Platform purification of six biosimilar molecules using Amsphere A3 – protein A resin

Currently more than 70 biosimilar mAbs (monoclonal antibodies) are under development and multiple originator mAbs are going off-patent in the next 3-4 years. Protein A resin remains the most important workhorse for the purification of monoclonal antibodies. Protein A resin has a high impact on both development and manufacturing cost, in particular during early stage clinical phases. This application note summarizes the key performance parameters for our high capacity protein A resin, Amsphere A3, for 6 biosimilar molecules of which five are mAbs and one is a Fc-fusion protein.

 

Materials and Methods

TABLE 1: MONOCLONAL ANTIBODIES AND FUSION PROTEIN— ALL BIOSIMILARS DERIVED FROM CHO CELL LINE CLARIFIED BY CENTRIFUGATION AND 0.22 MICROMETER FILTRATION

 

FIGURE 1: TEST CONDITIONS OF CHROMATOGRAPHY PROCESS

 

AmsphereTM A3

Amsphere A3 is a new protein A resin designed with a surface modified base bead and alkali-resistant optimized ligand.

 

Protein A ligand

• High DBC via controlled conformation and orientation

• High alkaline stability from protein engineering Surface modification

• Low HCP levels by surface hydrophilization

Base bead formulation

• High DBC at high flow rate

• Excellent pressure and flow properties via rigid crosslinking

 

 

 

 

 

 

 

 

 

TABLE 2: EXPERIMENTAL METHODS AND MATERIALS

 

Results

 

FIGURE 2: PLATFORM PURIFICATION OF FIVE BIOSIMILAR ANTIBODIES USING AMSPHERE A3

 

 

 

 

 

 

 

 

 

 

 

Figure 2 depicts the dynamic binding capacity (DBC) for 4 mAbs and 1 Fc-fusion protein. The DBC for mAb5 (Rituximab) was not determined.

The DBC was consistently between 43 g/L and 57 g/L at a residence time of 5 min (except for Fc-fusion protein).

 

TABLE 3: HCP REMOVAL

 

 

 

 

 

 

 

 

 

 

 

Table 3 shows the HCP removal which was consistently more than 2 LRV (Log Reduction Value) for all mAbs. The Fc-fusion protein showed approximately 1.8 LRV.

 

FIGURE 3: LEACHED PROTEIN A

 

 

 

 

 

 

 

 

 

 

 

 

In Figure 3, leached protein A was between 10 and 20 ppm for all mAb feedstocks. The Fc-fusion protein showed higher leaching at approximately 58 ppm. In all cases leached protein A is expected to drop after a subsequent polishing step.

 

Discussion and Conclusions

Amsphere A3 consistently delivers high binding capacities and excellent impurity clearance for multiple biosimilar molecules as highlighted in this application note. Amsphere A3 excels with comparably high DBC demonstrating its potential to improve the process economics for biosimilar development and manufacturing. In addition, the data underlines the suitability of Amsphere A3 as a platform, since the same protocol was used for all investigated mAbs, consistently yielding excellent performance results with standard wash and elution buffers.

 

 

AmsphereTM is a global trademark of JSR Corporation

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