Process for the purification of recombinant antibody fragments

A process for purification of recombinant humanized (rHu) Ranibizumab from a recombinant host cell expressed as inclusion bodies, said process comprising;

• (a) solubilizing inclusion bodies containing light and heavy chains of rHu Ranibizumab from a recombinant host cell to obtain a solubilized rHu Ranibizumab;
• (b) refolding the solubilized rHu Ranibizumab of step (a) to obtain a refolded rHu Ranibizumab;
• (c) concentrating the refolded rHu Ranibizumab of step (b) by ultra-filtration to obtain a concentrated and refolded rHu Ranibizumab;
• (d) subjecting the concentrated and refolded rHu Ranibizumab of step (c) to a first multimodal chromatography process, said first multimodal chromatography process comprising:
• (i) binding concentrated and refolded rHu Ranibizumab of step (c) having pH ranging from 4.0 to 10.0 onto a first multimodal chromatography resin to capture the rHu Ranibizumab and eliminate impurities, with a buffer comprising 20 mM to 50 mM Tris HCl and 0 mM to 150 mM NaCl, or 20 mM to 50 mM acetate and 0 mM to 150 mM NaCl, with pH ranging from 4.0 to 10.0,
• (ii) eluting rHu Ranibizumab in the presence of an elution buffer comprising 20 mM to 50 mM Tris HCl and 0 mM to 1000 mM sodium chloride, or 20 mM to 50 mM acetate and 0 mM to 1000 mM sodium chloride, with pH ranging from 4.0 to 10.0 into an elution pool;
• wherein the rHu Ranibizumab purified in step (d) has less than 5% of unfolded or misfolded forms of rHu Ranibizumab and less than 2% of aggregated form of rHu Ranibizumab;
• (e) subjecting the rHu Ranibizumab of step (ii) of step (d) to a second multimodal chromatography process, said second multimodal chromatography process comprising:
• (iii) feeding rHu Ranibizumab from the elution pool of step (ii) of step (d) having pH ranging from 4.5 to 6.5 onto a second multimodal chromatography resin to capture rHu Ranibizumab with a buffer comprising 20 mM to 50 mM acetate and 0 mM to 150 mM NaCl having pH ranging from 4.0 to 6.5;
• and
• (iv) eluting rHu Ranibizumab with an elution buffer comprising 20 mM to 50 mM acetate and 0 mM to 1000 mM NaCl having pH ranging from 4.0 to 6.5;
• wherein the rHu Ranibizumab purified in step (e) has less than 0.1% of unfolded or misfolded forms of rHu Ranibizumab and less than 0.3% of aggregated form of rHu Ranibizumab.