Robust IgG measurement in bioprocessing: Rapid, accurate and robust IgG measurement in the presence of cells

 By: Dr Leah Quinn, Product Development Scientist & Dr Hannah Byrne,Head of Biologics

Why is the presence of cells problematic in standard IgG quantification techniques?

Monoclonal antibodies (mAbs) have established themselves as the leading biopharmaceutical therapeutic modality. The establishment of robust manufacturing platforms are key for antibody drug discovery efforts to seamlessly translate into clinical and commercial successes. The accurate and reliable measurement of mAb (e.g. IgG) titer is essential in the development and subsequent manufacture to ensure optimal cell culture performance for the production of all biologics. The ability to reliably monitor protein titer in real time throughout a bioprocess allows operators to rapidly adjust the process conditions for maximum protein output while minimizing the process time. Quick access to titer data also enables earlier decisions regarding preparation of downstream processes (e.g. the preparation of purification systems (e.g., protein A HPLC)), further reducing the production timeline.

Of the various technologies currently employed by the biopharmaceutical industry to quantify mAbs, the gold standard Protein A HPLC, Bio-layer Interferometry, Enzyme-linked Immunosorbent (ELISA) and Immunoturbidimetric assays are common methods. They all have distinct features including: cost per test, cost of hardware and experience of staff required to perform the method; etc. Importantly, some of these techniques require various steps to prepare the samples for analysis, such as centrifugation or dilution to remove whole cells, cellular debris and contaminants. This is because mAbs (e.g. IgG) are contained within cell culture supernatants, which are complex and heterogenous mixtures, that can interfere with these techniques and the accuracy of the output data e.g. mechanical issues in the HPLC instrument, high particulate matter leading to non-homogenous samples and ultimately high variation with ELISAs etc. As such, centriguation is commonly performed to isolate cell supernatants containing the IgG’s of interest prior to quantification by Protein A HPLC or ELISA, while immunoturbidimetric assays require dilution of the test samples.

Despite their widespread adoption in industry, the high cost (Protein A HPLC), sensitivity to cellular contamination leading to variability in results, susceptibility to human error, labour intensive workflow (ELISA) and slow times-to-result (>3 hours in some cases) remain as big hurdles for users looking to adopt Protein A HPLC and ELISA throughout their bioprocessing workflows for the quantification of IgG.

How are Valitacell tackling the issue of cellular contamination in IgG quantification?

Valitacell Ltd., have developed and established a successful IP protected platform, namely Quantum, built on the application of fluorescence polarisation (FP) for the analysis of protein: protein or protein: peptide interactions. Utilising this technology, our aim is to develop novel, smart analytics to improve and enhance screening throughout the biomanufacturing process. We develop assays which are rapid, simple, cost-effective, easyto-use and require limited workflow steps and reagents. To address the drawbacks  associated with current IgG quantification techniques, specifically high cost, labour intensive workflows and a requirement for sample pre-preparation prior to analysis, utilising our Quantum platform technology, we developed Valita®TITER and Valita®TITER Plus. Valita®TITER and Valita®TITER Plus are 96 or 384-well IgG quantification tools with a wide functional range (2.5mg/L – 2000mg/L) to facilitate rapid IgG quantification throughout the drug development process. The assays, although relative quantification tools, compare closely with that of the gold standard Protein A HPLC (Figure 1) with regards to performance for the quantification of IgG, without the associated cost or sample preparation requirements.

Figure 1: Valita®TITER versus Protein A HPLC for the quantification of human IgG.A comparative study was carried out to compare the performance of Valita®TITER versus the gold standard, Protein A HPLC, for IgG quantification. Both data sets are plotted in mg/L, with Valitacell’s Valita®TITER assay comparing closely with Protein A HPLC (R2 > 0.9). However, there will always be an element of bias between the two (~20%), with variance in the data vs HPLC usually introduced as a result of subtle differences between molecules (e.g. IgG isotype, LC isotype).

Advantages of Valita®TITER and Valita®TITER Plus for IgG Quantification

Valita®TITER and Valita®TITER Plus are rapid, high-throughput assays, which are based on the detection of IgG-Fc interactions with a fluorescently labelled derivative of protein G using fluorescence polarisation (FP). The assay plates come pre-coated with the fluorescently labelled IgG Fc-specific probe, which the end user reconstitutes prior to IgG test sample addition. 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 (e.g. unbound fluorescently labelled Fc-specific probe) tumble faster in solution than larger molecules (fluorescently labelled Fc-specific probe: IgG complex). Hence, the change of size of molecules, with an associated fluorophore, can be measured using the degree of light de-polarization. Consequently, when the fluorescently labelled IgG Fc-specific probe is unbound, it tumbles rapidly and depolarizes the light more than when it is bound to the Fc of an IgG (which is ~20 times larger). 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).

Figure 2: Assay Principle: The assay applies fluorescence polarization to quantify IgG. Small, unbound molecules rotate rapidly in solution (top), while large, bound molecules rotate slowly (bottom).

Figure 3: The effects of high cell numbers on the performance of the Valita®TITER assay. The performance of our assay is not affected by the presence of cells in test samples, even at a density of 15 x 106 per mL.

Both assays have a two-step workflow and a 5 minute associated incubation time allowing the preparation and quantification of 96 test IgG samples in <20 minutes (based on manual off-line preparation of plates).

Figure 4: Schematic of Valita®TITER and Valita®TITER Plus assays for IgG quantification using fluorescence polarization. Each well of the plate is pre-coated with a fluorescently labelled Fc-specific probe. An IgG sample binds to the probe (B). Binding is measured via fluorescence polarization and rotational diffusion (C).

These simple, one-step, user-friendly, cost-effective and robust assays will not only increase the speed at which MAbs are selected (based on titre), but also help to ensure life-saving biologic medicines reach patients in the shortest time-frame possible.

Expensive, time-consuming, complex? Not if we can help it.

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