Purification of HIV-1 gag virus-like particles and separation of other extracellular particles

Abstract

Enveloped virus-like particles (VLPs) are increasingly used as vaccines and immunotherapeutics. Frequently, very time consuming density gradient centrifugation techniques are used for purification of VLPs. However, the progress towards optimized large-scale VLP production increased the demand for fast, cost efficient and scale able purification processes. We developed a chromatographic procedure for purification of HIV-1 gag VLPs produced in CHO cells. The clarified and filtered cell culture supernatant was directly processed on an anion-exchange monolith. The majority of host cell impurities passed through the column, whereas the VLPs were eluted by a linear or step salt gradient; the major fraction of DNA was eluted prior to VLPs and particles in the range of 100-200nm in diameter could be separated into two fractions. The earlier eluted fraction was enriched with extracellular particles associated to exosomes or microvesicles, whereas the late eluting fractions contained the majority of most pure HIV-1 gag VLPs. DNA content in the exosome-containing fraction could not be reduced by Benzonase treatment which indicated that the DNA was encapsulated. Many exosome markers were identified by proteomic analysis in this fraction. We present a laboratory method that could serve as a basis for rapid downstream processing of enveloped VLPs. Up to 2000 doses, each containing 1×10(9) particles, could be processed with a 1mL monolith within 47min. The method compared to density gradient centrifugation has a 220-fold improvement in productivity.

Separation of HIV‐1 gag virus‐like particles from vesicular particles impurities by hydroxyl‐functionalized monoliths

“…This amount was equivalent to 1.4 × 10 12 or 2.1 × 10 12 HIV-1 gag VLPs per milliliter of monolith. This is within a similar range as reported for enveloped viruses and VLPs on monoliths [9,16]. Although the pressure limit was reached, it was possible to elute HIV-1 gag VLPs (Table 1) and a consistent process performance, compared to processes with a loading volume of 50 mL (Fig.…”

Endothelial Extracellular Vesicles—Promises and Challenges

“…Additionally, there are commercially available kits that advertise fast and easy EV isolation by precipitation (Momen-Heravi et al, 2013), although often leading to low purity and altered functionality of EVs (Van Deun et al, 2014; Gámez-Valero et al, 2016). Since purification of virus-like particles (VLPs) has come into focus for large-scale purification for potential therapeutic applications, and since EVs to some extent behave similar to VLPs (Steppert et al, 2016), it can also be imagined that these areas might be cross-talking for establishing the widely necessary and hoped-for standardized purification techniques. One method for the isolation of viruses makes use of polyethylene glycol (PEG) and can be adapted for purification of EVs.…”

Exosomes Enter Vaccine Development: Strategies Meeting Global Challenges of Emerging Infections

“…Currently exosomes are considered as potential new biopharmaceutical for treatment of several diseases and also for vaccination. It has also been shown that virus like particles and viruses may be contaminated with exosomes and other extracellular particles . Even they can be used for bioprocess monitoring, because the exosome content reflects the metabolic state of the cells .…”