Application of mechanistic modelling in membrane and fiber chromatography for purification of biotherapeutics - A review.

Mechanistic modelling is a simulation tool which has been effectively applied in downstream bioprocessing to model resin chromatography. Membrane and fiber chromatography are newer approaches that offer higher rates of mass transfer and consequently higher flow rates and reduced processing times. This review describes the key considerations in the development of mechanistic models for these unit operations. Mass transfer is less complex than in resin columns, but internal housing volumes can make modelling difficult, particularly for laboratory-scale devices. Flow paths are often non-linear and the dead volume is often a larger fraction of the overall volume, which may require more complex hydrodynamic models to capture residence time distributions accurately. In this respect, the combination of computational fluid dynamics with appropriate protein binding models is emerging as an ideal approach.

Engineered Design of the E-Helix Structure on Ferritin Nanoparticles.

Insertion of an immunogenic epitope at the C-terminus of ferritin has shown the potential to produce a stable and efficacious vaccine. There is however limited understanding of how C-terminus insertion affects ferritin protein stability. The E-helix at the C-terminus has attracted interest because there are contradictory reports as to whether it has a role in protein stabilization. Here, we report, for the first time, combining molecular dynamics simulation (MDS) with experiment to engineer the design of the E-helix at the C-terminus of engineered human ferritin heavy chain (F1) inserted with Epstein-Barr nuclear antigen 1 (EBNA1, E1) and flexible linker (L3) residues (to afford F1L3E1). Hot spots on the E-helix of the C-terminus were predicted by MDS at aa 167 (Glu) and aa 171 (Asp). Five (5) variants of F1L3E1 were constructed by considering hot spots and alteration of electrostatic or hydrophobic interfaces, namely, (1) C1, hot spots substituted with noncharged residue Gln; (2) C2, hot spots substituted with positively charged residue Arg; (3) C3, hydrophobic residues substituted with the most hydrophobic residues Val and Ile; (4) C4, hydrophobic residues substituted with the most hydrophilic residues Gln and Asn; and (5) C5, a heptad repeat structure in the E-helix disrupted by substituting "a" and "d" heptad residues with noncharged polar residue Gln. It was found that the E-helix is essential to maintain integrated protein stability and that changing the hydrophobic interface (C3 and C4) had more significant effects on protein folding and stability than changing the electrostatic interface (C1 and C2). It was confirmed by both MDS and experiment that variants C1, C2, and C5 were able to fold to form stable conformational structures with protein surface hydrophobicity similar to that of F1L3E1. However, they are less thermally stable than F1L3E1. Significant changes in hydrophobicity drove significant protein aggregation for variants C3 and C4. It is concluded that the molecular design of the C-terminus in engineered ferritin, especially the E-helix, is important to ensure the epitope-based chimeric vaccine is safe (aggregate free) and efficacious.

Mechanism Study of Thermally Induced Anti-Tumor Drug Loading to Engineered Human Heavy-Chain Ferritin Nanocages Aided by Computational Analysis.

Diverse drug loading approaches for human heavy-chain ferritin (HFn), a promising drug nanocarrier, have been established. However, anti-tumor drug loading ratio and protein carrier recovery yield are bottlenecks for future clinical application. Mechanisms behind drug loading have not been elaborated. In this work, a thermally induced drug loading approach was introduced to load anti-tumor drug doxorubicin hydrochloride (DOX) into HFn, and 2 functionalized HFns, HFn-PAS-RGDK, and HFn-PAS. Optimal conditions were obtained through orthogonal tests. All 3 HFn-based proteins achieved high protein recovery yield and drug loading ratio. Size exclusion chromatography (SEC) and transmission electron microscopy (TEM) results showed the majority of DOX loaded protein (protein/DOX) remained its nanocage conformation. Computational analysis, molecular docking followed by molecular dynamic (MD) simulation, revealed mechanisms of DOX loading and formation of by-product by investigating non-covalent interactions between DOX with HFn subunit and possible binding modes of DOX and HFn after drug loading. In in vitro tests, DOX in protein/DOX entered tumor cell nucleus and inhibited tumor cell growth.

Immunogenicity study of engineered ferritins with C- and N-terminus insertion of Epstein-Barr nuclear antigen 1 epitope.

Human ferritin heavy chain, an example of a protein nanoparticle, has recently been used as a vaccine delivery platform. Human ferritin has advantages of uniform architecture, robust thermal and chemical stabilities, and good biocompatibility and biodegradation. There is however a lack of understanding about the relationship between insertion sites in ferritin (N-terminus and C-terminus) and the corresponding humoral and cell-mediated immune responses. To bridge this gap, we utilized an Epstein-Barr Nuclear Antigen 1 (EBNA1) epitope as a model to produce engineered ferritin-based vaccines E1F1 (N-terminus insertion) and F1E1 (C-terminus insertion) for the prevention of Epstein-Barr virus (EBV) infections. X-ray crystallography confirmed the relative positions of the N-terminus insertion and C-terminus insertion. For N-terminus insertion, the epitopes were located on the exterior surface of ferritin, while for C-terminus insertion, the epitopes were inside the ferritin cage. Based on the results of antigen-specific antibody titers from in-vivo tests, we found that there was no obvious difference on humoral immune responses between N-terminus and C-terminus insertion. We also evaluated splenocyte proliferation and memory lymphocyte T cell differentiation. Both results suggested C-terminus insertion produced a stronger proliferative response and cell-mediated immune response than N-terminus insertion. C-terminus insertion of EBNA1 epitope was also processed more efficiently by dendritic cells (DCs) than N-terminus insertion. This provides new insight into the relationship between the insertion site and immunogenicity of ferritin nanoparticle vaccines.

Stability of Engineered Ferritin Nanovaccines Investigated by Combined Molecular Simulation and Experiments.

Human ferritin is regarded as an attractive and promising vaccine platform because of its uniform structure, good plasticity, and desirable thermal and chemical stabilities. Besides, it is biocompatible and presumed safe when used as a vaccine carrier. However, there is a lack of knowledge of how different antigen insertion sites on the ferritin nanocage impact the resulting protein stability and performance. To address this question, we selected Epstein-Barr nuclear antigen 1 as a model epitope and fused it at the DNA level with different insertion sites, namely, the N- and C-termini of ferritin, to engineer proteins E1F1 and F1E1, respectively. Protein properties including hydrophobicity and thermal, pH, and chemical stability were investigated both by molecular dynamics (MD) simulation and by experiments. Both methods demonstrate that the insertion site plays an important role in protein properties. The C-terminus insertion (F1E1) leads to a less hydrophobic surface and more tolerance to the external influence of high temperature, pH, and high concentration of chemical denaturants compared to N-terminus insertion (E1F1). Simulated protein hydrophobicity and thermal stability by MD were in high accordance with experimental results. Thus, MD simulation can be used as a valuable tool to engineer nanovaccine candidates, cutting down costs by reducing the experimental effort and accelerating vaccine design.

Engineered Human Heavy-Chain Ferritin with Half-Life Extension and Tumor Targeting by PAS and RGDK Peptide Functionalization.

Ferritin, one of the most investigated protein nanocages, is considered as a promising drug carrier because of its advantageous stability and safety. However, its short half-life and undesirable tumor targeting ability has limited its usage in tumor treatment. In this work, two types of functional peptides, half-life extension peptide PAS, and tumor targeting peptide RGDK (Arg-Gly-Asp-Lys), are inserted to human heavy-chain ferritin (HFn) at C-terminal through flexible linkers with two distinct enzyme cleavable sites. Structural characterizations show both HFn and engineered HFns can assemble into nanoparticles but with different apparent hydrodynamic volumes and molecular weights. RGDK peptide enhanced the internalization efficiency of HFn and showed a significant increase of growth inhibition against 4T1 cell line in vitro. Pharmacokinetic study in vivo demonstrates PAS peptides extended ferritin half-life about 4.9 times in Sprague Dawley rats. RGDK peptides greatly enhanced drug accumulation in the tumor site rather than in other organs in biodistribution analysis. Drug loaded PAS-RGDK functionalized HFns curbed tumor growth with significantly greater efficacies in comparison with drug loaded HFn.

Immunogenicity and Vaccine Efficacy Boosted by Engineering Human Heavy Chain Ferritin and Chimeric Hepatitis B Virus Core Nanoparticles.

Human heavy-chain ferritin (HFn) and hepatitis B virus core (HBc) are both nanoparticle proteins presenting a well-oriented architecture with constant size and shape, which can be engineered to carry epitopes on the surface of the nanoparticle protein cage, enabling vaccine design. This study aims to investigate the immunogenicity differences between engineered HFn and chimeric HBc bearing the same epitope. As a proof of concept, the model epitope Epstein-Barr nuclear antigen 1 (EBNA1) is inserted at the N-terminus of the HFn and HBc subunit to produce two vaccine candidates named EBNA1-HFn (E1F1) and EBNA1-HBc (E1H1), respectively. From in vivo immunogenicity studies, E1H1 demonstrates the capability to prompt significant humoral and cell-mediated immune responses in adjuvant-free formulation. When formulated with the aluminum hydroxide adjuvant, E1H1 produces approximately 5× higher titer and 2× stronger proliferation index (PI) than E1F1. These results confirm that the HBc carrier induces a stronger humoral immune response than HFn. On the other hand, from lymphocyte activation experiments, E1F1 induces a stronger cell-mediated immune response indicated by 5× more CD8+T cells and 2× more effector memory T cells in the E1F1 group versus the E1H1 group. Through this study, HFn and HBc are shown to be potentially effective vaccine carrier nanoparticles having subtly different immunological responses.