AF.2A1 allows Removal of Aggregation Precursors to Suppress Aggregation of Therapeutic Monoclonal Antibodies during Production and Storage

AF.2A1 is a 25-residue artificial protein engineered to discriminate between the native and non-native three-dimensional structures of Immunoglobulin G (IgG). AF.2A1 specifically binds to the constant (Fc) region of non-native, misfolded, or stressed, IgG conformers, while ignoring correctly folded, native IgG. Because AF.2A1 can target misfolded antibodies with nanomolar affinity, this small protein is useful as a valuable new tool in biomanufacturing and pharmaceutical research.

Therapeutic antibodies can degrade or aggregate during manufacturing and storage due to chemical or physical stress. This degradation decreases clinical efficacy and risks triggering adverse immune responses in patients. Mitigating the immunogenicity of misfolded, or stressed, IgG conformers is important for the development and manufacture of safe therapeutic IgG preparations.

Since AF.2A1 binds to non-native IgGs induced by various types of physical stress at neutral pH, it acts as a sensitive analytical probe to detect trace amounts of non-native IgG that conventional size-based separation or spectroscopic techniques often miss.

When conjugated to carrier materials such as magnetic beads, AF.2A1 functions as a specific adsorbent, selectively binding and removing non-native monomers and small, nanometer-scale aggregation precursor molecules (<100 nm) from antibody solutions. Removing these precursors prevents IgGs from seeding larger aggregates, enabling the long-term, stable storage of antibody-based therapeutic drugs.

AF.2A1 can be fluorescently labeled and introduced into antibody-producing cells, such as Chinese hamster ovary (CHO) cells, allowing live-cell imaging to detect and analyze where and how intracellular IgG aggregation occurs during the recombinant production process.

AF.2A1 enables the design of homogeneous assays, such as the AlphaScreen assay, to rapidly evaluate antibody stability under various physical stressors, including low pH, heat, or stirring, in a high-throughput format, without requiring complex separation steps.

AF.2A1 is a specialized molecular tool designed to monitor, filter, and ensure the structural integrity of therapeutic antibodies.

Typical Applications

Quality Control and Monitoring during Therapeutic Antibody Production

• High-Throughput Aggregation Assays
• Stability and Accelerated Aging Tests
• Real-Time Production Monitoring

For Purification and Removal

• Precursor Removal
• Selective Adsorption

For Research and Visualization

• Live-Cell Imaging
• Conformational Analysis

Synthesis

Automated solid phase peptide synthesis allows the synthesis of the protein AF.2A1, biotinylated AF.2A1 as well as a fluorescent labeled conjugate useful for imaging and diagnostics. The biotinylated protein is useful for pull down processes. Since AS.2A1 is one of the smallest self-folding proteins, using a folding buffer allows the production of the correctly folded small protein.

Structural models of AF.2A1 and its conjugates

Synthesis of Conjugates

AF.2A1 -> AF.2A1-C12 -> AF.2A1-C12

Replacing arginine 12 with a cysteine enables the tagging of the protein with a fluorophore without interrupting the correct folding pattern for the production of sensitive analytical probe to detect trace amounts of non-native IgG that conventional size-based separation or spectroscopic techniques often miss.

AF.2A1 does not bind native IgG conformers but binds efficiently to the Fc region of non-native IgG conformers.

Watanabe et al. 2016 developed a biosensing method for the quality control of therapeutic antibodies based on AF.2A1, which discriminates between the native and the non-native three-dimensional structures of immunoglobulin G (IgG). AF.2A1 specifically recognized non-native IgG spiked into a solution of native IgG, allowing the detection of contamination by a small amount of non-native IgGs. AF.2A1 binds to non-native IgGs induced by physical stresses at neutral pH. A peptide probe based on AF.2A1 recognizes acid-stressed human IgG of any subclass. The protein recognizes a non-native structure in the CH3 domain. A CH3 mutant with five deleted residues also binds to AF.2A1. AF.2A1 binds to an exposed hydrophobic core centered on a C-terminal tryptophan on the heavy chain Fc region. The CH3 domain is the third constant domain of the heavy chain in IgGs, IgAs, and IgDs, and plays a critical role in the structure and function of the antibody.

Senga et al. (2022) evaluated intracellular IgG aggregates in living cells using the AF.2A1 protein as an AF.2A1-fluorescein conjugate to monitor intracellular IgG aggregates in antibody-producing CHO cells with the help of confocal microscopy to evaluate the presence of aggregates. This method allowed tracking changes in the amount of intracellular IgG aggregates produced in CHO cells.

Reference

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Saphire EO, Parren PW, Pantophlet R, Zwick MB, Morris GM, Rudd PM, Dwek RA, Stanfield RL, Burton DR, Wilson IA. Crystal structure of a neutralizing human IGG against HIV-1: a template for vaccine design. Science. 2001 Aug 10;293(5532):1155-9. [PubMed]

Senga Y, and Honda S; Suppression of Aggregation of Therapeutic Monoclonal Antibodies during Storage by Removal of Aggregation Precursors Using a Specific Adsorbent of Non-Native IgG Conformers. 2018 Bioconjugate Chemistry 29, 10, 3250-3261. [ACS]

Senga Y, Doi M, Onitsuka M, Honda S. Live-cell imaging to analyze intracellular aggregation of recombinant IgG in CHO cells. Cell Chem Biol. 2022 Jan 20;29(1):120-132.e4. [PubMed, Cell]

Senga Y, Imamura H, Miyafusa T, Watanabe H, Honda S. AlphaScreen-based homogeneous assay using a pair of 25-residue artificial proteins for high-throughput analysis of non-native IgG. Sci Rep. 2017 Sep 29;7(1):12466. [PMC]

Watanabe H, Yageta S, Imamura H, Honda S. Biosensing Probe for Quality Control Monitoring of the Structural Integrity of Therapeutic Antibodies. Anal Chem. 2016 Oct 18;88(20):10095-10101. [ACS]

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