At-line multi-angle light scattering detector for faster process development in enveloped virus-like particle purification

Abstract

At-line static light scattering and fluorescence monitoring allows direct in-process tracking of fluorescent virus-like particles. We have demonstrated this by coupling at-line multi-angle light scattering and fluorescence detectors to the downstream processing of enveloped virus-like particles. Since light scattering intensity is directly proportional to particle concentration, our strategy allowed a swift identification of product containing fractions and rapid process development. Virus-like particles containing the Human Immunodeficiency Virus-1 Gag protein fused to the Green Fluorescence protein were produced in Human Embryonic Kidney 293 cells by transient transfection. A single-column anion-exchange chromatography method was used for direct capture and purification. The majority of host-cell protein impurities passed through the column without binding. Virus-like particles bound to the column were eluted by linear or step salt gradients. Particles recovered in the step gradient purification were characterized by nanoparticle tracking analysis, size exclusion chromatography coupled to multi-angle light scattering and fluorescence detectors and transmission electron microscopy. A total recovery of 66% for the fluorescent particles was obtained with a 50% yield in the main product peak. Virus-like particles were concentrated 17-fold to final a concentration of 4.45 × 10¹⁰ particles/mL. Simple buffers and operation make this process suitable for large scale purposes.

Keywords: enveloped bionanoparticles; fluorescent virus-like particles; monoliths; nanoparticle tracking analysis.

Figure 1

Chromatogram of the linear gradient purification of HIV‐1 Gag‐GFP VLP using a QA monolith. The loading material was 100 mL of clarified and 0.8 µm filtered HEK 293 cell culture supernatant. Bars represent the area under the curve of the light scattering intensity (grey) and fluorescence (green) at‐line measurements. FT: flow‐through; W: wash; P1‐P5: polled fractions for peaks 1–5

Figure 2

(A) SDS‐PAGE and (B) Western blot analysis of the pooled fractions from the linear gradient purification (Figure 1). M: molecular weight marker; S: cell culture supernatant; L: loading material; FT: flow‐through; W: wash; P1‐P5: pooled fractions for peaks 1–5

Figure 3

Chromatogram of the step gradient purification of HIV‐1 Gag‐GFP VLP using a QA monolith. The loading material was 100 mL of clarified and 0.8 µm filtered HEK 293 cell culture supernatant. FT: flow‐through; W: wash; P1‐P4: pooled fractions for peaks 1–4

Figure 4

A: SDS‐PAGE and B: Western blot analysis of the pooled fractions from the step gradient purification (Figure 3). (C), (D), and (E) electron microscopy micrographs of loading material (L) and fractions P2 and P3, respectively. M: molecular weight marker; S: cell culture supernatant; L: loading material; FT: flow‐through; W: wash; P1‐P4: pooled fractions for peaks 1–4

Figure 5

Analysis of the fractions P1‐P4 from the step gradient purification (Figure 3) by analytical size exclusion chromatography coupled to MALS and fluorescence detectors (SEC‐MALS‐FL). (A) P1; (B) P2; (C) P3; (D) P4