Purification Strategies to Process 5 g/L Titers of Monoclonal Antibodies - Altering the order of operations, using new resins, and increasing dynamic binding capacity can obviate the need for major fa

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Purification Strategies to Process 5 g/L Titers of Monoclonal Antibodies
Altering the order of operations, using new resins, and increasing dynamic binding capacity can obviate the need for major facilty changes.


BioPharm International Supplements


Benefits of Increased Resin Capacity

One approach that allows greater amounts of antibody to be purified without increasing column volume is to increase the binding capacity of the chromatography step. Increasing the load capacity means the overall amount of processed antibody can be increased without increasing column size or volume. Since buffer volumes are usually based on column volume, increasing the load mass without increasing the column volume requires no increase in buffer volumes. The same applies for elution conditions where the pool volume will only be affected slightly by overall protein mass when using a final optical density (OD) cut off, or not affected at all when using constant volume pooling criteria.


Table 1. Maximum expression level of a MAb that can be purified as a function of Protein A dynamic binding capacity. This assumes no other modifications were made to the facility, equipment, or operating conditions.
For Protein A chromatography, Table 1 shows the increase in titers that can be tolerated in an existing facility without increasing the size of the chromatography column. At a dynamic binding capacity (DBC) of 15 g/L, a maximum titer of 1 g/L can be accommodated in the facility. If the DBC can be doubled to 30 g/L, then the maximum recoverable titer increases to 3 g/L and a DBC of 45 g/L accommodates titers of up to 5 g/L. An increase in DBC can be achieved in several ways: by lowering the flow rate at constant bed height, increasing the bed height at constant flow rate to increase residence time, and/or by changing to a different Protein A resin with higher capacity.4 In addition, increasing the maximum manufacturing load density from low breakthrough levels (0.5–2% DBC) to slightly higher levels (3–5% DBC) also enables the purification of >5 g/L titers and most likely has an insignificant impact on step yield. Differences in DBC resulting from packing or resin lot-to-lot variability are considered negligible.


Figure 2
Protein A resins all demonstrate higher DBCs with decreasing flow rate, although the slope of the curve may vary from resin to resin. As shown with the DBC curve for resin A in Figure 2, the DBC can be increased from 17 g/L at 40 column volumes (CV)/h to 38 g/L by decreasing the flow rate to 10 CV/h, therefore doubling the amount of antibody recovered with no other process changes. Processing time and facility throughput need to be assessed to determine whether reducing the flow rate also reduces overall capacity.5 This may be addressed in part by reducing the flow rate only for the loading phase of the Protein A step and then increasing the flow rate for the other phases such as equilibration, wash, elution, and regeneration. Unpublished data from our laboratories indicates that step yield is not affected by operating these other phases at higher flow rates. Another potential strategy is to conduct the load phase at two flow rates, and decrease the flow rate during the final stages of the load phase.6


Table 2. Maximum expression level of a MAb that can be purified as a function of the dynamic binding capacity of cation exchange chromatography. This assumes no other modifications were made to the facility, equipment, or operating conditions.
Table 2 shows how increasing the DBC affects a cation exchange chromatography step for a particular antibody and highlights the bottlenecks in an existing facility. In this example, a DBC of 50 g/L can accommodate a 1 g/L titer without constraints. A titer of 2 g/L is constrained by the buffer tank volume (the equilibration buffer in this case), and if this limitation is removed (perhaps by in-line dilution), the constraint becomes the pool tank volume at a titer of 3 g/L. Increasing the binding capacity of the cation exchange resin to 75 g/L or 100 g/L allows titers of 3 g/L and 4 g/L to be accommodated, respectively. In both cases, the buffer tank limitation occurs at lower titers than the pool tank volume limitation. This assumes that all other process parameters remain unchanged when the capacity increases. However, one key consideration during process development is the separation of the antibody and impurities at these higher load densities or with different (higher capacity) resins that may have different selectivities.


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