Innovation in rProtein A ligand design for manufacturing high-performing affinity chromatography media for implementing in mAb purification platforms.

Monoclonal antibodies (mAbs) are immune system proteins that bind to target molecules with high specificity. Since their discovery in the late 1970s, mAbs have become essential in treating diseases such as cardiovascular disorders, autoimmune diseases, cancers, multiple sclerosis, Crohn’s disease and for preventing organ transplant rejection. To maximise therapeutic efficacy and minimise adverse effects, mAbs must be delivered with high purity. After production, mAbs are accompanied by host cell proteins (HCP) and DNA, requiring a robust purification process.

mAb purification typically involves centrifugation, filtration and chromatographic techniques, depending on the molecular complexity and impurities present. Typically, mAb purification includes Protein A affinity chromatography followed by one or two ion-exchange steps or multi-modal chromatography.

Affinity chromatography for mAb purification

Affinity chromatography is a critical step in mAb purification. Protein A resins are widely used, enabling purification of mAbs and bispecific molecules, increasing purity from 60-70% to 90-95% in a single step. Recombinant expression technology has enhanced Protein A production, while genetic modifications have improved its alkaline stability during use.

At Sunresin, we undertook the challenge of optimising Protein A affinity resin and developed a novel recombinant Protein A (rProtein A) for mAb purification. This development involved a multi-disciplinary team of biologists, biochemists and polymer chemists, resulting in a new approach to affinity resin design.

Development of rProtein A

The newly developed rProtein A is a six-C domain fusion protein ligand based on the natural Protein A peptide sequence. After extensive amino acid mutation screening, 180 potential protein sequences were identified from an amino acid sequence library, with 14 fusion protein candidates showing promising results. One fusion protein was ultimately selected as the primary candidate for further development.

The selected rProtein A has a molecular weight of approximately 40 kDa and an isoelectric point of 5.5-5.6. Produced through E. coli fermentation, it undergoes a two-step chromatographic purification process using Ni-Seplife® FF (IDA) affinity resin and Seplife MA Large Scale multi-modal resin. This process results in a 95% pure rProtein A, designed to be covalently coupled to agarose chromatographic resin via a single-point covalent bond.

The coupling process utilises a thiol group at the C-terminus of the protein, which reacts with epoxy groups on the resin, forming a stable covalent bond. The linker, located in a specific sequence domain, minimises steric hindrance and improves antibody capture efficiency and load capacity. This innovation led to the submission of a patent (CN115850408A, 2022).

Features of rProtein A Seplife Suno Resin

The agarose beads used in the resin are designed in-house, featuring 4% cross-linked agarose with a long, flexible dextran linker, followed by rProtein A attached via a single-point covalent bond. This design reduces steric hindrance during interactions with large IgG or mAb molecules.

The hydrophilic nature of the agarose beads, combined with a large pore size and narrow particle size distribution (40-100 µm), results in a dynamic binding capacity (DBC) of 70 g hIgG/L of rProteinA Seplife Suno resin at five-minute retention time (RT). The resin exhibits excellent flow properties and high stability under cleaning-in-place (CIP) conditions, tolerating NaOH concentrations up to 1.5M without significant loss in binding capacity.

In tests with 240 repeated CIP cycles using 0.5M NaOH, the resin retained over 80% of its 10% DBC at five-minute RT. Similarly, with 1M NaOH, the resin retained around 88% of its initial DBC after 100 cycles. This exceptional stability allows for extended usage in manufacturing processes.

Purification performance

Lifetime cycling of the rProtein A Seplife Suno was evaluated, showing that the resin maintained 90% of its DBC after 100 purification cycles with human IgG molecules. The yield exceeded 85%, and the purity remained above 95%, with CIP performed after each cycle using 0.5M NaOH.

Properties of rProtein A Seplife Suno resin

Product Name rProtein A Seplife Suno

Matrix Highly cross-linked 4% agarose

Ligand High alkaline-tolerant rProtein A (E. coli)

Ligand Density (mg/mL) 8-9

Max Flow Rate (cm/h) 420

Max Pressure (MPa) 0.3

Particle Size (µm) 40-100

DBC, RT=5 min (mg hIgG/mL) >70

CIP Stability 0.5-1.0 M NaOH

Leached rProtein A (ppm) <20

mAb purification chromatography workflow

rProtein A Seplife Suno resin specifically binds to the Fc region of mAbs and Fc-fusion proteins, selectively retaining these molecules on the column. Elution occurs by reducing the pH to 3.0-3.5, a process that can also inactivate viruses.

To achieve mAb purity levels above 99%, additional purification steps like ion exchange chromatography (IEX), hydrophobic interaction chromatography (HIC), multi-modal chromatography (MM) or size exclusion chromatography (SEC) may be applied, depending on the impurities.

Sunresin offers a broad selection of agarose resins suitable for large biomolecule separation, including various particle sizes for capture, intermediate purification and polishing. These resins include sulfopropyl, quaternary amine with or without dextran extenders, carboxymethyl, DEAE, phenyl, butyl, multimodal and gel filtration resins. These options can be paired with the rProtein A affinity step to achieve mAb purity above 99%.

Conclusion

The new rProtein A Seplife Suno agarose affinity resin developed by Sunresin offers high dynamic binding capacity, excellent CIP stability and high tolerance to alkaline solutions (up to 1M NaOH and up to 1.5M in specific applications). The resin demonstrates excellent affinity for the Fc region of mAbs, maintains stable yield and purity across 100 cycles and can be seamlessly integrated with additional chromatography techniques such as IEX, HIC, or SEC to achieve drug-level purity.

Sunresin is a trusted partner for biomolecule purification, solid phase synthesis, enzymatic catalysis, ion exchange and adsorption, providing a comprehensive range of resins and expert technical support.

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*Sunresin New Materials, Sunresin Park, No. 135 Jinye Rd, Xi’an Hi-tech Industrial Development Zone, Shaanxi, China. Contact e-mail: simona.serban@sunresin.com