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Silvia Vidali, PhD I Application Scientist I Molecular Devices

Mark McPate I Senior Application Scientist I Molecular Devices

Introduction

In the dynamic landscape of biopharmaceutical development, the demand for efficient and highthroughput tools in the generation and analysis of culture samples is ever-growing. The industry’s shift towards automation and miniaturization is evident, particularly in the screening of hundreds to thousands of clones during cell line development. Operating within the constraints of limited sample volumes in 96-well and 384-well plates, the need for swift and insightful results is paramount. Addressing this complexity, the Fc PAIA Titer assay emerges as a robust solution, offering a fast, cost-effective, and automatable technology that caters to the biopharmaceutical industry’s quest for assays with streamlined workflows and minimal sample requirements. This plate-based assay, designed for monitoring titers for Fc-containing molecules such as monoclonal antibodies, aligns seamlessly with the goals of bioprocess development.

Molecular Devices SpectraMax® Multi-Mode Microplate Readers are versatile, configurable detection instruments adaptable to any application and budget. Dual monochromators enable users to find the best excitation and emission wavelengths for optimal signal detection, reducing background noise and negating the need for filters. SoftMax® Pro Software provides configurable protocols for fast data analysis, with the option of SoftMax Pro GxP Software for GLP/GMP-compliant regulated laboratories. This combination of cutting-edge technologies ensures the ease of handling routine and complex assays and provides excellent sensitivity for fluorescence-based applications. Together, the PAIA assay and SpectraMax microplate readers represent a total solution for meeting the challenges of high-throughput Fc quantification, from cell line development to bioprocess optimization.

Assay principle

PAIA assays are immunoassays conducted in single-use 384-well plates utilizing a fluorescent, bead-based format. Each well has a black bottom with transparent protrusion serving to separate bead-bound markers from unbound fluorescent markers (Figure 1). Specifically, the Fc titer assay kit is engineered for the swift quantification of all Fc-domain-containing proteins, such as IgGs, in sample volumes ranging from 5 to 15 µL. This assay employs capture beads with immobilized IgG and a reagent containing fluorescence-labeled Fc protein A (PAIA mix) as the marker. Operating as a competitive assay, the analyte in the sample binds to the fluorescentlylabeled Protein A in the reagent. Any remaining Protein A in the reagent binds to the beads, and there is a resulting reduction of fluorescence intensity due to the beads sitting at the bottom of the well, out of the way of the light path. Consequently, higher analyte concentrations correspond to higher fluorescence intensities, whereas lower concentrations yield lower fluorescence intensities. With this assay it is possible to detect from 6 µg/mL up to 400 µg/mL of Fc-domain-containing proteins, and cell culture supernatants can be used in the assay without requiring dilution. Finally, is not necessary to remove cells from the sample (Figure 1).

Materials

• IgG standard (Sigma catalog #I2511)
• Assay diluent – may be cell culture medium
• SpectraMax i3x Multi-Mode Microplate Reader (Molecular Devices)
• SpectraMax iD5 Multi-Mode Microplate Reader (Molecular Devices)
• PAIA Fc Titer Assay kit (PA-104-01)

Method

A serial dilution of IgG standards in microcentrifuge tubes was performed using culture media as the diluent. 5 µL of each concentration was then dispensed into each well of the PAIA plate along with 35 µL of the PAIA mix, giving final concentration ranges of 6.25 to 400 µg/mL (in quadruplicate). The plate was then shaken at 1400 rpm for 15 minutes (as per the PAIA protocol) to allow mixing of the sample with the beads. This was then followed by a 2-minute centrifugation step at ca. 230 x g.

PAIA microplates were loaded into the microplate readers and the data were acquired using the optimized acquisition settings shown in Table 1.

Figure 1. Assay workflow summary: addition of 35 µL of PAIA mix reagent in each well (1); addition of 5 µL of cell culture supernatant (2); shaking on orbital shaker at 1400 RPMI for 15 minutes (critical step) (3); Letting beads settling 5 minutes or centrifugation for 1 minute at 230 xg (4); Bottom reading on SpectraMax i3x or iD5 Multi-Mode Microplate Reader (5).

Table 1. Acquisition settings for the PAIA assay on SpectraMax i3x and iD5. Both plate readers setting referred to monochromator readings.

Results

The SpectraMax i3x and iD5 readers have the ability to run an optional microplate optimization step. This optimization step allows for further improvement of the data quality by improving the alignment of the read head of the microplate reader to the center of each well of the microplate. This was done on the PAIA microplates prior to initial data acquisition, with the new plate definition stored in the plate library for future experiments. This optimization step requires the four corner wells of the microplate to have a positive sample loaded so that a heat map image can be generated to assist in the alignment process (Figure 2).

Using the SpectraMax i3x and iD5 readers, robust IgG standard curves were generated with the PAIA microplates. Figure 3 shows sample concentration-response curves generated on the SpectraMax i3x and iD5 readers using the built-in monochromator. The data were fitted with a 4-parameter logistic curve in SoftMax Pro software. The assay showed excellent performance on both microplate readers with low %CV values (Table 2).

Figure 2. Representation of the microplate optimization wizard in SoftMax Pro software. For each corner well, the user aligns a crosshair icon on the center of the image, enabling the software to update the plate definition so that the center of each well is read.

Figure 3. IgG standard curves generated on SpectraMax i3x (left) and SpectraMax iD5 (right) readers. RFU, relative fluorescence units.

Table 2. IgG standard curve results for SpectraMax i3x and iD5 readers

Conclusions

This application note confirms the SpectraMax i3x and iD5 readers as highly effective microplate readers for use with the PAIA Fc Titer Assay Kit. The readers prove to be adept at rapidly screening large numbers of cell culture samples (384 samples in less than 4.5 minutes for iD5, with autoPMT; less than 3.5 minutes for i3x), demonstrating their suitability for high-throughput IgG quantitation. Robust read-outs, characterized by low coefficients of variation (CVs), were obtained using both readers. The SpectraMax readers’ performance, with the versatility of monochromators and Auto PMT gain, has proven to be optimal for executing the PAIA Fc titer assays.

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