Label-Free and Labeled Technology for Protein Characterization and Quantitation - Use it label-free, or add labels to detect contaminants in solution. - BioPharm International

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Label-Free and Labeled Technology for Protein Characterization and Quantitation
Use it label-free, or add labels to detect contaminants in solution.


BioPharm International
Volume 23, Issue 9

SELECTED APPLICATIONS

Label-free techniques are powerful tools in bioprocess monitoring for quality management applications and in R&D for applications such as antibody screening, epitope binning and mapping, and affinity and kinetic studies. LSPR technology fits into these categories based on recent published results.4–6,8 Concentration monitoring and kinetic analyses, two applications of LSPR technology, are discussed below.


Figure 2. Examples to illustrate the depth of the LSPR detection. A) Dose-response of a Protein A surface for human IgG binding, ranging from ~7 μg/mL to 10 mg/mL. 60 μL of IgG is introduced at 30 μL/min to the LSPR surface, followed by a rinse of PBS. The green (25 sec) and blue (120 sec) arrows indicate reading time for generating the calibration curves in panel B. B) Shifts after 25 sec (green line) and 120 sec (blue line) are plotted against the IgG concentrations. The dashed line represents a fit to the logistic model and is used as a calibration curve. C) Illustration of the influence of the matrix on the performance of the LSPR sensor. The binding properties for a Protein A/IgG binding are measured for IgG spiked in PBS and in cell culture media of non-expressing cells at an identical concentration (performed at a customer site). There is a close relationship between the two media, indicating only a minimal perturbation of the media on the performance of the LSPR sensor. D) Illustration of the LSPR capability to compute kinetic parameters. The model used here has caboxybenzene sulfonamide (CBS) on the surface and bovine carbonic anhydrase II (CAII), a 29 kDa molecule, in solution at concentrations of 100, 33.3, 11.1, 3.33, and 1.11 μg/mL. The gray line is the response of CAII on a reference surface lacking CBS on the surface. Fits with Scrubber 2 software to yield a KD of ~1.5 μM, consistent with results reported using SPR techniques.9–10
The high degree of binding reproducibility traces can be used to evaluate dose response or the titer of a particular analyte. In Figure 2A, the response of a Protein A biosensor is measured when exposed to different concentrations of human IgG ranging from 7 μg/mL to 10 mg/mL. There are three overlapping repeats at 500 μg/mL. For quantitation, the LSPR sensor shift is read as a function of the analyte concentration at arbitrarily set check times. Figure 2B reports the reading after 25 and 120 sec for each concentration. The readings can be fit with a logistic model as shown by the dashed lines through the data points. This approach establishes a calibration curve for the sensor or batch of sensors, and is used for determining the test antibody concentration by comparing the shift produced by the test antibody with the calibration curve.

Figure 2C compares the dose-response for human IgG measurements performed in PBS and crude media, and indicates only a marginal difference. In fact, LSPR technology has been used to quantify the amount of IgG expressed by Chinese hamster ovary cells and in media obtained from a fermenter within a few percent of the value obtained by HPLC. More generally, LSPR technology is compatible with various matrices, including crude media, whole blood, cell lysates, and other complex buffers containing up to 10% dimethyl sulfoxide (DMSO).

Similarly, LSPR technology is particularly useful for kinetic analyses because in injection and rinse steps there is negligible bulk effect, which minimizes the need for data manipulation, a potentially major source of variance. Figure 2D represents the kinetic data for the binding and dissociation of bovine carbonic anhydrase II, a 29 kDa protein, to immobilized sulfonamide ligands. Various repeats show that the responses can be reproduced, and allow the kinetic parameters to be computed using Scrubber 2 software, designed for data analysis and used for SPR. The rate constants (kon, koff, and KD) are compatible with values reported in the literature using SPR.9–10


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