High Capacity, Highly NaOH-Resistant Protein A Sorbent, for the Purification of Monoclonal Antibodies
KANEKA KanCapA chromatography sorbent is an industry-scalable Protein A affinity sorbent that increases the overall productivity of monoclonal antibody (MAb) processes through provision of high dynamic binding capacity and long service life. KANEKA KanCapA sorbent is ideal for a MAb capture step and may be used in either a single column batch operation or in a continuous process using a multicolumn chromatography system.
Proprietary recombinant Protein A (rProtein A) ligand with improved selectivity
High binding capacity aligned to current MAb expression levels
Mild pH elution to protect MAb aggregation or denaturation
Good NaOH stability to prolong service time
KANEKA KanCapA sorbent comprises a rigid cellulose matrix to which the rProtein A ligand is covalently attached to ensure stability over cycles, providing high flow performance at low pressure so is compatible with the process requirements for large scale MAb manufacture. KANEKA KanCapA sorbent is available in a variety of scouting configurations including ScreenExpert RoboColumns♦ and PRC prepacked columns designed for rapid method optimization or small-scale preparative work. The sorbent is supplied in bulk as a slurry in 20% (v/v) ethanol in several pack sizes ranging from 25 mL to 10 L.
Protein A Ligand
Native Protein A has a high affinity for the Fc region of immunoglobulins, but also exhibits an undesired affinity for the Fab region. In order to disrupt the Fab-Protein A interaction, a low pH is often required that can lead to unwanted MAb aggregation. The Protein A ligand used in KANEKA KanCapA sorbent has been modified and is a pentamer of the mutated C domain of the native Protein A molecule. The C domain is known to exhibit NaOH resistance and as a consequence, the KANEKA KanCapA sorbent has good NaOH stability, requires mild elution conditions and has no detectable Fab binding. The rProtein A is expressed in bacteria and is free of animal content.
Рисунок 1 Schematic Representation of KANEKA's New Protein A Ligand Construction
A regulatory support file is available under a confidential disclosure agreement. The entire production process is free from animal derived components and is certified BSE-TSE-free.
♦ KANEKA KanCapA is a trademark of Kaneka Corporation.
Protein A sorbents are well-known for their selectivity towards MAbs. KANEKA KanCapA sorbent has been shown to further improve the MAb purity by reducing residual HCP in the eluent. As shown in Figure 7, the HCP content is very dependent on the MAb feedstock. By including some additional wash steps following the load to remove some residual HCP, it was possible to improve the MAb purity during elution.
Рисунок 7 Comparison of Host-Cell Protein (HCP) Level in Elution Pool Measured by HCP ELISA
Stable in solutions commonly used in affinity chromatography
Working pH range
pH 2 ~ 13
0.1 ~ 0.5 M sodium hydroxide 15 min contact time
Operational flow rate
Up to 500 cm/hr (bed height 20 ~ 25 cm)
≥ 3 min (4 ~ 6 min is recommended)
1 5% Dynamic binding capacity determined by frontal analysis at 3 minute residence time
Operating Flow Rates
KANEKA KanCapA sorbent can be readily packed and unpacked in laboratory, pilot and production-scale columns, and supports high flow rates consistent with the requirements of the latest production processes.
Figure 2 presents pressure versus flow rate data for KANEKA KanCapA sorbent obtained in columns ranging from 4.4 cm to 45 cm I.D., with a bed height of approximately 20 cm, using water. At 500 cm/hr, the pressure drop is 1.5 bar g (22 psi g) which is consistent to process systems routinely used today. Packing performance is reliable and scalable from laboratory to large scale (60 cm I.D.) columns. Typical packing data is > 4,000 plates per meter (1% acetone pulse at 60 cm/hr) and asymmetry factors (AF) are of 1 – 1,2.
Рисунок 2 Pressure vs. Flow Rate for Columns from 4.4 cm to 45 cm I.D. and 20 cm to 23 cm Bed Height
The pressures generated by packed beds are calculated by subtracting the system pressure from total pressure.
MAb expression levels have steadily increased over recent years and titers > 5 g/L are now common place. One key parameter that affects overall productivity of MAbs is the dynamic binding capacity (Figure 3). KANEKA KanCapA sorbent exhibits capacities > 50 g/L at 6 minute residence time which meets the performance expectations of a leading Protein A chromatography sorbent. Dynamic binding capacites for two MAbs confirm the good properties of the sorbent (Figure 4).
Рисунок 3 Residence Time vs. Dynamic Binding Capacity (Polyclonal human IgG)
Рисунок 4 Dynamic Binding Capacity of KANEKA KanCapA Sorbent for Two MAb Molecules Expressed in CHO Cell Culture Supernatant
Excellent NaOH resistance leads to a long service life
KANEKA KanCapA sorbent can be cleaned with 0,1 or 0,5 N NaOH. It can be effectively used up to at least 200 cycles with periodic CIP using 0.1 M NaOH (15 minute contact time) with no significant loss of capacity after 100 cycles (Figure 5). A small loss of ~ 20% is observed after 300 cycles (data not shown). If required, higher concentrations of NaOH of up to 0,5 M can be used for periodic CIP of KANEKA KanCapA sorbent. Reusability of a sorbent is especially important when considering continuous processing using multicolumn chromatography, and the NaOH resistance exhibited by KANEKA KanCapA sorbent positions it well in this emerging area of bioprocessing. Leakage of Protein A is < 10 ppm and is below the limit of detection (1 ppm) after few CIP cycles.
Рисунок 5 Life Cycle Study with Repeated Alkaline CIP of KANEKA KanCapA Sorbent
Remaining 5% Dynamic Binding Capacity after repeated alkaline CIP with 0.1 M or 0.5 M NaOH (A); yield and Protein A ligand leaching in the cycle use study with 0,1 M NaOH (B). Contact time to NaOH in all experiments: 15 minutes.
Milder elution pH
Protein A affinity sorbents are well-known to require an acidic pH for the elution of bound MAbs. Unfortunately, exposure of the MAb to an acidic pH often leads to denaturation and aggregation of which affects the overall process yield. The modified rProtein A ligand used in KANEKA KanCapA sorbent enables elution under milder conditions of around pH 3.5 compared with a pH 3.0 or lower required for the more traditional Protein A ligands. These milder acidic solutions are effective with multiple MAbs with high yield (Figure 6).
Рисунок 6 Elution pH Values of Five MAbs
Concentration of MAb loaded: 1 mg/mL sorbent. Elution pH determined using a pH gradient study with 50 mM citrate from pH 6 to pH 2, 20 column volumes (CV).