Dual-Affinity Ratiometric Quenching (DARQ) Assay for the Quantification of Therapeutic Antibodies in CHO-S Cell Culture Fluids

Abstract

More than 100 monoclonal antibodies (mAbs) are in industrial and clinical development to treat myriad diseases. Accurate quantification of mAbs in complex media, derived from industrial and patient samples, is vital to determine production efficiency or pharmacokinetic properties. To date, mAb quantification requires time and labor-intensive assays. Herein, we report a novel dual-affinity ratiometric quenching (DARQ) assay, which combines selective biorecognition and quenching of fluorescence signals for rapid and sensitive quantification of therapeutic monoclonal antibodies (mAbs). The reported assay relies on the affinity complexation of the target mAb by the corresponding antigens and Protein L (PrL, which targets the Fab region of the antibody), respectively, labeled with fluorescein and rhodamine. Within the affinity complex, the mAb acts as a scaffold framing the labeled affinity tags (PrL and antigen) in a molecular proximity that results in ratiometric quenching of their fluorescence emission. Notably, the decrease in fluorescence emission intensity is linearly dependent upon mAb concentration in solution. Control experiments conducted with one affinity tag only, two tags labeled with equal fluorophores, or two tags labeled with fluorophores of discrete absorbance and emission bands exhibited significantly reduced effect. The assay was evaluated in noncompetitive (pure mAb) and competitive conditions (mAb in a Chinese Hamster Ovary (CHO) cell culture harvest). The "DARQ" assay is highly reproducible (coefficient of variation ∼0.8−0.7%) and rapid (5 min), and its sensitivity (∼0.2−0.5 ng•mL −1 ), limit of detection (75−119 ng•mL −1 ), and dynamic range (300−1600 ng•mL −1 ) are independent of the presence of CHO host cell proteins.

Continuous monitoring of IgG using immobilized fluorescent reporters

“…Binding‐induced fluorescence change in labeled Fc‐binding ligands has also been reported elsewhere (Aoyagi et al, 2003, 2002; Thompson et al, 2017; Turner et al, 2020); however, the longer response time and offline monitoring techniques used in these works make these approaches less attractive for column monitoring. We have demonstrated an approach for continuous human IgG detection by fluorescence intensity, using immobilized reporters, which can be reused at least 100 times.…”

“…A total of 5 g/L human IgG in buffer A was loaded (1.25 ml/min) onto a prepacked 5 ml MabSelect SuRe protein A and the column effluent continuously flowed into a monitoring column as described above, while fluorescence was continuously monitored (Figure 5a). There has also been reported elsewhere (Aoyagi et al, 2003(Aoyagi et al, , 2002Thompson et al, 2017;Turner et al, 2020); however, the longer response time and offline monitoring techniques used in these works make these approaches less attractive for column monitoring. We have demonstrated an approach for continuous human IgG The approach could potentially be extended to other biopharmaceutical products such as non-Fc proteins and therapeutic viruses, and IgG monitoring also could find applications in bioreactor and membrane integrity monitoring.…”

One-Step Quantification of anti-Covid-19 Antibodies via Dual Affinity Ratiometric Quenching Assays

“…Prior work introduced DARQ as a simple mix-andread assay for the quantification of therapeutic monoclonal antibodies Trastuzumab and Adalimumab�respectively used in fighting breast cancer and rheumatoid arthritis�in bioprocess fluids. 17 Our results showed that DARQ technology is endowed with numerous advantages compared to ELISA, BLI, and SPR assays (Table S1), 18−25 chiefly, high reproducibility, speed and ease of execution (mix-and-read), flexibility, robustness, and low cost of goods. This work demonstrates the application of DARQ technology to the quantification of anti-SARS-CoV-2 antibodies in a variety of complex samples.…”

Resorbable elastomers for implantable medical devices: highlights and applications

“…1,2 IMDs are being developed for drug delivery, 3 tissue regeneration 4 and biochemical sensing. 5 Despite their benefits, the application of IMDs has been forestalled by materials with inadequate mechanical properties, the risk of infection 1,2 and the need for removal surgery at the end of implant life. To overcome the limitations of traditional implants for short-term applications, a movement has emerged aiming at developing transient electronics that can dissolve or be degraded in situ.…”

“…Implantable medical devices (IMDs) provide functions ranging from cardiac pacing to neurostimulation, and enable seamless physiological monitoring and therapeutic delivery 1,2 . IMDs are being developed for drug delivery, 3 tissue regeneration 4 and biochemical sensing 5 . Despite their benefits, the application of IMDs has been forestalled by materials with inadequate mechanical properties, the risk of infection 1,2 and the need for removal surgery at the end of implant life.…”