Measurement Reproducibility of a Short Pathlength Cell using the High-Throughput Circular Dichroism System

Download PDF November 16, 2017

Introduction

HTCD Automated High-Throughput CD

The evaluation of secondary and tertiary structure is important in the quality testing of protein and peptide based biologics. CD’s sensitivity to a biomolecule’s asymmetry is ideal for pharmaceutical stability and processing studies, where even slight changes to the molecule or its environment can induce structural changes, altering its function. While CD measurements are known to be quick and easy, the high-throughput system dramatically increases the amount of data obtained with automated measurements from two, 96-well microplates.

Since CD is an absorption technique based on Beer’s Law, the sample concentration and pathlength are critical to obtaining good CD data. Strongly absorbing samples and/or buffers can be measured by either decreasing the concentration or cell pathlength. However, for some samples the working concentration cannot be modified. The 0.2 mm pathlength cell allows for strongly absorbing samples to be accurately and reproducibly measured in the far-UV with the high-throughput CD system.

This application note demonstrates the measurement reproducibility of the 0.2 mm pathlength cell using the HTCD system.

Experimental

Measurement Conditions
Data Pitch0.1 nmD.I.T.4 sec
Bandwidth1 nmScanning Speed100 nm/min
Accumulations3Concentration0.5 mg/mL
Syringe Pump SettingsWash Settings
1st Air Volume200 µLWash 1200 µL x 2 times (0.5% detergent)
Sample Volume100 µLWash 2200 µL x 2 times (Water)
Waiting Time5 secWash 3300 µL x 2 times (Ethanol)
Syringe Speed for Load/DrainMediumDry Time120 sec
Syringe Speed for FlushMedium

184 aliquots of a 0.5 mg/mL BSA (bovine serum albumin) solution were distributed in the sample wells marked by the green S in Figure 1. The orange B’s designate the wells used to obtain baseline measurements of water.

Figure 1. The rack display field indicates the placement of samples and baselines on the two microplates.

Keywords

High-throughput Circular Dichroism (HTCD), Pharmaceuticals, Biochemistry

Results

The far-UV CD spectra of the 184 BSA samples are shown in Figure 2. Tables 1-3 illustrate the measurement reproducibility of the HTCD system at 192.5, 208, and 210 nm, using a 0.2 mm pathlength cell.

Figure 2. Far-UV spectra of 184 bovine serum albumin samples.

Table 1. HTCD measurement reproducibility at 192.5 nm.

Samples1-2324-4647-6970-9293-115116-138139-161162-1841-184
Average34.6035734.74134.5285534.1176734.5052934.145934.5005234.554734.58715
Maximum35.296135.723334.044435.071635.264935.834835.771435.197435.8348
Minimum33.44333.519833.952133.116333.788933.98333.308532.465932.4659
S.D.0.4164720.442730.3284910.4180490.4062040.4189350.4676220.5358320.502431
RSD0.0120360.0127440.0095140.0122530.0117720.011920.0135540.0155070.014527

Table 2. HTCD measurement reproducibility at 208 nm.

Samples1-2324-4647-6970-9293-115116-138139-161162-1841-184
Average-19.26734-19.21747-19.44108-19.75571-19.63827-19.31892-19.36812-19.26523-19.40902
Maximum-18.9835-18.4109-19.204-19.1672-19.1612-18.8735-18.6892-18.1287-18.1287
Minimum-19.4804-19.7724-20.1067-20.4039-20.3477-19.8898-19.7292-.196747-20.4039
S.D.0.1364460.2451010.206230.3562290.2251210.2336980.2382880.3223880.307592
RSD-0.007082-0.012754-0.010608-0.018032-0.011463-0.012097-0.012303-0.016734-0.015848

Table 3. HTCD measurement reproducibility at 210 nm.

Samples1-2324-4647-6970-9293-115116-138139-161162-1841-184
Average-19.98659-19.87717-20.09943-20.48393-20.33072-20.00834-20.16703-19.97124-20.11556
Maximum-19.7322-19.0526-19.8503-19.9427-19.8844-19.497-19.5355-18.7035-18.7035
Minimum-20.2018-20.4108-20.6848-21.248-21.0578-21.05693-20.6122-20.3677-21.248
S.D.0.129060.2622190.1932070.3422510.2292450.240140.2676630.3481320.319071
RSD-0.006457-0.01319-0.009613-0.016708-0.011276-0.01200-0.013272-0.017432-0.015862

About the Author

Leah Pandiscia received her PhD from Drexel University where she studied Biophysical Chemistry. She is a Spectroscopy Applications Scientist at JASCO.