SUNRESIN
Stock Code 300487
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Innovation in mAb Purification Using Affinity Chromatography Resins Based on Proprietary rProtein A
Alessandra Basso, Simona Serban, Tong Nian Gu, Long Liu, Yan Jun Li
Sunresin New Materials, Sunresin Park, No. 135 Jinye Rd, Xi’an Hi-tech Industrial Development Zone, Shaanxi, China
Contact email: simona.serban@sunresin.com
This article focuses on the innovative design of recombinant Protein A (rProtein A) ligands for developing high-performing affinity chromatography media in monoclonal antibody (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 for treating cardiovascular disorders, autoimmune diseases, cancers, multiple sclerosis, Crohn’s disease, and organ transplant rejection. For therapeutic efficacy, mAbs need to be delivered in a highly purified form. After production, mAbs are often accompanied by host cell proteins (HCP) and DNA, which require removal through a robust purification process.
The typical mAb purification process involves centrifugation, filtration, and chromatographic techniques tailored to molecular complexity and impurities. A common workflow includes Protein A affinity chromatography followed by one or two ion-exchange steps or multimodal chromatography.
Affinity chromatography, especially with Protein A resins, is a critical step in mAb purification. Protein A resins facilitate purification of mAbs and bispecific molecules, improving purity from 60-70% to 90-95% in a single step. Advances in recombinant expression technology have enhanced Protein A production, while genetic modifications have improved its alkaline stability for multiple uses.
Sunresin has undertaken the challenge of optimizing Protein A affinity resin by developing a novel recombinant Protein A (rProtein A) for mAb purification. A multidisciplinary team of biologists, biochemists, and polymer chemists collaborated to create an innovative approach to affinity resin design.
The new rProtein A is a six-C domain fusion protein ligand based on the natural Protein A peptide sequence. Through extensive mutation screening, 180 potential sequences were identified from an amino acid sequence library. Fourteen fusion proteins showed promising results, with one candidate selected for further development.
This rProtein A has a molecular weight of approximately 40 kDa and an isoelectric point of 5.5-5.6. It is produced via E. coli fermentation and purified through a two-step chromatographic process using Ni-Seplife® FF (IDA) affinity resin and Seplife MA Large Scale multimodal resin. The resulting rProtein A, with 95% purity, is designed to be covalently coupled to agarose resin via a single-point covalent bond.
The coupling process utilizes a thiol group at the C-terminus of the protein, which reacts with epoxy groups on the resin, forming a stable bond. A linker minimizes steric hindrance, improving antibody capture efficiency and load capacity. This innovation has been patented (CN115850408A, 2022).
The agarose beads used in the resin, designed in-house, are 4% cross-linked with a long, flexible dextran linker, followed by single-point covalently bonded rProtein A. This design reduces steric hindrance, enhancing interactions with large IgG or mAb molecules.
The resin's hydrophilic agarose beads have a large pore size and narrow particle size distribution (40-100 µm), resulting in a dynamic binding capacity (DBC) of 70 g hIgG/L of rProtein A Seplife Suno resin at a 5-minute retention time (RT). It offers excellent flow properties and stability under cleaning-in-place (CIP) conditions, tolerating NaOH concentrations up to 1.5M without significant loss in binding capacity.
In a test with 240 repeated CIP cycles using 0.5M NaOH, the resin retained over 80% of its 10% DBC at 5-minute RT. Under 1M NaOH conditions, it maintained around 88% of the initial DBC after 100 cycles. This exceptional stability makes it ideal for extended use in manufacturing processes.
The lifetime of rProtein A Seplife Suno resin was evaluated, showing 90% retention of its DBC after 100 purification cycles with human IgG molecules. The yield remained above 85%, with a purity of over 95%. CIP was performed after each cycle using 0.5M NaOH.
| 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 |
rProtein A Seplife Suno resin binds specifically to the Fc region of mAbs and Fc-fusion proteins, selectively retaining them on the column. Elution is achieved by lowering the pH to 3.0-3.5, a process that also inactivates viruses. To achieve purity levels above 99%, additional steps like ion exchange chromatography (IEX), hydrophobic interaction chromatography (HIC), multimodal chromatography (MM), or size exclusion chromatography (SEC) can be applied.
Sunresin offers a wide range of agarose resins suitable for large biomolecule separation, including different particle sizes for capture, intermediate purification, and polishing. These resins can be paired with the rProtein A affinity step to achieve mAb purity above 99%.
The rProtein A Seplife Suno resin developed by Sunresin offers high dynamic binding capacity, excellent CIP stability, and tolerance to alkaline solutions (up to 1M NaOH, with specific applications up to 1.5M). It demonstrates excellent affinity for the Fc region of mAbs, maintaining stable yield and purity across 100 cycles. This resin can be seamlessly integrated with additional chromatographic techniques to achieve drug-level purity.
Sunresin is a trusted partner in biomolecule purification, solid-phase synthesis, enzymatic catalysis, ion exchange, and adsorption, offering a comprehensive range of resins and expert support.
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