Precision and accuracy of protein size determination using the ActiPix TDA200 Nano-Sizing System Keywords: Hydrodynamic radius, diffusion coefficient, sizing, Taylor dispersion analysis, protein, antibodies, quality control, protein folding Summary There is an increasing need within the biopharmaceutical industry for techniques which complement or enhance currently available techniques such as dynamic light scattering (DLS), size exclusion chromatography (SEC) and transmission electron microscopy (TEM) for monitoring the amount and extent of aggregation of therapeutic proteins and monoclonal antibodies. All of the techniques previously mentioned have limitations, e.g. it is not possible to determine size for proteins without dilution, they cannot be used in conjunction with in-line systems, e.g. GE AKTA explorers and also have limited application in the analysis of membrane proteins. This application note outlines results with a test protein, myoglobin, using a technique developed by Paraytec for determining the hydrodynamic radius of species in solution. Repeatability and intermediate precision are reported, and the key factor influencing the accuracy of the method is documented. The technique measures the extent of Taylor dispersion of a plug of sample moving through a buffer solution. The extent of dispersion correlates directly with the hydrodynamic radius of the species. Theory behind Taylor Dispersion Analysis Band broadening due to Taylor dispersion has been well characterized in previous literature. 1,2,3 In the method developed by Paraytec, UV area imaging is used to detect the peak of a slug of sample as it migrates through a fused silica capillary past two detection windows. The absorbance versus time data is processed to obtain the peak centre times at the first and second window, t 1 and t 2 respectively, and the corresponding standard deviations, 1 and 2, and variances, 1 2 and 2 2. These values are used to calculate the hydrodynamic radius, R h, using Equation 1 R 4k T r t t h 2 2 2 B 2 1 2 1 Equation 1 where k B is the Boltzmann constant, T the absolute temperature, the viscosity of the solution, and r the capillary radius. For dilute solutions used in these experiments, the viscosity of the solution may be assumed to be that of water at that temperature. Further details concerning Equation 1 and data analysis are available separately. 4 The ActiPix software automatically locates the peaks and uses Equation 1 to calculate the hydrodynamic radius of the sample as seen in Figure 1. 2010 Paraytec Ltd 1
Figure 1. Example of experimental data from Taylor dispersion analysis. Output is using Gaussian fitting function available with Paraytec software. Summary of Technique Paraytec have developed a system for rapid determination of protein radii known as the ActiPix TDA200 Nano-Sizing System (Figure 2). This technology has previously been applied to determine its applicability for rapid quality control of quantum dots. 5 For the purposes of this application note, a brief summary of the technique is given below. The ActiPix Nano-Sizing System is a high precision nano-sizing system consisting of a precision nano-injector, autosampler and detector. Samples are typically stored in the autosampler prior to injection of a few nanolitres of each sample into a fused capillary. A plug of the sample, typically 20-100 nl, is injected at the capillary inlet of a specially designed sizing cartridge (see Figure 3) and driven by application of external pressure along the capillary. UV absorption of the sample is recorded in the first and the second detection window using the integrated UV area imaging detector (ActiPix D100, Paraytec Ltd). Whilst the area of the peak is the same, the widths of both peaks are different: the signal from the second window has a greater width and lower amplitude due to Taylor dispersion. The peaks are fitted with an appropriate peak fitting function using software supplied with the system. The area under the peak corresponds to the amount of the sample injected. The standard deviations are used to calculate the hydrodynamic radius of the sample. 2010 Paraytec Ltd 2
Figure 2. ActiPix TDA200 Nano-Sizing System. Figure 3. ActiPix Nano-Sizing Cartridge. Experimental: Instrumentation and Materials ActiPix TDA200 HT Nano-Sizing System with ActiPix NanoSizing software (Paraytec Ltd) ActiPix cartridge for sizing application (Paraytec Ltd) 214 nm wavelength filter (Paraytec Ltd) Capillary dimensions: 75 m nominal ID, 360 m OD (Polymicro Technologies) Capillary length: 130.2 cm total length, 44.7 cm to 1st window, 40.1 cm between windows Protein standard: myoglobin (Sigma Aldrich) Buffer: phosphate buffered saline (PBS), ph 7.4 (Sigma Aldrich) Sample solutions: 1 mg/ml solution prepared by dissolving a weighed amount of protein in the buffer 0.1 mg/ml solution prepared by ten-fold dilution of the 1 mg/ml stock solution into PBS Run sequence Rinse: PBS; 1000 mbar, 5 min Inject: sample; 65 mbar, 12 s Dip: PBS; 0 mbar, 12 s Run: PBS; 65 mbar, 24 min Wash: 0.1 M NaOH; 1000 mbar, 5 min 2010 Paraytec Ltd 3
An alkaline wash was applied after every run to ensure the internal surface of the capillary was clean. A dip step was applied to remove any trace of sample on the outside of the capillary. The capillary was re-equilibrated with buffer prior to starting data collection. Stock solutions of myoglobin standard were prepared at 1 mg/ml and 0.1 mg/ml. Using the protocol described in the previous section (Run sequence), 9 consecutive runs with injection of the 1 mg/ml solution were made on each of 4 consecutive days. This enabled repeatability and intermediate precision to be determined for Paraytec s Taylor dispersion analysis method. Drift and limit of detection were also calculated using the data obtained. On one of the days, 9 consecutive runs with injection of the 0.1 mg/ml solution were carried out to evaluate repeatability at 0.1 mg/ml. Results Repeatability Figure 4 below is an overlay of primary data obtained from 9 consecutive runs with injection of 1 mg/ ml myoglobin. Excellent concordance is seen between consecutive runs. Figure 4. Overlay of 9 consecutive runs with injection of 1 mg/ml. 2010 Paraytec Ltd 4
Hydrodynamic radius - repeatability and intermediate precision Table 1 below gives a summary of hydrodynamic radii obtained from 9 consecutive runs with injection of 1 mg/ml myoglobin. The mean of the average radii was 2.220 ± 0.020 nm with an RSD of 0.88%. Very good repeatability and intermediate precision from different lots of sample on consecutive days was seen, demonstrating the robustness of this technique. Hydrodynamic radius (nm) Run Day 1 Day 2 Day 3 Day 4 1 2.24 2.20 2.21 2.24 2 2.21 2.23 2.19 2.25 3 2.22 2.21 2.17 2.28 4 2.21 2.21 2.21 2.24 5 2.22 2.21 2.20 2.24 6 2.24 2.21 2.20 2.27 7 2.26 2.20 2.20 2.22 8 2.22 2.20 2.20 2.22 9 2.25 2.21 2.20 2.21 AVERAGE 2.230 2.209 2.198 2.241 STD DEV 0.017 0.009 0.011 0.022 Peak area reproducibility RSD (%) 0.76 0.40 0.52 0.97 Table 1. Repeatability and intermediate precision using Taylor dispersion analysis. A unique feature of determining hydrodynamic radius using Taylor dispersion analysis with UV area imaging in the Paraytec TDA200 instrument is that absolute peak areas are generated. Since peak areas are proportional to the amount of protein injected, this readily enables quantification of the proteins of interest. Table 2 below shows peak area for the data set obtained for Day 2. Peak area repeatability for this data set is concordant with the previous data set, and enables a peak area reproducibility of <1% to be specified. c1 Fit area (mau s) c2 Fit area (mau s) c1area/ c2area Filename Day 2 001 916.67 905.7 1.0121 Day 2 002 928.95 920.38 1.0093 Day 2 003 923.54 915.47 1.0088 Day 2 004 923.91 915.1 1.0096 Day 2 005 928.45 920.19 1.0090 Day 2 006 924.34 915.08 1.0101 Day 2 007 921.55 912.11 1.0103 Day 2 008 925.02 915.8 1.0101 Day 2 009 922.66 913 1.0106 Table 2. Peak area repeatability using Taylor dispersion analysis. Average 923.90 914.76 1.0100 SD 3.66 4.40 0.0010 RSD (%) 0.40 0.48 0.0990 2010 Paraytec Ltd 5
Noise Figure 5 below shows a 6 minute sample of baseline to enable calculation of system baseline noise. A peak to peak value for noise of 0.080 mau was determined at 214 nm using a 2 s time constant. Figure 5. Noise performance for TDA200 instrument. Analysis down to 0.1 mg/ml myoglobin Figure 6. Primary data file for 0.1 mg/ml myoglobin obtained using TDA200 software. 2010 Paraytec Ltd 6
The analysis report shown in Figure 6 shows data with no filtering. The TDA200 software has smoothing functionality. In Figure 7 below, the same data is processed with a 2 s time constant. Figure 7. Primary raw data file from Figure 7 after reprocessing with a 2 s filter. Note that the net result of the filtering function is greatly reduced baseline noise. However, the value reported for the hydrodynamic radii are the same before (2.1753 nm) and after filtering (2.1752 nm). Hydrodynamic radius: repeatability at 1 mg/ml and 0.1 mg/ml 9 repeats each of 1 mg/ml and 0.1 mg/ml of myoglobin standard were run on the first day of the repeatability study. For the 1 mg/ml sample, a hydrodynamic radius value of 2.230 ± 0.017 nm was obtained with RSD = 0.79%. For the 0.1 mg/ml sample, a hydrodynamic radius value of 2.165 ± 0.047 nm was obtained with RSD = 2.16%. Good concordance is seen for the radii at both concentrations, demonstrating that the hydrodynamic radius of myoglobin is independent of concentration over the concentration range 0.1-1 mg/ml. Accuracy of Taylor dispersion technique Taylor dispersion analysis is an absolute method for determination of hydrodynamic radius. The factor most likely to be the source of any systematic error in application of Equation 1 is the capillary radius, r. The capillary diameter (2r) is specified by the manufacturer as 75 ± 3 µm. This leads to a potential systematic error of 8% in the measured hydrodynamic radius. The actual radius of the capillary was determined using a gravimetric method, by measuring the weight changes in both the inlet and outlet vials after pressure driven flow of water at 1000 mbar for 20 minutes. These weight differences enabled determination of the actual radius to be 77.6 µm. Full details of the method and the calculation will be provided in a separate technical note. Table 3 gives recalculated hydrodynamic radii using the actual capillary radius of 77.6 µm. The accurate value of the hydrodynamic radius, using data obtained over three days, is 2.066 ± 0.014 nm. This shows that for accurate measurement of the hydrodynamic radius it is critical to know the actual radius of the capillary used. In using the nominal value for capillary radius, a systematic error of 7% is present in the hydrodynamic radius. 2010 Paraytec Ltd 7
Day 1 Day 2 Day 3 2.1066 2.0797 2.0324 2.0644 2.0666 2.0565 2.0708 2.0669 2.0513 2.0727 2.0616 2.0579 2.0938 2.0598 2.0624 2.1012 2.0563 2.0564 2.0873 2.0546 2.0569 2.0608 2.0519 2.0542 2.0743 2.0594 2.0558 Average SD RSD Average 2.0813 2.0619 2.0538 2.0656 0.0142 0.6858 SD 0.0165 0.0084 0.0085 RSD 0.7908 0.4055 0.4156 Table 3. Hydrodynamic radii re-calculated for myoglobin at 1 mg/ml using actual measured capillary radius of 77.6 µm, and data for radii reported to 5 significant figures. Limit of quantification Using raw data obtained for myoglobin at 1 mg/ml (for day 2, n=9), the peak amplitudes at each windows were found to be: First window: Second window: 20.994 ± 0.087 mau 16.432 ± 0.063 mau By combining these values with the measured peak to peak noise of 0.08 mau, the signal to noise ratios are S/N > 16/0.08 i.e. > 200. Thus quantification and size determination is readily measurable, assuming S/N = 10 down to 0.05 mg/ml. This is below the limit of 0.1 mg/ml normally required as the lower bound to measurements when carrying out protein sizing experiments. Drift Drift was determined using one of the data sets for myoglobin at 1 mg/ml. Using day 2 (n=9) data, drift was calculated at 0.3 ± 0.1 mau/hour. Conclusions Our study demonstrates very good repeatability and intermediate precision in determining the hydrodynamic radius of myoglobin (RSDs better than 1% at protein concentration 1 mg/ml) using Taylor dispersion analysis. Measurement of hydrodynamic radius is readily achieved at 0.1 mg/ml (RSD 2.2%), suggesting radius determination with acceptable precision down to at least 0.05 mg/ml. There is also very good repeatability in peak area. Taylor dispersion analysis shows that both protein concentration and protein radius can be determined in a single run using Paraytec s TDA200 Nano-Sizing System. 2010 Paraytec Ltd 8
References 1. Bello, M. S.; Rezzonico, R.; Righetti, P. G., Science 1994, 266, 773-776. 2. Cottet, H.; Martin, M.; Papillaud, A.; Souaïd, E.; Collet, H. Commeyras, A., Biomacromolecules 2007, 8, 3235-3243. 3. Cottet, H.; Biron J.-P.; Martin, M., Anal. Chem. 2007, 79, 9066-9073. 4. Paraytec Technical Note TN001: Hydrodynamic radius using Taylor dispersion. 5. Paraytec Application Note AN005: Rapid sizing of quantum dots and nanoparticles. 2010 Paraytec Ltd 9