Non-protein A purification platform for continuous processing of monoclonal antibody therapeutics.

Protein A capture chromatography, the core of a mAb purification platform, is known to account for more than 50% of downstream processing costs along with other limitations including lack of complete stability to alkaline cleaning solutions, relatively lower binding capacity, and ligand leaching. Researchers have explored alternatives to protein A chromatography, both chromatographic and non-chromatographic, but with limited success. In this paper, we propose a non-protein A purification platform for continuous processing of monoclonal antibodies (mAbs). The proposed platform consists of precipitation in coiled flow inverter reactor, cation exchange chromatography for capture, multimodal chromatography and a salt tolerant anion exchange membrane as polishing steps. The versatility of the proposed platform has been successfully demonstrated for three different mAbs. In all cases, acceptable process yield was obtained (70-80 %) and the product quality attributes of the final unformulated drug substance were consistent and well within accepted limits (HCP < 100 ppm, DNA < 10 ppb, % aggregate content < 1%) along with desired charge variant composition.

Postmitotic nuclear pore assembly proceeds by radial dilation of small membrane openings.

The nuclear envelope has to be reformed after mitosis to create viable daughter cells with closed nuclei. How membrane sealing of DNA and assembly of nuclear pore complexes (NPCs) are achieved and coordinated is poorly understood. Here, we reconstructed nuclear membrane topology and the structures of assembling NPCs in a correlative 3D EM time course of dividing human cells. Our quantitative ultrastructural analysis shows that nuclear membranes form from highly fenestrated ER sheets whose holes progressively shrink. NPC precursors are found in small membrane holes and dilate radially during assembly of the inner ring complex, forming thousands of transport channels within minutes. This mechanism is fundamentally different from that of interphase NPC assembly and explains how mitotic cells can rapidly establish a closed nuclear compartment while making it transport competent.