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Silvia Vidali, PhD | Application Scientist | Molecular Devices

Cathy Olsen, PhD | Sr. Application Scientist | Molecular Devices

Introduction

Biopharmaceuticals and biologics are utilized to treat a wide range of diseases; recently, monoclonal antibodies (mAbs) have been employed to improve treatment outcomes for a wide variety of human diseases, including cancer, autoimmune diseases, infections, transplantation rejection, and inflammatory disorders. However, accessibility is limited due to high costs. Furthermore, patients require large doses when undergoing antibody therapies, therefore it is essential to establish reliable approaches to produce cell lines which have high antibody expression levels.

During the process of cell line development, hundreds to thousands of hybridoma clones are typically screened in order to identify those clones that are antigen-specific, have optimal growth, and have high productivity, as represented by Figure 1. High-throughput screening and automation methods have been widely adopted within the biopharmaceutical industry for the improvement of cell culture conditions and monoclonal antibody (mAb) identification, as well as optimization of the purification process. These methods have enabled the screening of larger numbers of samples, using smaller volumes, with 96-well and 384-well microplate assay formats offering a high-throughput format that saves time and costs.

Here we demonstrate the use of a fully optimized rapid, robust, and accurate IgG titer platform combining Beckman Coulter Life Science’s ValitaTiter IgG quantitation assay with a suite of Molecular Devices fluorescence polarization (FP) configured microplate readers. The ValitaTiter 384-well assay measures IgG concentrations across a range of 2.5 to 100 mg/L, with a simple add-andread protocol. The ValitaTiter assay binds all Fc-containing IgG-based mAbs. It has been optimized for human IgG and validated for rabbit IgG.

ValitaTiter assay plates come precoated with a fluorescently labeled, target-specific probe that the user reconstitutes prior to IgG test sample addition. The assay is performed in less than 15 minutes and can be incorporated easily into the bioprocess workflow. Its 384-well format is high throughput and can be fully automated. Analysis can be carried out in crude cell culture media containing up to 10 x 106 cells/mL with a low sample volume and little or no test sample pre-preparation. Assay detection can be performed using FP mode on the following Molecular Devices microplate readers:

• SpectraMax® iD5 Multi-Mode Microplate Reader (equipped with appropriate filters)
• SpectraMax i3x Multi-Mode Microplate Reader (with FP detection cartridge)
• SpectraMax Paradigm Multi-Mode Microplate Reader (with FP detection cartridge)
• SpectraMax M5e Multi-Mode Microplate Reader (SpectraMax M5 and FlexStation® 3 Multi-Mode Microplate Readers have the same FP functionality and produce FP data equivalent to that of M5e.)

Assay principle

In contrast to other quantitative approaches, the ValitaTiter assay uses fluorescence polarization (FP), a homogeneous technique that streamlines IgG quantitation by eliminating time-consuming wash steps and cumbersome sample preparation methods. FP has been widely used to monitor binding events in solution. It can be used to assess biomolecular interactions, including protein-antibody binding and DNA hybridization, as well as enzyme activity, and it has been adapted to basic research as well as high-throughput screening.

A small, fluorescently labeled molecule (tracer) that is excited with plane-polarized light emits mostly depolarized light because the tracer tumbles rapidly during the time between excitation and emission. However, when the tracer binds a much larger molecule, it rotates more slowly, and the emitted light remains largely polarized. FP measurements are obtained via an optical system which sends polarized light into a sample and detects the polarization of the light emitted. An increase in binding of a fluorescent tracer to a much larger molecule, such as IgG, is detected by an increase in the polarization of the light emitted by the sample, as illustrated in Figure 2.

Figure 1. Cell line development process from cell transfection to scale-up. This process is highly complex, tedious, and time consuming. The screening and selection for clones with high productivity, quality and stable long-term expression is the slowest step in developing a new mAb with therapeutic potential. Making this step fast, easy to perform, and scalable is very important to the bio-pharmaceutical industry.

Figure 2. Representation of FP in the absence and presence of IgG.

Materials

• ValitaTiter assay microplate, 384-well (Beckman cat. #VAL0013)
• Sigma IgG standard (Sigma cat. #I2511)
• XP Media™ CHO Growth A (Molecular Devices cat. #K8860), supplemented with 4 mM L-glutamine
• SpectraMax iD5 Multi-Mode Microplate Reader
• Set of 2 Fluorescence Polarization Filters 485 nm BW 25 nm Polarized Vertical & Horizontal (Molecular Devices cat. #6590-0136)
• Set of 2 Fluorescence Polarization Filters 535 nm BW 25 nm Polarized Vertical & Horizontal (Molecular Devices cat. #6590-0137)
• SpectraMax i3x Multi-Mode Microplate Reader
• Fluorescence Polarization (FP-FLUO) Detection Module (Molecular Devices cat. #0200-7009)
• SpectraMax Paradigm Multi-Mode Microplate Reader
• Fluorescence Polarization (FP-FLUO) Detection Module (Molecular Devices cat. #0200-7009)
• SpectraMax M5e Multi-Mode Microplate Reader

Method

1. A serial dilution of IgG standards was performed, using XP Media/L-glutamine as diluent, at concentrations from 2.5–100 mg/L (ValitaTiter).
2. 40 μL of medium was pipetted into each well of the ValitaTiter plate to reconstitute the probe.
3. 40 μL of prepared standards were then added to the appropriate wells in quadruplicate.
4. Well contents were mixed by gently pipetting up and down three times, avoiding the introduction of bubbles.
5. Assay plates were incubated in the dark for at least five minutes at room temperature prior to measurement on a Molecular Devices microplate reader, using the optimized settings outlined in Table 1

Note: Example read heights are listed in Table 1, but read height should be optimized for each SpectraMax i3x or Paradigm reader. Additionally, the microplate should be optimized upon first assay setup to ensure the center of each well is read. Optimizations can be selected in ‘More Settings’ in SoftMax Pro Software.

Figure 3. Each well of the assay plate is precoated with a fluorescently labeled Fc-specific probe (1). An IgG sample binds to the probe (2). Binding is measured via fluorescence polarization (3)

Table 1. Optimal instrument settings for ValitaTiter assay fluorescence polarization measurement on Molecular Devices microplate readers. SpectraMax i3x and Paradigm readers require the FP-FLUO detection module, the SpectraMax iD5 reader requires specific FP filters listed in Materials. Settings not required by a reader are indicated by ‘---’. (SpectraMax M5 and FlexStation 3 Multi-Mode Readers use the same settings as M5e.)

Results

Several tests were carried out in order to identify the optimal parameters for detecting the ValitaTiter 384 assay on different Molecular Devices multi-mode microplate readers. IgG standard curves were prepared and analyzed using a simple add-and-read method, with no sample or plate pre-preparation or wash steps required, and an easy workflow.

With the SpectraMax iD5 reader, superior results were obtained using a 485 nm FP filter set for excitation and a 535 nm FP filter set for emission (Figure 4A). With the SpectraMax i3x (Figure 4B) and Paradigm (Figure 4C) readers, best results were obtained using the Fluorescence Polarization Fluorescein (FP-FLUO) detection module with the Stop and Go read setting (Table 1). With all four instruments, IgG standards from 2.5 mg/L to 100 mg/L were plotted with a 4-parameter curve fit in SoftMax® Pro Software. Comparable data across the readers can be found in Table 2. A preconfigured software protocol automated the calculation of mP values from relative fluorescence units (RFU), as well as standard curve plotting.

Figure 4. Standard curves for the ValitaTiter 384 assay read on (A) SpectraMax iD5, (B) i3x, (C) Paradigm, and (D) SpectraMax M5e readers. Curves were plotted using a quadratic curve fit in SoftMax Pro Software (r2 = 0.999 for all four readers).

Table 2. Standard delta mP, average standard deviation (StDev) and %CV (n = 4 to 5) for ValitaTiter standards read on Molecular Devices readers.

Conclusions

To guarantee an optimal cell culture performance for the production of different biologics, an accurate and reliable measurement of IgG titer is essential to different stages of development and manufacture. Assays that are fast, easy to handle but able to guarantee accurate results are critical to success. Here, we successfully demonstrate that ValitaTiter 384-well assays, combined with Molecular Devices microplate readers, enable quantitation of IgG across a wide functional range.

The ValitaTiter assay is a homogeneous, high-throughput method for precise and rapid quantitation of IgG in crude samples, without the requirement of sample preparation or purification steps. This 384-well assay has been fully validated on the SpectraMax iD5, i3x, Paradigm, and M5e* readers to ensure reliable results. SoftMax Pro Software minimizes setup time for detection and automates standard curve fitting and sample quantitation.

* SpectraMax M5 and FlexStation 3 Multi-Mode Microplate Readers have the same FP hardware as the SpectraMax M5e reader and are likewise suitable for use with the ValitaTiter 384-well assay.

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