This process is highly flexible because the material can flow either way. The CEX and the HCI resins can be used for either
capture or a polish step, without any need for an in-process tangential flow filtration (TFF). Using CEX chromatography for
capture takes advantage of the higher pI of human antibodies (typically higher than 8), which allows their capture or binding
at a pH close to neutral (Ex. 6.2). The HCI resin can accommodate a wide range of conductivity values, provided that the pH
of the load is maintained close to or above neutral. The viral inactivation step can be implemented between chromatography
steps or after the second column. The convenient place to carry out viral filtration is at the end of the process, before
product formulation, when volumes are smaller, more manageable, and the product is fully purified. This method has proven
to be scalable (from about 1 mL to 10,000 mL column size, when CEX was used for capture) with reproducible results for different
MEP HYPERCEL: THE FLEXIBLE RESIN
The unique properties of MEP HyperCel (Pall Corp., East Hills, NY) make process simplification possible. MEP HyperCel, a HCI-based
resin is dependent on binding pH (neutral and above) but is independent of the conductivity of the load (up to 1 M NaCl equivalent).
This is what makes it extremely flexible. When MEP HyperCel is used for capture, the resin is insensitive to the high conductivity
of the cell culture supernatant, allowing straight loading of the harvest. Pre-conditioning the feed during the recovery TFF
can extend the life of the resin as a capture step.
The tolerance of MEP HyperCel to a wide range of conductivity during sample load is particularly useful when fed a neutralized
CEX eluate. Consequently, this resin allowed for interchangeability in the proposed purification scheme because it can be
applied either to capture or polish.
An additional benefit provided by this resin is seen during elution. As the pH is reduced, product is recovered due to electrostatic
repulsion between the ligand and the antibody, and conductivity has no significant influence in this elution process. Therefore,
when MEP HyperCel resin is used for capture, the eluate can be collected at low conductivity to allow for effective binding
onto the CEX column. Consequently, product can flow directly from the HCI column to the CEX column without needing any in-process
TFF. As a polishing step, there is more flexibility in the conductivity of the elution buffer, because HCI is now the final
chromatography step. In addition, MEP Hypercel can be viewed as both an alternative to TFF and a chromatography step. An interesting
option would be to use the formulation buffer when HCI is being used as a polish.
OPTIMIZATION OF HCL CHROMATOGRAPHY
Several studies were performed on the HCI protocol to minimize HCPs during elution. Decreasing the conductivity during elution
and increasing the elution pH from 3–4 to 4.8–5.2 favored a preferential elution of the product with a minimal relative amount
of HCPs during a capture chromatography study (Figure 1). Also, we achieved higher purity by decreasing the elution flow rate
(Figure 2). Other parameters such as elution at low conductivity and post-load washes with phosphate buffers also helped to
exclude HCP from the purified antibody.
Finally, we observed that the binding capacity of this resin can be manipulated not only by the product's residence time in
the column, but also by manipulating buffer species during equilibration and load. For example, when the strength of phosphate-based
buffers was reduced from 35 mM to 10 mM, binding capacity was increased by about 170%, from 13 mg/mL to 22 mg/mL, for a specific