Ranibizumab is a fragment antigen binding (Fab) antibody fragment. It is used for the treatment of Wet Age-related Macular Degeneration (AMD), a common form of age-related vision loss. It is generally produced intracellular in E.coli as inclusion bodies. Refolding and purification of Fab antibody fragment has been a major bottleneck in downstream processing due to its poor refolding yield and corresponding complex purification process. In the current study, we have optimized refolding and purification process for ranibizumab. An in-house clone expressing ranibizumab as inclusion bodies were used for the study. Refolding optimization was carried out using a design of experiment (DoE) study. Post refolding, a 3 step chromatography process comprising of hydrophobic interaction chromatography (HIC)-cation exchange chromatography (CEX)-anion exchange chromatography (AEX) was developed for purification. HIC was used in bind and elute mode as the capture step. Most of the impurities related to high molecular weight (HMW) and residual light and heavy chains were alleviated by using HIC. For the polishing step, CEX was performed for removal of product charge variants. After CEX, AEX in flow-through mode was utilized for further clearance of HCP and DNA. The developed purification process results in product purity &gt;99% and yield &gt;70% along with acceptable product quality attributes HMW &lt;0.1%, HCP &lt;100 ppm, DNA &lt;10 ppb. Further identification of native ranibizumab was carried out using electrospray ionization-quadrupole-time of flight mass spectrometer (ESI Q-TOF-MS) confirming a molecular weight 48.3 kDa. The optimized purification process supports the efficient separation of antibody fragments and is useful for industrial processing.

Downstream processing and characterization of ranibizumab expressed in E.coli

2019 Spring National Meeting

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