Nanoparticles reduce monocytes within the lungs to improve outcomes after influenza virus infection in aged mice.

Older people exhibit dysregulated innate immunity to respiratory viral infections, including influenza and SARS-CoV-2, and show an increase in morbidity and mortality. Nanoparticles are a potential practical therapeutic that could reduce exaggerated innate immune responses within the lungs during viral infection. However, such therapeutics have not been examined for effectiveness during respiratory viral infection, particular in aged hosts.

Tolerogenic Immune-Modifying Nanoparticles Encapsulating Multiple Recombinant Pancreatic β Cell Proteins Prevent Onset and Progression of Type 1 Diabetes in Nonobese Diabetic Mice.

Type 1 diabetes (T1D) is an autoimmune disease characterized by T and B cell responses to proteins expressed by insulin-producing pancreatic β cells, inflammatory lesions within islets (insulitis), and β cell loss. We previously showed that Ag-specific tolerance targeting single β cell protein epitopes is effective in preventing T1D induced by transfer of monospecific diabetogenic CD4 and CD8 transgenic T cells to NOD. mice.

ONP-302 Nanoparticles Inhibit Tumor Growth By Altering Tumor-Associated Macrophages And Cancer-Associated Fibroblasts.

In this study, we evaluated the ability of negatively charged bio-degradable nanoparticles, ONP- 302, to inhibit tumor growth. Therapeutic treatment with ONP-302 in vivo resulted in a marked delay in tumor growth in three different syngeneic tumor models in immunocompetent mice. ONP- 302 efficacy persisted with depletion of CD8+ T cells in immunocompetent mice and also was effective in immune deficient mice.

Exploring the linkage between cell culture process parameters and downstream processing utilizing a plackett-burman design for a model monoclonal antibody.

Linkage of upstream cell culture with downstream processing and purification is an aspect of Quality by Design crucial for efficient and consistent production of high quality biopharmaceutical proteins. In a previous Plackett-Burman screening study of parallel bioreactor cultures we evaluated main effects of 11 process variables, such as agitation, sparge rate, feeding regimens, dissolved oxygen set point, inoculation density, supplement addition, temperature, and pH shifts. In this follow-up study, we observed linkages between cell culture process parameters and downstream capture chromatography performance and subsequent antibody attributes.

The Effect of Oseltamivir on the Disease Progression of Lethal Influenza A Virus Infection: Plasma Cytokine and miRNA Responses in a Mouse Model.

Lethal influenza A virus infection leads to acute lung injury and possibly lethal complications. There has been a continuous effort to identify the possible predictors of disease severity. Unlike earlier studies, where biomarkers were analyzed on certain time points or days after infection, in this study biomarkers were evaluated over the entire course of infection.

Improved Stability of a Model IgG3 by DoE-Based Evaluation of Buffer Formulations.

Formulating appropriate storage conditions for biopharmaceutical proteins is essential for ensuring their stability and thereby their purity, potency, and safety over their shelf-life. Using a model murine IgG3 produced in a bioreactor system, multiple formulation compositions were systematically explored in a DoE design to optimize the stability of a challenging antibody formulation worst case. The stability of the antibody in each buffer formulation was assessed by UV/VIS absorbance at 280 nm and 410 nm and size exclusion high performance liquid chromatography (SEC) to determine overall solubility, opalescence, and aggregate formation, respectively.

Modern analytics for synthetically derived complex drug substances: NMR, AFFF-MALS, and MS tests for glatiramer acetate.

Glatiramer acetate (GA) is a mixture of synthetic copolymers consisting of four amino acids (glutamic acid, lysine, alanine, and tyrosine) with a labeled molecular weight range of 5000 to 9000 Da. GA is marketed as Copaxone™ by Teva for the treatment of multiple sclerosis. Here, the agency has evaluated the structure and composition of GA and a commercially available comparator, Copolymer-1.

Marketplace Analysis of Conjugated Estrogens: Determining the Consistently Present Steroidal Content with LC-MS.

Conjugated estrogens purified from pregnant mares urine has been used as estrogen hormone replacement therapy since 1942. Previously, methods were proposed to identify and quantify the components of this complex mixture but ultimately were withdrawn due to incomplete characterization of the product and difficulties in transferring the method between laboratories. The aim of the current study is to develop a LC method that can reliably detect multiple steroidal components in conjugated estrogen tablets and measure their relative amount.

Performance metrics for evaluating system suitability in liquid chromatography--Mass spectrometry peptide mass mapping of protein therapeutics and monoclonal antibodies.

The use of liquid chromatography--mass spectrometry (LC-MS) for the characterization of proteins can provide a plethora of information related to their structure, including amino acid sequence determination and analysis of posttranslational modifications. The variety of LC-MS based applications has led to the use of LC-MS characterization of therapeutic proteins and monoclonal antibodies as an integral part of the regulatory approval process. However, the improper use of an LC-MS system, related to intrinsic instrument limitations, improper tuning parameters, or poorly optimized methods may result in the production of low quality data.

Primary Sequence Confirmation of a Protein Therapeutic Using Top Down MS/MS and MS(3).

Mass spectrometry has gained widespread acceptance for the characterization of protein therapeutics as a part of the regulatory approval process. Improvements in mass spectrometer sensitivity, resolution, and mass accuracy have enabled more detailed and confident analysis of larger biomolecules for confirming amino acid sequences, assessing sequence variants, and characterizing post translational modifications. This work demonstrates the suitability of a combined approach using intact MS and multistage top down MS/MS analyses for the characterization of a protein therapeutic drug.

Comparison of Traditional 2-AB Fluorescence LC-MS/MS and Automated LC-MS for the Comparative Glycan Analysis of Monoclonal Antibodies.

Monoclonal antibody therapeutics are a heterogeneous mixture of glycoforms. Multiple methods exist for defining the glycan composition and relative abundance of species present. In the current report, two MS-based methods were compared for their ability to both identify glycans and monitor differences in the glycoprofile.

Bioreactor process parameter screening utilizing a Plackett-Burman design for a model monoclonal antibody.

Consistent high-quality antibody yield is a key goal for cell culture bioprocessing. This endpoint is typically achieved in commercial settings through product and process engineering of bioreactor parameters during development. When the process is complex and not optimized, small changes in composition and control may yield a finished product of less desirable quality.

Liquid Chromatography-High Resolution Mass Spectrometry for Peptide Drug Quality Control.

A liquid chromatography-high resolution mass spectrometry (LC-HRMS) method was developed using three peptide drugs: salmon calcitonin, bivalirudin, and exenatide as model systems to assess the suitability of this approach for monitoring peptide drug product quality. Calcitonin and its related impurities displayed linear responses over the range from 0.1 to 10 μM (R (2) values for calcitonin salmon, Glu(14)-calcitonin, and acetyl-calcitonin were 0.

Direct site-specific glycoform identification and quantitative comparison of glycoprotein therapeutics: imiglucerase and velaglucerase alfa.

Gaucher disease, the most common lysosomal metabolic disorder, can be treated with enzyme replacement therapy (ERT). Recombinant human glucocerebrosidase imiglucerase (Cerezyme(®)), produced in Chinese hamster ovary cells, has been used for ERT of Gaucher disease for 20 years. Another recombinant glucocerebrosidase velaglucerase alfa (VPRIV), expressed in a human fibroblast cell line, was approved by the US Food and Drug Administration in 2010.

Development and validation of a liquid-chromatography tandem mass spectrometry method to determine in vitro and in vivo histamine release.

Histamine is an important biogenic amine involved in regulating numerous physiological and pathophysiological processes in humans and animals. To date, there have been very few studies focused on developing and validating sensitive liquid-chromatography-tandem mass spectrometric (LC-MS/MS) assays capable of quantitative trace level histamine analysis in biological matrices. In the present study, a rapid and sensitive LC-MS/MS assay, amenable to high throughput analysis was developed and validated to characterize in vitro and in vivo histamine release.

Pharmacodynamic evaluation of the activities of six parenteral vancomycin products available in the United States.

A recent report found that generic parenteral vancomycin products may not have in vivo efficacies equivalent to those of the innovator in a neutropenic murine thigh infection model despite having similar in vitro microbiological activities and murine serum pharmacokinetics. We compared the in vitro and in vivo activities of six of the parenteral vancomycin products available in the United States. The in vitro assessments for the potencies of the vancomycin products included MIC/minimal bactericidal concentration (MBC) determinations, quantifying the impact of human and murine serum on the MIC values, and time-kill studies.

Modern analytics for naturally derived complex drug substances: NMR and MS tests for protamine sulfate from chum salmon.

This work describes orthogonal NMR and MS tests for the structure and composition of the drug protamine sulfate derived from chum salmon. The spectral response pattern obtained by 1D-(1)H-NMR and MS methods from salmon protamine, a mixture of four predominant peptide chains, is dependent on the amino acid sequence and abundance of each peptide. Thus, an assay was developed based on the ratios of alanine, glycine and arginine amino acid residue NMR peaks (relative to the arginine CδH proton signal) in this mixture that are unique to the salmon source.

Post-translational structural modifications of immunoglobulin G and their effect on biological activity.

The size, heterogeneity, and biological production process of protein therapeutics like monoclonal antibodies create unique challenges for their analysis and regulation compared with small molecules. Complete structural characterization of a molecule 1000-fold heavier than aspirin is no small feat. Biological post-translational modifications such as glycosylation further complicate their characterization and regulation.

More methemoglobin is produced by benzocaine treatment than lidocaine treatment in human in vitro systems.

The clinical use of local anesthetic products to anesthetize mucous membranes has been associated with methemoglobinemia (MetHba), a serious condition in which the blood has reduced capacity to carry oxygen. An evaluation of spontaneous adverse event reporting of MetHba submitted to FDA through 2013 identified 375 reports associated with benzocaine and 16 reports associated with lidocaine. The current study was performed to determine the relative ability of benzocaine and lidocaine to produce methemoglobin (MetHb) in vitro.

Identification of site-specific heterogeneity in peptide drugs using intact mass spectrometry with electron transfer dissociation.

Rationale: Protamine sulfate is a peptide drug product consisting of multiple basic peptides. As traditional high-performance liquid chromatography (HPLC) separation methods may not resolve these peptides, as well as any possible peptide-related impurities, a method utilizing top-down mass spectrometry was developed for the characterization of complex peptide drug products, including any low-level impurities, which is described in this study.

Methods: Herring protamine sulfate was used as a model system to demonstrate the applicability of the method.

Analytical techniques and bioactivity assays to compare the structure and function of filgrastim (granulocyte-colony stimulating factor) therapeutics from different manufacturers.

The FDA has approved more than 100 protein and peptide drugs with hundreds more in the pipeline (Lanthier et al. in Nat Rev Drug Discov 7(9):733-737, 2008). Many of these originator biologic products are now coming off patent and are being manufactured by alternate methods than the innovator as follow-on drugs.

Analyses of marketplace tacrolimus drug product quality: bioactivity, NMR and LC-MS.

Tacrolimus (FK506) is a potent, narrow therapeutic index, immunosuppressive drug used to avoid organ rejection in patients that have undergone organ transplantation. Recent clinical reports suggested a significant reduction in the tacrolimus concentration/dose ratio in the plasma of liver and kidney recipients when the reference listed drug was substituted with a generic drug. In response to these concerns about switching between tacrolimus from different approved manufacturers during treatment, the FDA initiated purity, potency and quality studies of the innovator and generic tacrolimus products available in the US marketplace.

Structural comparison of two anti-CD20 monoclonal antibody drug products using middle-down mass spectrometry.

Liquid chromatography-mass spectrometry (LC-MS) is an information rich analytical tool that can provide fast, robust and sensitive characterization of protein therapeutics for quality assurance and structural comparison. Herein, structural characterization of two anti-CD20 monoclonal antibodies obtained from two different sources was performed using a middle-down LC-MS strategy to determine if they can be analytically differentiated. Through the use of a specific enzymatic digestion method using IdeS with subsequent LC-MS analysis, we show that the anti-CD20 monoclonal antibody that has been approved by the FDA can be partially characterized and differentiated analytically from an Indian sourced product that lacks FDA approval.

Direct approach for qualitative and quantitative characterization of glycoproteins using tandem mass tags and an LTQ Orbitrap XL electron transfer dissociation hybrid mass spectrometer.

The application of multiplexed isobaric tandem mass tag (TMT) labeling and an LTQ Orbitrap XL ETD (electron transfer dissociation) hybrid mass spectrometer as a direct approach for qualitative and quantitative characterization of glycoproteins is reported. Bovine fetuin was used as a model glycoprotein in this study. For online liquid chromatography-mass spectrometry (LC-MS) analysis, high-resolution, mass accurate full scan MS spectra were acquired in the Orbitrap mass analyzer followed by data-dependent tandem mass spectrometry (MS/MS) with alternating collision-induced dissociation (CID), ETD, and higher-energy collisional dissociation (HCD) scans.