One-Step Identification of Antibody Degradation Pathways Using Fluorescence Signatures Generated by Cross-Reactive DNA-Based Arrays

Abstract

Therapeutic antibodies are prone to degradation via a variety of pathways during each stage of the manufacturing process. Hence, a low-cost, rapid, and broadly applicable tool that is able to identify when and how antibodies degrade would be highly desirable to control the quality of therapeutic antibody products. With this goal in mind, we have developed signature-based sensing system to discriminate differently degraded therapeutic antibodies. The use of arrays consisting of conjugates between nanographene oxide and fluorophore-modified single-stranded DNAs under acidic pH conditions generated unique fluorescence signatures for each state of the antibodies. Multivariate analyses of the thus obtained signatures allowed identifying (i) common features of native, denatured, and visibly aggregated antibodies, (ii) complicated degradation pathways of therapeutic omalizumab upon time-course heat-treatment, and (iii) the individual compositions of differently degraded omalizumab mixtures. As the signature-based sensing has the potential to identify a broad range of degraded antibodies formed by different kinds of realistic stress types, this system may serve as the basis for high-throughput assays for the screening of antibody manufacturing processes.

A Multi-Fluorescent DNA/Graphene Oxide Conjugate Sensor for Signature-Based Protein Discrimination

“…To construct a multi-fluorescent ssDNAs/nGO sensor, we designed three fluorophore-modified ssDNAs ( Figure 2 A); P1 -FAM: a quadraplex-formative sequence with FAM (λ ex max /λ em max = 495 nm/518 nm); P2 -TAMRA: a simple repeated sequence with TAMRA (λ ex max /λ em max = 555 nm/575 nm); P3 -Cy5: a hairpin-structure-formative sequence with Cy5 (λ ex max /λ em max = 645 nm/660 nm). These ssDNAs bear different sequences, and two of these can fold into different higher-order structures, which were expected to impart the individual elements of the sensor system with differential cross-reactivity [ 38 , 39 ]. In addition, well-separated absorption and emission spectra allow the readout of independent emissions of the fluorophores ( Figure 2 B).…”

“…This accessibility and tunability should create the structurally-diverse sensing elements necessary for high levels of discrimination. Recently, Pei et al [ 38 ] and our group [ 39 ] have developed arrays of fluorophore-modified ssDNA quenched via noncovalent complexation with nano-graphene oxide (nGO) for the identification of proteins based on measurements of spatially separated conjugates. Encouraged by these studies, we have used conjugates between nGO and three ssDNAs with different sequences and different fluorophores in order to construct a sensing system that affords unique protein fluorescence signatures from a single microplate well ( Figure 1 ).…”

A Multichannel Pattern-Recognition-Based Protein Sensor with a Fluorophore-Conjugated Single-Stranded DNA Set

“…Thus, fluorophore-conjugated ssDNAs are capable of generating differential optical signals following interactions with different proteins. There are a number of sensing systems that employ complexes of ssDNAs and other materials, such as gold nanoparticles [ 29 , 30 , 31 ], metal oxide nanoparticles [ 32 , 33 ], graphene oxide nanosheets [ 34 , 35 , 36 , 37 , 38 ], and metal oxide nanosheets [ 38 , 39 ].…”

Noninvasive Fingerprinting-Based Tracking of Replicative Cellular Senescence Using a Colorimetric Polyion Complex Array

“…In general, fingerprints are generated with arrays of cross-reactive molecules that can interact in different ways with analytes. This technique has been employed for the detection of proteins that are solubilized in dilute solutions − or in biological matrixes. − A distinct advantage of fingerprint-based sensing is that it can also be applied to recognize the composition of complex matrixes themselves, such as serum ,, and cell lysate, ,, even though not all components present may be known …”