We have previously identified extensive glycation, bound fatty acids and increased quantities of protein aggregates in commercially available recombinant HSA (rHSA) expressed in Oryza sativa (Asian ...rice) (OsrHSA) when compared to rHSA from other expression systems. We propose these differences may alter some attributes of nanoparticles fabricated with OsrHSA, as studies have associated greater quantities of aggregates with increased nanoparticle diameters. To determine if this is the case, nanoparticles were fabricated with OsrHSA from various suppliers using ethanol desolvation and subsequent glutaraldehyde cross-linking. All nanoparticles fabricated with OsrHSA showed larger diameters of approximately 20 to 90nm than particles fabricated with either defatted bovine serum albumin (DF-BSA) (100.9 ± 2.8nm) or human plasma albumin (pHSA) (112.0 ± 4.0nm). It was hypothesized that the larger nanoparticle diameters were due to the presence of bound fatty acids and this was confirmed through defatting OsrHSA prior to particle fabrication which yielded particles with diameters similar to those fabricated with pHSA. For additional conformation, DF-BSA was incubated with dodecanoic acid prior to desolvation yielding particles with significantly larger diameters. Further studies showed the increased nanoparticle diameters were due to the bound fatty acids modulating electrostatic interactions between albumin nanoparticles during the desolvation and not changes in protein structure, stability or generation of additional albumin oligomers. Finally the presence of dodecanoic acid was shown to improve doxorubicin loading efficiency onto preformed albumin nanoparticles.
The use of different expression systems to produce the same recombinant human protein can result in expression-dependent chemical modifications (CMs) leading to variability of structure, stability ...and immunogenicity. Of particular interest are recombinant human proteins expressed in plant-based systems, which have shown particularly high CM variability. In studies presented here, recombinant human serum albumins (rHSA) produced in Oryza sativa (Asian rice) (OsrHSA) from a number of suppliers have been extensively characterized and compared to plasma-derived HSA (pHSA) and rHSA expressed in yeast (Pichia pastoris and Saccharomyces cerevisiae). The heterogeneity of each sample was evaluated using size exclusion chromatography (SEC), reversed-phase high-performance liquid chromatography (RP-HPLC) and capillary electrophoresis (CE). Modifications of the samples were identified by liquid chromatography-mass spectrometry (LC-MS). The secondary and tertiary structure of the albumin samples were assessed with far U/V circular dichroism spectropolarimetry (far U/V CD) and fluorescence spectroscopy, respectively. Far U/V CD and fluorescence analyses were also used to assess thermal stability and drug binding. High molecular weight aggregates in OsrHSA samples were detected with SEC and supplier-to-supplier variability and, more critically, lot-to-lot variability in one manufactures supplied products were identified. LC-MS analysis identified a greater number of hexose-glycated arginine and lysine residues on OsrHSA compared to pHSA or rHSA expressed in yeast. This analysis also showed supplier-to-supplier and lot-to-lot variability in the degree of glycation at specific lysine and arginine residues for OsrHSA. Both the number of glycated residues and the degree of glycation correlated positively with the quantity of non-monomeric species and the chromatographic profiles of the samples. Tertiary structural changes were observed for most OsrHSA samples which correlated well with the degree of arginine/lysine glycation. The extensive glycation of OsrHSA from multiple suppliers may have further implications for the use of OsrHSA as a therapeutic product.
Microflow digital imaging (MDI) has become a widely accepted method for assessing sub-visible particles in pharmaceutical formulations however, to date; no data have been presented on the utility of ...this methodology when formulations include opaque vaccine adjuvants. This study evaluates the ability of MDI to assess sub-visible particles under these conditions. A Fluid Imaging Technologies Inc. FlowCAM® instrument was used to assess a number of sub-visible particle types in solution with increasing concentrations of AddaVax™, a nanoscale squalene-based adjuvant. With the objective (10X) used and the limitations of the sensor resolution, the instrument was incapable of distinguishing between sub-visible particles and AddaVax™ droplets at particle sizes less than 5 μm. The instrument was capable of imaging all particle types assessed (polystyrene beads, borosilicate glass, cellulose, polyethylene protein aggregate mimics, and lysozyme protein aggregates) at sizes greater than 5 μm in concentrations of AddaVax™ up to 50% (vol:vol). Reduced edge gradients and a decrease in measured particle sizes were noted as adjuvant concentrations increased. No significant changes in particle counts were observed for polystyrene particle standards and lysozyme protein aggregates, however significant reductions in particle counts were observed for borosilicate (80% of original) and cellulose (92% of original) particles. This reduction in particle counts may be due to the opaque adjuvant masking translucent particles present in borosilicate and cellulose samples. Although the results suggest that the utility of MDI for assessing sub-visible particles in high concentrations of adjuvant may be highly dependent on particle morphology, we believe that further investigation of this methodology to assess sub-visible particles in challenging formulations is warranted.
The importance and fast growth of therapeutic monoclonal antibodies, both innovator and biosimilar products, have triggered the need for the development of characterization methods at high resolution ...such as nuclear magnetic resonance (NMR) spectroscopy. However, the full power of NMR spectroscopy cannot be unleashed without labelling the mAb of interest with NMR-active isotopes. Here, we present strategies using either
Komagataella phaffii
(
Pichia pastoris
) or
Escherichia coli
that can be widely applied for the production of the antigen-binding fragment (Fab) of therapeutic antibodies of immunoglobulin G1 kappa isotype. The
E. coli
approach consists of expressing Fab fragments as a single polypeptide chain with a cleavable linker between the heavy and light chain in inclusion bodies, while
K. phaffii
secretes a properly folded fragment in the culture media. After optimization, the protocol yielded 10–45 mg of single chain adalimumab-Fab, trastuzumab-Fab, rituximab-Fab, and NISTmAb-Fab per liter of culture. Comparison of the 2D-
1
H-
15
N-HSQC spectra of each Fab fragment, without their polyhistidine tag and linker, with the corresponding Fab from the innovator product showed that all four fragments have folded into the correct conformation. Production of
2
H-
13
C-
15
N-adalimumab-scFab and
2
H-
13
C-
15
N-trastuzumab-scFab (>98% enrichment for all three isotopes) yielded NMR samples where all amide deuterons have completely exchanged back to proton during the refolding procedure.
The development of reference standards for nanoparticle sizing allows for cross laboratory studies and effective transfer of particle sizing methodology. To facilitate this, these reference standards ...must be stable upon long-term storage. Here, we examine factors that influence the properties of cross-linked albumin nanoparticles, fabricated with an ethanol desolvation method, when reconstituted from a lyophilized state. We demonstrate, with nanoparticle tracking analysis, no significant changes in mean particle diameter upon reconstitution of albumin nanoparticles fabricated with bovine serum albumin loaded with dodecanoic acid, when compared to nanoparticles fabricated with a fatty acid-free BSA. We attribute this stability to the modulation of nanoparticle charge-charge interactions at dodecanoic acid specific binding locations. Furthermore, we demonstrate this in a lyophilized state over six months when stored at - 80 °C. We also show that the reconstitution process is readily transferable between technicians and laboratories and further confirm our finding with dynamic light scattering analysis.
The incidence of Lyme disease (LD) in Canada and the United States has risen over the last decade, nearing 480,000 cases each year.
sensu lato, the causative agent of LD, is transmitted to humans ...through the bite of an infected tick, resulting in flu-like symptoms and often a characteristic bull's-eye rash. In more severe cases, disseminated bacterial infection can cause arthritis, carditis and neurological impairments. Currently, no vaccine is available for the prevention of LD in humans.
In this study, we developed a lipid nanoparticle (LNP)-encapsulated DNA vaccine encoding outer surface protein C type A (OspC-type A) of
Vaccination of C3H/HeN mice with two doses of the candidate vaccine induced significant OspC-type A-specific antibody titres and borreliacidal activity. Analysis of the bacterial burden following needle challenge with
(OspC-type A) revealed that the candidate vaccine afforded effective protection against homologous infection across a range of susceptible tissues. Notably, vaccinated mice were protected against carditis and lymphadenopathy associated with Lyme borreliosis.
Overall, the results of this study provide support for the use of a DNA-LNP platform for the development of LD vaccines.
Product excipients are used to confer a number of desirable properties on the drug substance to maintain or improve stability and facilitate drug delivery. This is especially important for products ...where the active pharmaceutical ingredient (API) is a recombinant protein. In this study, we aimed to determine if excipients and formulation conditions affect the structure and/or modulate the dynamics of the protein API of filgrastim products. Samples of uniformly labeled 15N-Met-granulocyte-colony stimulating factor (GCSF) were prepared at 100 μM (near formulation concentration) with various concentrations of individual components (polysorbate-20 and -80, sorbitol) and three pH values. Nuclear magnetic resonance (NMR) spectroscopy techniques were applied to measure chemical shift perturbation (CSP) to detect structural changes, and relaxation parameters (T 1, T 2, and heteronuclear Overhauser effect) were measured to probe the effects on protein backbone motions. In parallel, the same solution conditions were subjected to protein thermal unfolding studies monitored by circular dichroism spectropolarimetry (CD). Detergents (polysorbate-20 and 80) do not induce any observable changes on the protein structure and do not modify its dynamics at formulation concentration. Lowering pH to 4.0, a condition known to stabilize the conformation of filgrastim, as well as the addition of sorbitol produced changes of the fast motion dynamics in the nanosecond and picosecond timescale. NMR-derived order parameters, which measure the local conformational entropy of the protein backbone, show that lowering pH leads to a compaction of the four-helix bundle while the addition of sorbitol relaxes helices B and C, thereby reducing the mobility of loop CD. CSPs and measurements of protein dynamics via NMR-derived order parameters provide a description in structural and motional terms at an atomic resolution on how formulation components contribute to the stabilization of filgrastim products.
In recent years, lipid nanoparticles (LNPs) have emerged as a revolutionary technology for vaccine delivery. LNPs serve as an integral component of mRNA vaccines by protecting and transporting the ...mRNA payload into host cells. Despite their prominence in mRNA vaccines, there remains a notable gap in our understanding of the potential application of LNPs for the delivery of DNA vaccines. In this study, we sought to investigate the suitability of leading LNP formulations for the delivery of plasmid DNA (pDNA). In addition, we aimed to explore key differences in the properties of popular LNP formulations when delivering either mRNA or DNA. To address these questions, we compared three leading LNP formulations encapsulating mRNA- or pDNA-encoding firefly luciferase based on potency, expression kinetics, biodistribution, and immunogenicity. Following intramuscular injection in mice, we determined that RNA-LNPs formulated with either SM-102 or ALC-0315 lipids were the most potent (all p-values < 0.01) and immunogenic (all p-values < 0.05), while DNA-LNPs formulated with SM-102 or ALC-0315 demonstrated the longest duration of signal. Additionally, all LNP formulations were found to induce expression in the liver that was proportional to the signal at the injection site (SM102: r = 0.8787, p < 0.0001; ALC0315: r = 0.9012, p < 0.0001; KC2: r = 0.9343, p < 0.0001). Overall, this study provides important insights into the differences between leading LNP formulations and their applicability to DNA- and RNA-based vaccinations.
Previous studies have demonstrated that liposome-protein interactions can result in changes to the thermal stability of the protein. We utilized far-UV circular dichroism spectropolarimetry and ...fluorescence spectroscopy to investigate the interaction of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) liposomes with two recombinant human serum albumins (rHSA). We demonstrate that rHSA expressed in Oryza sativa (OsrHSA) has improved secondary structure thermal stability compared to rHSA expressed in Pichia pastoris (PprHSA). A similar stability profile was observed when comparing bovine serum albumin (BSA) and defatted bovine serum albumin (dfBSA), suggesting the presence of fatty acids may be responsible for the improved stability of OsrHSA. Addition of DPPC liposomes reduced the thermal stability of both OsrHSA and BSA, but not of PprHSA or dfBSA. DPPC liposomes may disrupt stabilizing native fatty acids on OsrHSA and BSA.
Human serum albumin (HSA) is a versatile and important protein for the pharmaceutical industry (Fanali et al., Mol. Aspects Med. 33(3) (2012) 209–290). Due to the potential transmission of pathogens ...from plasma sourced albumin, numerous expression systems have been developed to produce recombinant HSA (rHSA) (Chen et al., Biochim. Biophys. Acta (BBA)—Gen. Subj. 1830(12) (2013) 5515–5525; Kobayashi, Biologicals 34(1) (2006) 55–59). Based on our previous study showing increased glycation of rHSA expressed in Asian rice (Frahm et al., J. Phys. Chem. B 116(15) (2012) 4661–4670), both supplier-to-supplier and lot-to-lot variability of rHSAs from a number of expression systems were evaluated using reversed phase liquid chromatography linked with MS and MS/MS analyses. The data are associated with the research article ‘Determination of Supplier-to-Supplier and Lot-to-Lot Variability in Glycation of Recombinant Human Serum Albumin Expressed in Oryza sativa’ where further analysis of rHSA samples with additional biophysical methods can be found (Frahm et al., PLoS ONE 10(9) (2014) e109893). We determined that all rHSA samples expressed in rice showed elevated levels of arginine and lysine hexose glycation compared to rHSA expressed in yeast, suggesting that the extensive glycation of the recombinant proteins is a by-product of either the expression system or purification process and not a random occurrence.
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