KANEKA KanCapA♦ 3G Affinity Sorbent product photo Primary L

KANEKA KanCapA♦ 3G Affinity Sorbent

  • Great dynamic binding capacity and long service life for improved process economy
  • Gentle elution at a mild pH to protect against mAb aggregation or denaturation
  • Guard against impurities with high selectivity for efficient HCP and aggregate removal

High Capacity Sorbent with Enhanced Impurity Removal for the Purification of Monoclonal Antibodies and Fc Related Molecules

KANEKA KanCapA 3G Protein A affinity sorbent achieves high binding capacity with enhanced selectivity to reduce host cell proteins (HCP) and aggregates while eluting at a mild pH which can reduce denaturation and improve process yields. These performance attributes combine to deliver a key element of a modern, scalable purification platform so you do not have to choose between purity and productivity.

KANEKA KanCapA 3G sorbent comprises a recombinant Protein A ligand covalently attached to a rigid cellulose matrix. This ensures stability over a high number of cycles and provides high flow performance at low pressure making the sorbent compatible with the process requirements for large scale mAb manufacture in both batch and continuous processing using multi-column chromatography solutions.

The sorbent is available in 1 and 5 mL PRC prepacked columns designed for rapid method optimization or small-scale preparative work and is also available in bulk as a slurry in 20% (v/v) ethanol in 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.

Figure 1

Schematic representation of KANEKA KanCapA 3G Protein A ligand

The Protein A ligand used in KANEKA KanCapA 3G sorbent is a mutated multimer of the C domain (C’’) of the native Protein A molecule which enables much milder pH elution to protect mAbs from aggregation. In addition, this high selectivity provides enhanced removal of HCP and aggregates. The ligand design is such that there is no detectable Fab binding and is alkali stable to meet the requirements of commercial manufacturing in terms of clean in place (CIP) using 0.1 M or 0.5 M NaOH. The rProtein A is expressed in bacteria and is free of animal derived content.

Table 1

Sorbent properties

 
Base matrix Highly cross-linked cellulose
Average particle size 65 - 85 μm
Ligand C” Modified engineered recombinant Protein A
Coupling chemistry Reductive amination
Dynamic binding capacity (DBC)* ≥ 58 mg human polyclonal IgG/mL packed resin
Chemical stability Stable in solutions commonly used in affinity
chromatography
Working pH range pH 2 - 13
CIP condition 0.1 M sodium hydroxide 15 min contact time
or 0.5 M for shorter periods of time
Operational flow rate Up to 500 cm/h (bed height 20 - 25 cm)
Residence time ≥ 3 min (4 - 10 min is recommended)
 
*5% DBC determined by frontal analysis at 6 minutes residence time

Operating Flow Rates

KANEKA KanCapA 3G sorbent is based on highly cross-linked cellulose matrix and can be readily packed and unpacked in laboratory, pilot and production-scale columns. The sorbent supports high flow rates consistent with the requirements of the latest production processes.

The pressure drop obtained at 500 cm/h in columns ranging from 4.4 cm to 60 cm internal diameter (ID) with a 20 cm bed height is no more than 1.5 barg (22 psig) which is consistent with routinely used process systems.



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KANEKA

List Price
$ 325.00
Description
  • Great dynamic binding capacity and long service life for improved process economy
  • Gentle elution at a mild pH to protect against mAb aggregation or denaturation
  • Guard against impurities with high selectivity for efficient HCP and aggregate removal

High Capacity Sorbent with Enhanced Impurity Removal for the Purification of Monoclonal Antibodies and Fc Related Molecules

KANEKA KanCapA 3G Protein A affinity sorbent achieves high binding capacity with enhanced selectivity to reduce host cell proteins (HCP) and aggregates while eluting at a mild pH which can reduce denaturation and improve process yields. These performance attributes combine to deliver a key element of a modern, scalable purification platform so you do not have to choose between purity and productivity.

KANEKA KanCapA 3G sorbent comprises a recombinant Protein A ligand covalently attached to a rigid cellulose matrix. This ensures stability over a high number of cycles and provides high flow performance at low pressure making the sorbent compatible with the process requirements for large scale mAb manufacture in both batch and continuous processing using multi-column chromatography solutions.

The sorbent is available in 1 and 5 mL PRC prepacked columns designed for rapid method optimization or small-scale preparative work and is also available in bulk as a slurry in 20% (v/v) ethanol in 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.

Figure 1

Schematic representation of KANEKA KanCapA 3G Protein A ligand

The Protein A ligand used in KANEKA KanCapA 3G sorbent is a mutated multimer of the C domain (C’’) of the native Protein A molecule which enables much milder pH elution to protect mAbs from aggregation. In addition, this high selectivity provides enhanced removal of HCP and aggregates. The ligand design is such that there is no detectable Fab binding and is alkali stable to meet the requirements of commercial manufacturing in terms of clean in place (CIP) using 0.1 M or 0.5 M NaOH. The rProtein A is expressed in bacteria and is free of animal derived content.

Table 1

Sorbent properties

 
Base matrix Highly cross-linked cellulose
Average particle size 65 - 85 μm
Ligand C” Modified engineered recombinant Protein A
Coupling chemistry Reductive amination
Dynamic binding capacity (DBC)* ≥ 58 mg human polyclonal IgG/mL packed resin
Chemical stability Stable in solutions commonly used in affinity
chromatography
Working pH range pH 2 - 13
CIP condition 0.1 M sodium hydroxide 15 min contact time
or 0.5 M for shorter periods of time
Operational flow rate Up to 500 cm/h (bed height 20 - 25 cm)
Residence time ≥ 3 min (4 - 10 min is recommended)
 
*5% DBC determined by frontal analysis at 6 minutes residence time

Operating Flow Rates

KANEKA KanCapA 3G sorbent is based on highly cross-linked cellulose matrix and can be readily packed and unpacked in laboratory, pilot and production-scale columns. The sorbent supports high flow rates consistent with the requirements of the latest production processes.

The pressure drop obtained at 500 cm/h in columns ranging from 4.4 cm to 60 cm internal diameter (ID) with a 20 cm bed height is no more than 1.5 barg (22 psig) which is consistent with routinely used process systems.

Applications

Application - mAbs Purification from CHO Supernatant

Cell culture supernatant was loaded onto 2 mL column at the optimum pH for the KANEKA KanCapA 3G sorbent and the Protein A agarose columns. The conditions described in Table 2 were applied. The HCP and aggregate contents are summarized in Figure 6.

Analytical method: HCP concentration of eluate as measured by ELISA using Cygnus kit F550.

DNA concentration of eluate was measured by PCR using Cygnus kit D555T.

Table 2

Chromatography steps on KANEKA KanCapA 3G and agarose based sorbents


Step Buffer CV
Equilibration 25 mM Tris-HCl pH 7.5 3
Load CCF containing mAb (IgG1) 30 mg/mL-sorbent
Wash 25 mM Tris-HCl + 150 mM
NaCl pH 7.5
5
Pre elution 25 mM Tris-HCl pH 7.5 2
Elution 60 mM acetate pH 4.0 or 3.9 5
Strip 0.1 M phosphoric acid 4
Wash 25 mM Tris-HCl pH 7.5 3
CIP 0.1 N NaOH 4
Re-equilibration 25 mM Tris-HCl pH 7.5 5

Figure 6

mAb purification on KANEKA KanCapA 3G and an agarose based sorbents*


*This data comes from one experiment with one sample, and results may differ further to the variability of the biological origin of the samples and related conditions of use. (Source: KANEKA Corporation)

Data shows the high purity of the mAb on KANEKA KanCapA 3G sorbent with low DNA and HCP content.

Specifications

Great Dynamic Binding Capacity

Figure 2

Capacities as a function of residence time (Polyclonal IgG)*

This data comes from one experiment with one sample, and results may differ further to the variability of the biological origin of the samples and related conditions of use. (Source: KANEKA Corporation)

mAb expression levels have steadily increased over recent years and titers >5 g/L are now common. One key parameter that affects overall productivity of Protein A sorbent is the dynamic binding capacity (DBC). Figure 2 shows a solution of 3 mg/mL of polyclonal IgG loaded on to a column at a range of residence times from 2 to 8 min. KANEKA KanCapA 3G sorbent has enhanced capacities versus KANEKA KanCapA sorbent.

Capacities higher than 70 g/L were obtained at 8 minute residence time which meets the performance expectations of a leading Protein A chromatography sorbent used at large commercial scale.


Alkali Stable for a Long Service Life


KANEKA KanCapA 3G sorbent can be effectively used up to at least 150 cycles with periodic CIP using 0.1 M NaOH (15 minutes contact time) with no significant loss of capacity (Figure 3). 97% of the initial capacity remains after 100 cycles and 94% after 150 cycles. Leakage of Protein A remains <10 ppm along cycles as shown in figure 3 

For more stringent cleaning, it is recommended to perform a 15 minute 0.5 M NaOH CIP every 10 cycles.


Figure 4

Gentle Elution at a Mild pH

pH elution values for 8 different mAbs and fusion proteins*

Elution pH was determined using a pH gradient study with 50 mM citrate buffer from pH 6 to pH 3, 20 column volumes (CV).

*This data comes from one experiment with one sample, and results may differ further to the variability of the biological origin of the samples and related conditions of use. (Source: KANEKA Corporation)

Protein A affinity sorbents are well-known to require an acidic pH for the elution of bound mAbs. Exposure of the mAb to an acidic pH may lead to denaturation and aggregation which affects the overall process yield.

The modified rProtein A ligand used in KANEKA KanCapA 3G sorbent enables elution under mild conditions. Fig 4 shows the elution pH related to the purification of a humanized IgG1, FC fusion protein and a chimeric IgG for KANEKA KanCapA 3G sorbent and an agarose based sorbent. Despite differences, due to the characteristics of each antibody, all are eluted at a milder pH using a KANEKA KanCapA 3G packed column.


Guard Against Impurities with High Selectivity

The selectivity of this unique ligand enables an enhanced purification. This is achieved through delayed elution of HCP and aggregates (Figure 5). This benefit may enable removal of impurities which might not be achieved with the standard ligand.

In the example in Figure 5, the selected elution pH was the optimum for both sorbents.

A Cygnus F550 ELISA test was used for HCP concentration measurement.

Figure 5

HCP and aggregate content analysis in elution fractions from KANEKA KanCapA sorbent and Protein A agarose based sorbent*

*This data comes from one experiment with one sample, and results may differ further to the variability of the biological origin of the samples and related conditions of use. (Source: KANEKA Corporation)

Additional Information

Regulatory

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.

Ordering Information
Part Number Description
26090-026 KANEKA KanCapA 3G sorbent 25 mL
26090-035 KANEKA KanCapA 3G sorbent 500 mL
26090-058 KANEKA KanCapA 3G sorbent 5 L
26090-064 KANEKA KanCapA 3G sorbent 10 L
Segment
Chromatography