular weight range can be determined.
Due to a special calibration procedure using a linear elution volume on the x-axis versus
a logarithmic molecular weight on the y-axis the demands on the instrumentation are
very stringent.
Small Change in elution volume ---->big change in molecular weight ---> excellent HPLC equipment required!
Title of Presentation
1 March, 2001
Agilent Restricted
000855P1.PPT

6. Steps in SEC Analysis Sample chromatogram10 20 30 8 12 14 16
Time (min.)
Sample chromatogram
Determination of separation parameters (eluent, column(s), column temp. etc.)
Analysis of narrow distributed calibration standards
Set-up of calibration curve
Analysis of sample
Calculation of molecular weight data and molecular weight distribution
Having selected an appropriate column and chromatographic conditions, narrow standards with known molecular weights are analyzed. The GPC-SEC software is then calculating the calibration curve based on the MW data and retention times of the used standards. If the standards are not the same polymer type than the sample a further calibration step can be performed by analyzing a polymer of the same type with known Mw and Mn data. In protein SEC commercially available, for the molecular weight well characterized proteins as bovine serum albumin and ovalbumin are available, for example from the Sigma company. Although proteins are used for the calibration, the calibrant and the sample proteins may have a different configuration in solution. This may result in a difference between the
molecular weight determined by SEC and for example by MS and sequencing (for a comparison refer to the table in figure 23).
Having finished the calibration the unknown polymer sample can be analyzed. The software then calculates the MW data and MW distribution curves and prints out the results.
Authoring Division Name File Name
Security Notice (if required)
Title of Presentation
1 March, 2001
Agilent Restricted

7. Column Selection in Protein SEC
Molecular Weight Range (based on globular proteins, eluent 0.3 M NaCl)
TSK SW2000xl (79911S3-597)
TSK SW3000xl (79912S3-597)
TSK SW (79913S3- 107)
The most important parameter for a proper SEC column is the pore size. The pore size should be such that it allows selective permeation of the compounds of interest. Ideally, the pore size range of the column should be such that the largest pore size is just larger than the largest sample molecule and the smallest pore size should be just smaller then the smallest sample molecule. Most frequently the pore size of a single column is insufficient to cover the full molecular weight range of the sample. In these cases a combination of several columns plumbed in series must be used. Typically a column or a column set which covers a wide molecular weight range is used. The table shows the pore size ranges of TSK SW columns in our cataloque, they are most widely used in protein sizing.
Agilent Technologies TSK SW columns for protein SEC
Authoring Division Name File Name
Security Notice (if required)
Title of Presentation
1 March, 2001
Agilent Restricted

8. SEC Applications with Proteins
Impurity testing *
Molecular weight characterization *
Expression and folding studies + ,
Separation of Reaction Components and Products, (esp., antibodies, fragments, and conjugates) +
Purification +
Desalting and Exchange of Sample Buffer
Most important application is impurity testing, for example testing of aggregation and degradation. Desalting and Exchange of sample buffer does not require molecular weight characterisation.
We recommend the system for customers in laboratories from basic research to manufacturing. You find them in pharmaceutical and biotech industries, in universities and medical research labs.
* SEC software (G2182AA) required, + recommended
Title of Presentation
1 March, 2001
Agilent Restricted

9. Life Science Applications with SEC and MW determination
Selfexplanatory
Title of Presentation
1 March, 2001
Agilent Restricted

10. Recommended System Isocratic pump w/ seal wash Vacuum degasser
Thermostatted autosampler
Thermostatted column compartment
VWD, MWD or DAD
FLD (for highest sensitivity)
ChemStation w/ GPC-SEC data analysis software(G2182AA)
Important features of the GPC-SEC data analysis software for protein chemists are:
- many calibration curve fits: linear, 2nd to 7th order, proprietory ones: PSS3, PSS5 and PSS7
- test of calibration curve fit by information on slope and deviation at each calibration point(numerically and graphically),
- user-configurable one-page report with all important acquisition, calibration and data analysis parameters
- molecular weight report with Mp (molecular weight at peak apex) and
polydispersity information. Polydispersity <1.01 is an indication of purity. Molecular weight averages Mn, Mw, Mz and Mv are not of interest for protein chemists since proteins have a disinct molecular weight and not a distribution of molecular weights as synthetic polymers.
- qualification procedures dedicated to SEC analysis: installation qualification, software validation and verification, SEC performance testing, OQ/PV, Start-up-kits
RID not required!
Title of Presentation
1 March, 2001
Agilent Restricted

11. Agilent 1100 Series SEC System
# 1 HPLC - worldwide
NEW! HP
1100 HPLC
Superior pump performance and thermostat stability for best retention time precision
Peltier-column thermostat with temperature range up to 80°C
Single valve injector for extended uptime, optional 4-40°C
Choice of VWD, MWD and DAD for sensitive UV detection
Sensitive fluorescence detector w/ “on the fly” spectra
Complete system solution for convenient operation
Hardware & software
Procedures for start up & system checkout
Columns
The 1100 Series SEC system is a complete solution to include the 1100 hardware modules and the ChemStation. Agilent offers columns, standards and services for SEC.
With respect to the hardware we offer an excellent isocratic pump that is appropriate for demanding SEC work. Together with the column thermostat it ensures the high retention time reproducibility required. Heat exchangers inside the column compartment are used to condition the solvent. The temperature can be raised to 80 oC. UV-detectors are most widely used in protein analysis at 214 and 280 nm. Single, multiple wavelength detection or UV/Vis spectra can offer selective information on certain compounds. Most proteins do also fluoresce due to the aromatic amino acids tryptophan and tyrosine. This yiels in most cases in increased sensitivity.
NOTE: Also the quarternary and binary pump can be used in isocratic mode and they offer additional flexibility for additional applications if desired.
Title of Presentation
1 March, 2001
Agilent Restricted

12. Conditions for Protein Analyses
Columns: TSK Gel SW 2000xl (p/n 79911S3-597) and TSK Gel SW 3000xl (p/n 79912S3-597) in series
Mobile phases A: 0.10 M Na-Sulfate, 0.1 M Na-phosphat, pH 6.7 (A) B: 0.40 M NaCl, 10mM Na-phosphat, pH 7.4 (B) C: 0.15 M NaCl, 20mM Na-phosphat, 0.01% Tween 20, pH 6.8 (C)
Detection: DAD (278, 20/360,40, 214, 4/360,40) FLD (220/350)
Flow: 0.8 ml/min
Sample conc. < 0.1 %
Column Temp.: 25 and 42 C Autosampler thermostat: 4 C
Calibration standards: Proteins, supplier Sigma Chemicals
Title of Presentation
1 March, 2001
Agilent Restricted

13. Optimized SEC separation of Proteins and Peptides
555
OVA 45 K
Lysozyme 14.3 K
Tyrosin 180
Leucin-Enkephalin
Cytochrome C 12.4 K
Carbonic Anhydrase 29 K
Insulin 5.8 K
Thyroglobulin 660K
Gamma-Globulin 150 K
BSA 66 K
The slide shows the separation of 9 proteins and the amino acid tyrosine with one TSK SW 2000xl and one TSK 3000xl in series - a molecular weight difference of a factor 1.5 (< 50000) or 2 (> 50000) is typically required for the separation. The peptide Leucin-Enkephalin, molecular weight 555 elutes after tyrosine, molecular weight 180, thus indicating a mixed SEC/IEC (IEC = Ion Exchange Chromatography) mechanism.
Eluent: 0.4 M NaCl, 10mM Na-phosphat, pH 7.4 Factor in molecular weight needed for separation
Title of Presentation
1 March, 2001
Agilent Restricted

14. Calibration Curve
Selected curve fit
Commercially available proteins with the molecular weights as specified by the supplier were used for the present publication.
To ensure a widely SEC based separation the correct conditions must be carefully determined for each standard and sample protein. For the determination of the best calibration curve it is extremely useful that the Agilent ChemStation SEC data analysis software offers a wide variety of curve fits: linear, 2nd to 7th fits and three special ones - PSS 3, PSS 5 and PSS 7.
The best curve fit for the data points was determined with the following, software displayed criteria: -.Curve shape must be physically meaningful
.Curve slope must be negative for all points (as predicted by theory)
.Small deviation between each measured data point and curve
A decision for the curve fit based just on an average parameter as for example the chi square value or the coefficient of regression is not recommended. Such a parameter is only an average value for all data points and not detailed information on the situation at a certain elution volume. Note: The least square fit chi square is calculated by the GPC-SEC data analysis software.
Deviation between data point and curve
Optimization of calibration standards and curve for each protein!!!
Title of Presentation
1 March, 2001
Agilent Restricted

15. Simultaneous Impurity Testing and Identiifcation: I. Impurity Testing
Impurity Determination: standard HPLC report
91.1 % Ovalbumin
The figure shows the SEC chromatogram of an ovalbumin sample with the software calculated area-%-data. The impurity is the ovalbumin dimer as identified easily by the molecular weight. The next Figure is the user configured molecular weight report for the sample. The software calculated molecular weight data of ovalbumin monomer and –dimer agree very well with the reference molecular weights (refer to figure 23). The software calculates besides the molecular weight Mp derived from the peak apex the polydispersity D and other molecular weight data which are derived from synthetic polymer characterization. Of interest in protein analysis is besides the molecular weight Mp the polydispersity value D. 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 present example the D value is with for the monomer indicating purity. The D-value of for the dimer is larger than the limit and indicates some impurity for this peak. For further examples refer to following slides.
Polydispersity D is calculated by the division of the weight average molecular weight Mw through the number average molecular weight Mn.
8.9 % dimer
Title of Presentation
1 March, 2001
Agilent Restricted

16. Simultaneous Impurity Testing and Identification: II. Identif
Simultaneous Impurity Testing and Identification: II. Identif. by Molecular Weight
Identification: SEC report
Ovalbumin?
Dimer of Ovalbumin?
Refer to previous slide
Mp = (OVA)
and 84400(dimer)!
Molecular weight data prove ovalbumin and dimer peak (A)! Reference molec. weight ovalbumin: 43000
Title of Presentation
1 March, 2001
Agilent Restricted

17. Purity Control and Molecular Weight Determination
Molecular weight data:
- Mn = Mw = 33727
- Polydisp. = 1.007
Mp = (ref ) Purity Proof: - Peak shape w/ three detectors
- Polydispersity D < 1.05
- Spectra Overlay
Proof:
Pure
Beta- Lactoglobulin
An analysis of a 100 % pure commercial beta-lactoglobulin including the identity confirmation by the molecular weight is shown.
Peak purity was confirmed threefold by:
a visual inspection of the peak shape,
a polydispersity D with significantly smaller than 1.01
the overlay of the peak spectra acquired at inflection points and apex (for an example of protein UV spectra refer to the erythropoietin (EPO) spectrum. Peak identity was confirmed by the molecular weight(Mp = 35600), which is in very good agreement with the reference value (M = 35000) from gel electrophoresis.
(C)
Title of Presentation
1 March, 2001
Agilent Restricted

18. Quality Control of Antibody w/ Fluorescence Det.
Molecular weight
Comparison: SEC: Bioanalyzer:
Antibody
Native(SEC) vs. denaturing method
High MW Impurity 0.2%
Impurity Testing and Molecular Weight Characterization of an Human Antibody
UV detection at 214 or 278 nm is most widely used in the analysis of proteins. Fluorescence detection (FLD)is also of interest because of improved selectivity and sensitivity. Protein fluorescence derives from the aromatic amino acids tryptophan and tyrosine, which we find in almost every protein.
The UV detector should be a diode-array detector(DAD) because it acquires several wavelengths and spectra at the same time. The Figure shows the SEC analysis of an human antibody sample with fluorescence detection at an excitation wavelength of 220 nm and an emission wavelength of 350 nm. The large antibody peak and a small impurity of only 0.2 % of the antibody’ s area can be seen. The impurity could not be detected with the DAD at 214 nm due to the small concentration. For a typical excitation and emission fluorescence protein spectrum obtained with the Agilent 1100 Series fluorescence detector refer to the EPO spectrum. The spectra were acquired without stopping the flow.
Eluent
Title of Presentation
1 March, 2001
Agilent Restricted

19. QA/QC-Analysis of Erythropoietin (EPO)
M = (ref. SEC ~ 55000) D = 1.007
Tyr (ISTD)
EPO
Erythropoietin (EPO) is the principal factor responsible for the regulation of red blood cell production during steady-state conditions and for accelerating recovery of red blood cell mass following hemorrhage. The primary site for EPO synthesis in adult organisms is the kidney. It is a 165 amino acid glycoprotein with a molecular weight of approximately 30,000 to Dalton. EPO can nowadays be made by a recombinant process and is used to treat anemia associated with chronic renal failure for patients on dialysis. EPO has also become widely known because of its forbidden usage by athletes for blood-doping in endurance sports.
The Figure shows the overlay of a simultaneous diode array (solid line) and fluorescence detector (dashed line) analysis of an EPO sample. For medical use it is a must that the sample is highly pure, it does not form aggregates and that the molecular weight is correct. Besides by the visual inspection of the chromatographic signals the purity was demonstrated for the investigated EPO sample by:
The overlay of the UV spectra acquired at inflection points and apex of
the EPO peak (refer to next Figure)
The polydispersity D = 1.007
The molecular weight Mp = agreed very well with the reference value Mp ~ which was determined in the supplier’s laboratory, also by SEC. It should be noted that is not the “true” molecular weight of this EPO sample. Since it is glycosilated by 40% and therefore larger and very hydrophilic it elutes earlier than an EPO without glycolization. With MALDI-MS a molecular weight of Da was determined. The disadvantage of any MS based technique in protein analysis is that aggregates cannot be distinguished from the monomeric protein.
(B)
100% monomer - proof by DAD- and fluorescence signal and low polydispersity. Good Mp agreement!
Title of Presentation
1 March, 2001
Agilent Restricted

20. EPO Purity by Spectra Overlay
Refer to previous text
No spectra difference between mono- and dimer!
Title of Presentation
1 March, 2001
Agilent Restricted

21. Fluorescence Spectra of EPO
Excitation spectrum
278 nm
340 nm
Emissiom spectrum
Refer to previous text
Title of Presentation
1 March, 2001
Agilent Restricted

22. Identification of Proteins in Human Plasma by Molecular Weight
Albumin Mp = 65500
Immumoglobuline Mp =
FLD
Protein Identification and Molecular Weight Characterization of Proteins in Human Plasma
The figure shows the DAD- and FLD-analysis (within one run) of an human plasma sample with eluent A and one TSK SW3000xl-column 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 of the limited resolution the largest peak could be easily identified as human albumin by molecular weight comparison. The SEC determined molecular weight of Dalton agrees very well with the reference value of Dalton. The first peak eluting is obviously an immunoglobuline with a molecular weight of Dalton. For obtaining a better separation, especially in the region between the immunoglobuline- and the albumin peak a further column, for example TSK SW 2000xl should be installed.
DAD
Improved separation required by further column TSK G2000SW
Title of Presentation
1 March, 2001
Agilent Restricted

23. Comparison of MW Results
The Table 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 difference between the MALDI-MS- and the SEC-value for EPO is explained with the 40% glycolization of the sample, which makes it larger and very hydrophilic. This strongly results with SEC 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 also by SEC. To obtain molecular weights with good precision from lab to lab the SEC parameters have to be optimized for each protein and published. For the optimization typically the molecular weight obtained by a reference method must be known.
Title of Presentation
1 March, 2001
Agilent Restricted

24. Features of Protein Analysis and Characterization by SEC
Not denaturing; collection of fractions; different detection principles
Eluent optimization for each protein required
Good accuracy - (typically < 2%) and precision (typically < 2% RSD)
Molecular weight and impurity testing simultaneously (peaks, polydispersity, spectra) in a single run
MW range: MD
Sensitivity (~ 8 ng/µl (100 µl sample) - S/N = 3/1)
Resolution: ~ 100 %(factor 2 in MW difference)
Selfexplanatory
Workhorse !
Title of Presentation
1 March, 2001
Agilent Restricted

25. Pharma-/Lifescience Customers in SEC
Pharmacia/Upjohn, Italy
Amgen, USA ---> 5 Systems in 2000
Roche Company, USA
Novartis, Switzerland
Bayer Pharma, Germany
Fibrinogen, USA
Hexal Company, Germany
BioSyntech, Canada
Angiotech Pharmaceutical, Canada
Astra - Zeneca, Sweden
The Master Workhorse
Protein expression-, QA/QC- analysis
Extract from > 300 customers (all industries)!
Company Confidential
Selfexplanatory
Title of Presentation
1 March, 2001
Agilent Restricted

26. Alternative Techniques - SDS PAGE
The strong features of each technique are always highlighted with red.
Polyacrylamide Gelelectrophoresis with sodiumdodecylsulfate is still most widely used by biochemists for historical reasons and the low equipment costs. But:
- it includes a number of laborious, time-consuming manual steps
- bad repeatability in quantitative analysis
- No fraction collection
- Denatures the protein
- Limited molecular weight range
Title of Presentation
1 March, 2001
Agilent Restricted

27. Alternative Techniques - CE -SDS
CE-SDS is not often used in protein analysis.
Title of Presentation
1 March, 2001
Agilent Restricted

28. Alternative Techniques - Bioanalyzer
The Agilent 2100 Bioanalyzer with the Protein 200 LabChip® kit offers a new technology to protein analysis. The bioanalyzer is a compact system for rapid and automated analysis of proteins. It eliminates the cumbersome equipment and labor-intensive, manual steps associated with gel electrophoresis. The Bioanalyzer was developed by Agilent Technologies in cooperation with Caliper Technologies. A distinct limitation of the Bioanalyzer is the reduced flexibility for proteins with different molecular weights and hydrophobicities since the number of chips for sizing is small.
Title of Presentation
1 March, 2001
Agilent Restricted