Milling solid proteins to enhance activity after melt-encapsulation

Abstract

Polymeric systems for the immobilization and delivery of proteins have been extensively used for therapeutic and catalytic applications. While most devices have been created via solution based methods, hot melt extrusion (HME) has emerged as an alternative due to the high encapsulation efficiencies and solvent-free nature of the process. HME requires high temperatures and mechanical stresses that can result in protein aggregation and denaturation, but additives and chemical modifications have been explored to mitigate these effects. This study explores the use of solid-state ball milling to decrease protein particle size before encapsulation within poly (lactic-co-glycolic acid) (PLGA) via HME. The impact of milling on particle dispersion, retained enzymatic activity, secondary structure stability, and release was explored for lysozyme, glucose oxidase, and the virus-like particle derived from Qβ to fully understand the impact of milling on protein systems with different sizes and complexities. The results of this study describe the utility of milling to further increase the stability of protein/polymer systems prepared via HME.

Cowpea Mosaic Virus Nanoparticle Vaccine Candidates Displaying Peptide Epitopes Can Neutralize the Severe Acute Respiratory Syndrome Coronavirus

“…Therefore, we also formulated the CPMV vaccine candidates as a slow-release polymer implant prepared by hot-melt extrusion, a highly scalable process technology suited for epidemic or pandemic response. Our previous research showed that VNPs can withstand hot-melt extrusion, yielding slow-release polymer melts that release structurally intact and biologically active VNPs; in previous work, virus-like particles (VLPs) derived from a bacteriophage were considered; − here, we apply these technologies to the plant virus CPMV.…”

Biocomposite Thermoplastic Polyurethanes Containing Evolved Bacterial Spores as Living Fillers to Facilitate Polymer Disintegration

“…Additional loading above these critical concentrations led to a decreased toughness improvement, which was likely due to aggregation of spores within the matrix. High temperature and shear applied during the HME process, together with high spore concentration, has been found to induce the aggregation of spores in a TPU matrix 51 . Interestingly, the critical spore concentration of BC TPU HST (0.8 w/w%) was higher than BC TPU WT (0.4 ∼ 0.6 w/w%), while the ultimate toughness improvement of BC TPU HST (37%) was also more remarkable than that of BC TPU WT (25%) at their respective critical spore concentrations.…”

Hot melt extrusion: An emerging manufacturing method for slow and sustained protein delivery

“…(e) End‐point titers of anti‐Qβ IgG indicating the implanted PLGA/Qβ devices immunize as effectively as repeated Qβ administration. Adapted with permission from Duque et al (2018), Ghalanbor et al (2010), P. W. Lee, Maia, and Pokorski (2017), P. W. Lee, Shukla, et al (2017), and Lee et al (2015)…”

“…PEGylation can result in reaction products that can be difficult to separate and require extensive filtration or chromatography to remove unreacted components, and cause low yield and waste (Freitas & Abrahão‐Neto, 2010). Lee, Maia, and Pokorski (2017) then sought an alternative method of using milling to reduce the size of protein before HME, making it easier for proteins to homogeneously disperse in a PLGA substrate. The implants were manufactured with a self‐built syringe extruder heating at 95°C for 10 min.…”