The accurate, rapid, and high-throughput measurement of IgG is essential in the development and manufacture of most therapeutic antibodies. Monoclonal antibodies are becoming increasingly dominant in biopharmaceuticals, where a vast number of samples must be screened for the development of each potential therapeutic. Here we present the Valita™TITER assay, a novel assay for the quantiﬁcation of IgG in cell culture supernatant.
Current workﬂows, which include HPLC protein A, ELISA, protein A surface interferometry, and HTRF (homogenous time resolved ﬂuorescence) have major drawbacks, which include costly, labour-intensive methods and long analysis times.
The ValitaTMTITER assay provides a very accurate and cost-effective solution to measure IgG in a range from 2.5-80mg/L. The ValitaTMTITER assay relies on the detection of IgG Fc interactions with Protein G by measurement of ﬂuorescence polarization (Fig. 1). The assay comes in a 96-well format and is simple, high-throughput, rapid, and fully automatable. Analysis can be carried out in cell culture media with a low sample volume and no complex preparation steps.
In this application note, we show the optimal protocol for performing IgG quantiﬁcation using ValitaTMTITER assay kits obtained by the PHERAstar® and the CLARIOstar® plate readers.
FP effectively analyzes changes in the size of molecules. “Fixed” ﬂuorophores that are excited by polarized light preferentially emit light in the same plane of polarization. However, rotation of the molecules between absorption and emission of the photon has the effect of “twisting” the polarization of the light. Small molecules tumble faster in solution than larger molecules. Hence, the change of size of molecules, with an associated ﬂuorophore, can be measured using the degree of light depolarization. Consequently, when ﬂuorescently labelled Protein G is unbound, it tumbles rapidly and depolarizes the light more than when it is bound to an IgG (which is 5 times larger) (Fig. 2). This change in polarization is used to measure the amount of IgG in the solution. FP is measured by exciting the solution with plane polarized light and measuring the intensity of light emitted in the plane parallel to the exciting light (polarized proportion) and perpendicular to the exciting light (depolarized portion). The FP is expressed as a normalized difference of these two intensities, which is typically in millipolarization units (mP).
Materials & Methods
- Valita™TITER Assay kit
- Valita™APP software (provided)
- IgG standard (IgG from Human Serum)
- PHERAstar, and CLARIOstar (BMG LABTECH)
Samples were prepared according to the respective product instruction for use. A standard curve was prepared using an IgG standard as per IFU. 60 μl of ValitaMab reconstitution buffer was pipetted into each well of the ValitaTMTITER plate, along with 60 μl of prepared standards and samples. Contents were mixed and incubated in the dark for 30 minutes before being read on the PHERAstar, and CLARIOstar plate readers using preconﬁgured protocols in the BMG LABTECH software (detailed settings below). Data was then analysed using exported .csv ﬁles from the raw data, using the ValitaAPP software.
Fluorescence Polarization, endpoint
|Optic module FP 485 520 520
Focus and gain adjusted prior to measurement (sample: 0 mg/ml)
|70 mP target mP
|200 flashes per well
|0.5 s settling time
Results & Discussion
A standard curve (2.5-80 mg/l) was quantiﬁed with the ValitaTMTITER assay on three FP capable multi-mode readers by BMG LABTECH (CLARIOstar, and PHERAstar). All readers showed low variation of replicate measurements (Fig. 3).
Finally, the ValitaTMTITER IgG quantiﬁcation assay was compared to a conventional IgG quantitation by HPLC and used a range of differentially conditioned cell culture media samples. Figure 4 shows the correlation of both methods and reveals similar precisions for both quantiﬁcation assays with ValitaTMTITER being much faster due its homogenous format with no need for complex sample preparation.
The ability to quickly determine IgG concentration from a mixed sample is of particular importance and value during the development and manufacture of these biopharmaceuticals. Here we report a novel, rapid, and simple ﬂuorescence polarization based assay for high-throughput titer measurement of IgG and FC-containing derivatives. The Valita™TITER assay allows the direct quantiﬁcation of IgG from process samples without sample preparation or puriﬁcation steps. The CLARIOstar, and PHERAstar exhibit excellent assay quality when used with the Valita™TITER assay. By comparison with alternatives, Valita™TITER is a high-throughput, simple, precise method for quantiﬁcation of IgG.
- Thompson, B. et al. (2017) High-throughput quantitation of Fc-containing recombinant proteins in cell culture supernatant by ﬂ uorescence polarization spectroscopy, Analytical Biochemistry 534: 49-55.