Two-dimensional liquid chromatography (2D-LC) isolation and purification of immunoglobulin G (IgG) and extracellular vesicles (EVs) from cell-culture supernatant using capillary-channeled polymer (C-CP) fiber columns.

There is a diversity of therapeutic agents which are manufactured through cellular processes. Prominent among these are the monoclonal antibodies (mAbs) with immunoglobulin G (IgG) being the most common variant. Newer entries to the biotherapeutic palette are the extracellular vesicles (EVs), nanosized membrane-bound particles that are released by cells into the extracellular environment, emerging as viable drug delivery vectors possessing low immunogenicity. IgG is commonly manufactured in bioreactors using Chinese hamster ovary (CHO) cells cultured in suspension, with its primary purification involving the passage of cell-culture supernatant through a Protein A (ProA) affinity chromatography column to isolate the antibodies. The waste stream from this process, however, contains valuable EVs that are presently discarded; thus, an opportunity exists for by-product valorization. We describe a two-dimensional liquid chromatography (2D-LC) platform that employs columns packed with capillary-channeled polymer (C-CP) fibers to isolate both IgG and EVs from CHO cell-culture supernatant. The first dimension utilizes ProA to isolate and quantify IgG, while hydrophobic interaction chromatography (HIC) is used in the second dimension to isolate EVs from the first-dimension effluent. Solvent incompatibility between the two dimensions was mediated using a loop-based modulation strategy and an in-line microfluidic mixer to recondition the first-dimension effluent prior to passage to 2D HIC. Quantitative recoveries of IgG were realized across a range of injected masses, with response linearity and performance comparable to a commercial ProA monolith column. EV recovery was verified by immunofluorescence using nano-flow cytometry while TEM confirmed preservation of vesicular morphology. Isolated EVs demonstrated a purity of 5.86 × 1010 particles µg-1 protein, exceeding the target values for high-purity isolates. Thus, we demonstrate a convenient framework for characterizing CHO cell cultures in terms of both IgG and EV production traits which points towards applications in process monitoring and perhaps new product recoveries.