24/7 BIOPHARMA - issue 1 / October 2024

SUNRESIN NEW MATERIALS

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

Figure 1. Lifetime cycling for the affinity purification of an IgG type molecule using rProteinA Seplife Suno. Operating conditions: column size 6.6x200mm, RT 5min, equilibration buffer 0.05M Tris-acetate with 0.15M NaCl, pH 7.4, elution 0.03M sodium acetate pH 3, CIP 0.5M NaOH (3CV).

and load capacity. This innovation led to the submission of a patent (CN115850408A, 2022).

Features of rProtein A seplife Suno resin

purity remained above 95%, with CIP performed after each cycle using 0.5M NaOH.

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.

Properties of rProtein a seplife Suno resin

Product Name

rProtein A Seplife Suno Highly cross-linked 4% agarose High alkaline-tolerant rProtein A ( E. coli)

Matrix

Ligand

Ligand Density (mg/mL) Max Flow Rate (cm/h) Max Pressure (MPa)

8-9 420

0.3

Particle Size (µm)

40-100

DBC, RT=5 min (mg hIgG/mL) >70 CIP Stability Leached rProtein A (ppm) <20

0.5-1.0 M NaOH

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.

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

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TWENTYFOURSEVENBIOPHARMA Issue 1 / October 2024