An Interlaboratory Study to Identify Potential Visible Protein-Like Particle Standards.

Visible protein-like particle standards may improve visual inspection and/or appearance testing practices used in the biotechnology industry. They may improve assay performance resulting in better alignment and more standardized training among different companies. The National Institute of Standards and Technology (NIST) has conducted an interlaboratory study to test whether the standards under development mimic typical proteinaceous particles found in biotherapeutics and if they can be implemented during the visual inspection process. Fourteen organizations from industry and government have participated. A total of 20 labs from these 14 organizations participated with analysts from 6 formulation, 7 analytical, 4 quality control, and 3 manufacturing labs. The circulated samples consisted of abraded ethylene tetrafluoroethylene (ETFE) particles or photolithographic particles. The results consist of qualitative ratings, which varied substantially among organizations and within labs. Polydisperse ETFE particle suspensions, containing particles enriched in greater than 150 µm in size, were rated more favorably than the photolithographic particles by formulation and analytical scientists. The largest monodisperse photolithographic particles (approximately 300 µm in size) were favored equally compared to ETFE by all scientists. Solution modifications to decrease the settling rate or to alter optical properties of the ETFE solutions yielded lower ratings by the analysts. Both particle types received mixed ratings for their usability and for their application for visual inspection and for training purposes. Industry feedback will assist NIST in developing reference material(s) for visible protein-like particles.

Exon edited dystrophin rods in the hinge 3 region.

We have studied the properties of a panel of proteins engineered to be end-products of envisioned exon skipping therapy by antisense oligonucleotides, AONs, directed at exon 51 applied to relevant dystrophin defects causing Duchenne muscular dystrophy, DMD. Exon skipping therapy is a leading therapeutic strategy being investigated for the treatment of this devastating genetic disease. AONs targeting exon 51 have progressed furthest in human clinical trials. Exon 51 skipping is applicable to a variety of dystrophin defects found in different patients. Due to the differences in original defect, the end result of the therapy will be different in each case. An open question is whether these differences will produce significant differences in the dystrophin protein so edited. In this study we have identified differences in the stability, structure and lipid binding properties of these end-product proteins produced by exon 51 skipping repair.

A biophysical map of the dystrophin rod.

We have conducted a biophysical scan of the rod region of dystrophin, targeting all 24 single spectrin type repeat, STR, motifs and 23 2-STR tandem motifs. Of these 47 targets, we were able to express and purify 39 and have characterized them with regard to various stability metrics: thermodynamic stability as assessed by thermal and solvent denaturation, as well as resistance to proteolysis. We find that while all measured parameters varied greatly throughout the rod, there was no general stabilization of the 2-STR motifs over single STR motifs. However, stabilization by thermodynamic interaction was seen in six regions: strongly in D16:17 and D21:22 and to a lesser extent in D2:3, D4:5, D6:7 and D20:21. This indicates that these STRs interact structurally. In the rest of the rod, no cooperativity was seen and STRs appear to be thermodynamically independent. Stability also varied widely along the rod, with some motifs that are barely stable, beginning to unfold at physiological temperatures; these are largely found in the central rod region from D7 to D15. Regions of high stability were found in the interacting motifs, as well as a general trend toward increasing stability at the C-terminus of the rod. Interestingly, the rod region nNOS binding site occurs at such an interacting, very stable site, D16:17. Overall this describes a highly heterogeneous rod region.

Characterizing and Minimizing Aggregation and Particle Formation of Three Recombinant Fusion-Protein Bulk Antigens for Use in a Candidate Trivalent Rotavirus Vaccine.

In a companion paper, the structural integrity, conformational stability, and degradation mechanisms of 3 recombinant fusion-protein antigens comprising a non-replicating rotavirus (NRRV) vaccine candidate (currently being evaluated in early-stage clinical trials) are described. In this work, we focus on the aggregation propensity of the 3 NRRV antigens coupled to formulation development studies to identify common frozen bulk candidate formulations. The P2-VP8-P[8] antigen was most susceptible to shaking and freeze-thaw-induced aggregation and particle formation. Each NRRV antigen formed aggregates with structurally altered protein (with exposed apolar regions and intermolecular ß-sheet) and dimers containing a non-native disulfide bond. From excipient screening studies with P2-VP8-P[8], sugars or polyols (e.g., sucrose, trehalose, mannitol, sorbitol) and various detergents (e.g., Pluronic F-68, polysorbate 20 and 80, PEG-3350) were identified as stabilizers against aggregation. By combining promising additives, candidate bulk formulations were optimized to not only minimize agitation-induced aggregation, but also particle formation due to freeze-thaw stress of P2-VP8-P[8] antigen. Owing to limited material availability, stabilization of the P2-VP8-P[4] and P2-VP8-P[6] was confirmed with the lead candidate P2-VP8-P[8] formulations. The optimization of these bulk NRRV candidate formulations is discussed in the context of subsequent drug product formulations in the presence of aluminum adjuvants.

Racial Diversity in Hepatocellular Carcinoma in a Predominately African-American Population at an Urban Medical Center.

PURPOSE: Surveillance, treatment, and outcomes for African-American (AA) populations with hepatocellular carcinoma (HCC) remain under evaluated. This study evaluated demographics, surveillance, therapy, and outcomes for a predominately AA population. METHODS: The electronic medical records of a large health-care provider were used to identify 274 patients with visits for HCC between 2010 and 2017. Tumor size at diagnosis was defined by imaging with ≤ 5 cm being defined as "small." Surveillance for HCC was defined based on ultrasound (US) assessments. RESULTS: Patients were primarily AA (78%) and male (76%) with an average age at diagnosis of 62 years. Hepatitis C virus (HCV) was more likely to be a risk factor for the development of HCC in AA as compared to non-AA (92% vs 67%; p < 0.005). Surveillance rates were low (16% for AA vs 7% for non-AA). An aspartate aminotransferase platelet ratio index (APRI) value > 0.7 within 2 years of tumor diagnosis was a strong predictor for the risk of the development of HCC (86% AA vs 79 % non-AA). In this study, race was not a factor in treatment or outcomes, and most patients received tumor ablative treatment. CONCLUSION: Given the low surveillance rates and the demonstrated increased survival for patients with small tumors, ways to increase surveillance must be initiated. The results of this study demonstrate the need for physician/patient education on the importance of surveillance US. Further, this study supports routine assessment of APRI in AA patients in an effort to identify patients in whom intensive surveillance will significantly improve earlier detection of tumors.

Recombinant Subunit Rotavirus Trivalent Vaccine Candidate: Physicochemical Comparisons and Stability Evaluations of Three Protein Antigens.

Although live attenuated Rotavirus (RV) vaccines are available globally to provide protection against enteric RV disease, efficacy is substantially lower in low- to middle-income settings leading to interest in alternative vaccines. One promising candidate is a trivalent nonreplicating RV vaccine, comprising 3 truncated RV VP8 subunit proteins fused to the P2 CD4+ epitope from tetanus toxin (P2-VP8-P[4/6/8]). A wide variety of analytical techniques were used to compare the physicochemical properties of these 3 recombinant fusion proteins. Various environmental stresses were used to evaluate antigen stability and elucidate degradation pathways. P2-VP8-P[4] and P2-VP8-P[6] displayed similar physical stability profiles as function of pH and temperature while P2-VP8-P[8] was relatively more stable. Forced degradation studies revealed similar chemical stability profiles with Met1 most susceptible to oxidation, the single Cys residue (at position 173/172) forming intermolecular disulfide bonds (P2-VP8-P[6] was most susceptible), and Asn7 undergoing the highest levels of deamidation. These results are visualized in a structural model of the nonreplicating RV antigens. The establishment of key structural attributes of each antigen, along with corresponding stability-indicating methods, have been applied to vaccine formulation development efforts (see companion paper), and will be utilized in future analytical comparability assessments.

Structural Characterization and Physicochemical Stability Profile of a Double Mutant Heat Labile Toxin Protein Based Adjuvant.

A novel protein adjuvant double-mutant Escherichia coli heat-labile toxin, LT (R192G/L211A) or dmLT, is in preclinical and early clinical development with various vaccine candidates. Structural characterization and formulation development of dmLT will play a key role in its successful process development, scale-up/transfer, and commercial manufacturing. This work describes extensive analytical characterization of structural integrity and physicochemical stability profile of dmLT from a lyophilized clinical formulation. Reconstituted dmLT contained a heterogeneous mixture of intact holotoxin (AB5, ∼75%) and free B5 subunit (∼25%) as assessed by analytical ultracentrifugation and hydrophobic interaction chromatography. Intact mass spectrometry (MS) analysis revealed presence of Lys84 glycation near the native sugar-binding site in dmLT, and forced degradation studies using liquid chromatography-MS peptide mapping demonstrated specific Asn deamidation and Met oxidation sites. Using multiple biophysical measurements, dmLT was found most stable between pH 6.5 and 7.5 and at temperatures ≤50°C. In addition, soluble aggregates and particle formation were observed upon shaking stress. By identifying the physicochemical degradation pathways of dmLT using newly developed stability-indicating analytical methods from this study, we aim at developing more stable candidate formulations of dmLT that will minimize the formation of degradants and improve storage stability, as both a frozen bulk substance and eventually as a liquid final dosage form.

Development of a candidate stabilizing formulation for bulk storage of a double mutant heat labile toxin (dmLT) protein based adjuvant.

This work describes the formulation design and development of a novel protein based adjuvant, a double mutant of heat labile toxin (dmLT), based on knowledge of the protein's structural integrity and physicochemical degradation pathways. Various classes of pharmaceutical excipients were screened for their stabilizing effect on dmLT during exposure to thermal and agitation stresses as monitored by high throughput analytical assays for dmLT degradation. Sucrose, phosphate, sodium chloride, methionine and polysorbate-80 were identified as potential stabilizers that protected dmLT against either conformational destabilization, aggregation/particle formation or chemical degradation (e.g., Met oxidation and Lys glycation). Different combinations and concentrations of the selected stabilizers were then evaluated to further optimize dmLT stability while maintaining pharmaceutically acceptable ranges of solution pH and osmolality. The effect of multiple freeze-thaw (FT) cycles on the physical stability of candidate bulk formulations was also examined. Increasing the polysorbate-80 concentration to 0.1% in the lead candidate bulk formulation mitigated the loss of protein mass during FT. This formulation development study enabled the design of a new bulk formulation of the dmLT adjuvant and provides flexibility for future use in combination with a variety of different vaccine dosage forms with different antigens.

Effect of acrylodan conjugation and forced oxidation on the structural integrity, conformational stability, and binding activity of a glucose binding protein SM4 used in a prototype continuous glucose monitor.

Continuous glucose monitoring (CGM) devices offer diabetes patients a convenient approach to assist in controlling blood glucose levels. A prototype CGM has been developed that uses the emission profile of a polarity-sensitive fluorophore (acrylodan) conjugated to a glucose/galactose-binding protein (SM4-AC) to measure the concentration of glucose in vivo. During development, a decrease in the devices signal intensity was observed in vivo over time, which was postulated to be result of oxidative degradation of SM4-AC. A comprehensive physicochemical analysis of SM4-AC was pursued to identify potential mechanisms of signal intensity loss in this CGM during in vitro forced oxidation studies. An assessment of the structural integrity and conformational stability of SM4-AC indicated a relatively decreased polarity and lower tertiary structure stability compared to unconjugated protein (SM4). The stability and polarity of SM4-AC was also altered in the presence of H2 O2 . Furthermore, a time-dependent loss in the fluorescence signal of SM4-AC was observed when incubated with H2 O2 . An LC-MS peptide mapping analysis of these protein samples indicated that primarily two Met residues in SM4-AC were susceptible to oxidation. When these two residues were genetically altered to an amino acid not prone to oxidation, the glucose binding ability of the protein was retained and no loss of acrylodan fluorescence was observed in the presence of H2 O2 . Genetic alteration of these two residues is proposed as an effective approach to increase the long-term stability of SM4-AC within this prototype CGM in vivo.

Preformulation Characterization, Stabilization, and Formulation Design for the Acrylodan-Labeled Glucose-Binding Protein SM4-AC.

This study describes the physicochemical characterization, stabilization, and formulation design of SM4-AC, an acrylodan-labeled glucose/galactose-binding protein for use in a continuous glucose monitoring device. The physical stability profile of SM4-AC as a function of pH and temperature was monitored using a combination of biophysical techniques and the resulting physical stability profile was visualized using an empirical phase diagram. Forced degradation chemical stability studies (Asn deamidation, Met oxidation) of SM4-AC were performed using a combination of capillary isoelectric focusing, peptide mapping, and reversed-phase HPLC. Differential scanning fluorimetry was then employed to screen various pharmaceutical excipients for their ability to physically stabilize SM4-AC. An optimized formulation of 20% sucrose and 2.5 mM calcium chloride in 10 mM MES buffer, 150 mM NaCl at pH 6.0 increased the conformational stability of SM4-AC by 15°C. Accelerated and real-time stability studies were setup to compare the SM4-AC protein's physicochemical stability and glucose-binding activity in 2 formulations for up to 12 months. SM4-AC in an optimized formulation (vs the original formulation) showed improved physical stability, and similar chemical stability and glucose binding activity profiles during storage up to 52 weeks at various temperatures.

Challenges and opportunities of using liquid chromatography and mass spectrometry methods to develop complex vaccine antigens as pharmaceutical dosage forms.

Liquid chromatographic methods, combined with mass spectrometry, offer exciting and important opportunities to better characterize complex vaccine antigens including recombinant proteins, virus-like particles, inactivated viruses, polysaccharides, and protein-polysaccharide conjugates. The current abilities and limitations of these physicochemical methods to complement traditional in vitro and in vivo vaccine potency assays are explored in this review through the use of illustrative case studies. Various applications of these state-of-the art techniques are illustrated that include the analysis of influenza vaccines (inactivated whole virus and recombinant hemagglutinin), virus-like particle vaccines (human papillomavirus and hepatitis B), and polysaccharide linked to protein carrier vaccines (pneumococcal). Examples of utilizing these analytical methods to characterize vaccine antigens in the presence of adjuvants, which are often included to boost immune responses as part of the final vaccine dosage form, are also presented. Some of the challenges of using chromatographic and LC-MS as physicochemical assays to routinely test complex vaccine antigens are also discussed.

Formulation and stabilization of recombinant protein based virus-like particle vaccines.

Vaccine formulation development has traditionally focused on improving antigen storage stability and compatibility with conventional adjuvants. More recently, it has also provided an opportunity to modify the interaction and presentation of an antigen/adjuvant to the immune system to better stimulate the desired immune responses for maximal efficacy. In the last decade, there has been a paradigm shift in vaccine antigen and formulation design involving an improved physical understanding of antigens and a better understanding of the immune system. In addition, the discovery of novel adjuvants and delivery systems promises to further improve the design of new, more effective vaccines. Here we describe some of the fundamental aspects of formulation design applicable to virus-like-particle based vaccine antigens (VLPs). Case studies are presented for commercially approved VLP vaccines as well as some investigational VLP vaccine candidates. An emphasis is placed on the biophysical analysis of vaccines to facilitate formulation and stabilization of these particulate antigens.

Glassy-state stabilization of a dominant negative inhibitor anthrax vaccine containing aluminum hydroxide and glycopyranoside lipid A adjuvants.

During transport and storage, vaccines may be exposed to temperatures outside of the range recommended for storage, potentially causing efficacy losses. To better understand and prevent such losses, dominant negative inhibitor (DNI), a recombinant protein antigen for a candidate vaccine against anthrax, was formulated as a liquid and as a glassy lyophilized powder with the adjuvants aluminum hydroxide and glycopyranoside lipid A (GLA). Freeze-thawing of the liquid vaccine caused the adjuvants to aggregate and decreased its immunogenicity in mice. Immunogenicity of liquid vaccines also decreased when stored at 40°C for 8 weeks, as measured by decreases in neutralizing antibody titers in vaccinated mice. Concomitant with efficacy losses at elevated temperatures, changes in DNI structure were detected by fluorescence spectroscopy and increased deamidation was observed by capillary isoelectric focusing (cIEF) after only 1 week of storage of the liquid formulation at 40°C. In contrast, upon lyophilization, no additional deamidation after 4 weeks at 40°C and no detectable changes in DNI structure or reduction in immunogenicity after 16 weeks at 40°C were observed. Vaccines containing aluminum hydroxide and GLA elicited higher immune responses than vaccines adjuvanted with only aluminum hydroxide, with more mice responding to a single dose.

Understanding the relevance of local conformational stability and dynamics to the aggregation propensity of an IgG1 and IgG2 monoclonal antibodies.

Aggregation of monoclonal antibodies is often a multi-step process involving structural alterations in monomeric proteins and subsequent formation of soluble or insoluble oligomers. The role of local conformational stability and dynamics of native and/or partially altered structures in determining the aggregation propensity of monoclonal antibodies, however, is not well understood. Here, we investigate the role of conformational stability and dynamics of regions with distinct solvent exposure in determining the aggregation propensity of an IgG1 and IgG2 monoclonal antibody. The temperatures employed span the pre-unfolding range (10-40°C) and the onset temperatures (T onset ) for exposure of apolar residues (≈ 50°C), alterations in secondary structures (≈ 60°C) and initiation of visible aggregate formation (≈ 60°C). Solvent-exposed regions were found to precede solvent-shielded regions in an initiation of aggregation for both proteins. Such a process was observed upon alterations in overall tertiary structure while retaining the secondary structures in both the proteins. In addition, a greater dynamic nature of solvent-shielded regions in potential intermediates of IgG1 and the improved conformational stability increased its resistance to aggregation when compared to IgG2. These results suggest that local conformational stability and fluctuations of partially altered structures can influence the aggregation propensity of immunoglobulins.

Development of a stable virus-like particle vaccine formulation against Chikungunya virus and investigation of the effects of polyanions.

Chikungunya virus (CHIKV) is an alphavirus that infects millions of people every year, especially in the developing world. The selective expression of recombinant CHIKV capsid and envelope proteins results in the formation of self-assembled virus-like particles (VLPs) that have been shown to protect nonhuman primates against infection from multiple strains of CHIKV. This study describes the characterization, excipient screening, and optimization of CHIKV VLP solution conditions toward the development of a stable parenteral formulation. The CHIKV VLPs were found to be poorly soluble at pH 6 and below. Circular dichroism, intrinsic fluorescence, and static and dynamic light scattering measurements were therefore performed at neutral pH, and results consistent with the formation of molten globule structures were observed at elevated temperatures. A library of generally recognized as safe excipients was screened for their ability to physically stabilize CHIKV VLPs using a high-throughput turbidity-based assay. Sugars, sugar alcohols, and polyanions were identified as potential stabilizers and the concentrations and combinations of select excipients were optimized. The effects of polyanions were further studied, and while all polyanions tested stabilized CHIKV VLPs against aggregation, the effects of polyanions on conformational stability varied.

Metronidazole-induced encephalopathy in a patient with end-stage liver disease.

Purpose. Metronidazole-induced encephalopathy (MIE) has been rarely reported. We report a case in a patient with end-stage liver disease (ESLD). Summary. A 63-year-old male with ESLD secondary to hepatitis C virus presented with progressively worsening fatigue, slurred speech, aphasia, vomiting, and left-sided facial droop after completing a 2-week course of metronidazole for recurrent Clostridium difficile-associated diarrhea. He completed a previous course of metronidazole 3 weeks prior to presentation. He is on the liver transplant waiting list and has known hepatic encephalopathy. MRI revealed hyperintense T2 signals involving the bilateral dentate nuclei, inferior colliculi and splenium of the corpus callosum, and increased diffusion restriction at the splenium of the corpus callosum. His neurological function improved over the next several days. He underwent liver transplantation 6 days after admission. A follow-up MRI 6 weeks after presentation revealed resolution of abnormalities; however, paresthesias persisted 6 months after MIE diagnosis. Conclusion. An ESLD patient with hepatic encephalopathy developed MIE after a relatively short course of metronidazole. Metronidazole has been shown to accumulate in patients with ESLD. Increased awareness for neurotoxicity when using metronidazole in ESLD patients is warranted, especially in those with potentially confounding hepatic encephalopathy.