Application of a Best Practice Approach Using Resonant Mass Measurement for Biotherapeutic Product Characterization.

Characterizing and quantifying subvisible particles in protein drug products is critical to ensuring product quality. A variety of analytical methods are used to detect and make meaningful measurements of subvisible particles. Resonant mass measurement (RMM) is a novel technology that characterizes the subvisible particle content of samples on a particle-by-particle basis. The technology presents great promise in the study of therapeutic protein products. As an emerging tool in the biopharmaceutical field, the best practices and limitations of RMM for protein products have not been well established. One key challenge of particle analysis is producing robust and reliable data, with high precision and accuracy, for particle characterization. In this study, we develop a set of possible best practices for RMM using a model protein system. We test the effects of these practices on the repeatability and reproducibility of particle measurements. Additionally, we present the data collected under a rigorously controlled set of operating conditions at 3 collaborating sites as well as a summary of the resulting optimal practices. In employing these practices, we successfully obtained improved relative standard deviation values and achieved high reproducibility and repeatability in both sizing and concentration measurement results over a broad range of sample volumes.

Impact of Sterilization Method on Protein Aggregation and Particle Formation in Polymer-Based Syringes.

The effects of sterilization methods on the storage stability of erythropoietin (EPO) in polymer-based syringes were assessed by quantifying protein oxidation, aggregation, and particle formation. Micro-particle counting and size exclusion chromatography coupled with a multi-angle light scattering detector demonstrated much lower levels of protein particles and aggregates for EPO stored for 12 weeks in steam-sterilized than in radiation (Rad)-sterilized syringes. Intermediate levels of damage were observed for EPO stored in ethylene oxide-sterilized syringes. HPLC analysis documented that the Rad-sterilized syringes caused increased oxidation of the protein during storage. In contrast, in the steam- and ethylene oxide-sterilized syringes EPO oxidation did not change. Analysis with electron spin resonance revealed that only Rad-sterilized syringes formed radicals in the syringe body, which persisted over the 12-week storage period. These results demonstrated that Rad-sterilization generated radicals in the syringes which in turn caused increased EPO oxidation, particle formation, and protein aggregation. Therefore, steam sterilization was shown to be a preferable sterilization method for the polymer-based syringe system when using biopharmaceutical drugs highly sensitive to oxidation, and particle formation and aggregation.

Characterization of Factors Affecting Nanoparticle Tracking Analysis Results With Synthetic and Protein Nanoparticles.

In many manufacturing and research areas, the ability to accurately monitor and characterize nanoparticles is becoming increasingly important. Nanoparticle tracking analysis is rapidly becoming a standard method for this characterization, yet several key factors in data acquisition and analysis may affect results. Nanoparticle tracking analysis is prone to user input and bias on account of a high number of parameters available, contains a limited analysis volume, and individual sample characteristics such as polydispersity or complex protein solutions may affect analysis results. This study systematically addressed these key issues. The integrated syringe pump was used to increase the sample volume analyzed. It was observed that measurements recorded under flow caused a reduction in total particle counts for both polystyrene and protein particles compared to those collected under static conditions. In addition, data for polydisperse samples tended to lose peak resolution at higher flow rates, masking distinct particle populations. Furthermore, in a bimodal particle population, a bias was seen toward the larger species within the sample. The impacts of filtration on an agitated intravenous immunoglobulin sample and operating parameters including "MINexps" and "blur" were investigated to optimize the method. Taken together, this study provides recommendations on instrument settings and sample preparations to properly characterize complex samples.

Characterization of Nanoparticle Tracking Analysis for Quantification and Sizing of Submicron Particles of Therapeutic Proteins.

Submicron particles may play important roles in therapeutic protein product quality, stability, and adverse effects in patients. However, quantitation of these particles has been challenging. Nanoparticle tracking analysis (NTA) is capable of both sizing and counting submicron particles. We investigated the effects of product and instrument parameters on NTA results for nanoparticle standards and therapeutic protein samples. To obtain proper particle size distributions, complete tracking numbers of at least 200 and 400 were required for latex nanobeads and protein nanoparticles, respectively. In addition, when set at suboptimal values, the minimum expected particle size parameter led to inaccurate sizing and counting for all particles types investigated. A syringe pump allowed for higher sampling volumes, and results were reproducible for nanoparticle sizing and counts at flow rates ≤7 µL/min. Finally, because therapeutic protein products are being formulated at relatively high protein concentrations, we investigated the effects of protein concentration on nanoparticle characterization. With high protein concentrations, nanoparticle sizing was not affected, whereas particle concentrations were significantly reduced. Linear relationships between particle count and dilution factor were obtained when a high protein concentration formulation was diluted into particle-free solutions at the same protein concentrations, but not when dilutions were made into buffer.

Small Molecule, NSC95397, Inhibits the CtBP1-Protein Partner Interaction and CtBP1-Mediated Transcriptional Repression.

Carboxyl-terminal binding protein (CtBP) is a transcriptional corepressor that suppresses multiple proapoptotic and epithelial genes. CtBP is overexpressed in many human cancers, and its overexpression increases stem cell-like features, epithelial-mesenchymal transition, and cancer cell survival. Knockdown of CtBP also increases apoptosis independent of p53 in cell culture. Therefore, targeting CtBP with small molecules that disrupt its interaction with transcription factor partners may be an effective cancer therapy. To elicit its corepressing effect, CtBP binds to a conserved peptide motif in each transcription factor partner. We developed an AlphaScreen high-throughput screening assay to monitor the interaction between CtBP and E1A (which mimics the interaction between CtBP and its transcriptional partners). We screened the LOPAC library of 1280 bioactive compounds and identified NSC95397, which inhibits the CtBP-E1A interaction (IC50 = 2.9 µM). The inhibitory activity of NSC95397 was confirmed using two secondary assays and a counterscreen. NSC95397 also behaved as a weak substrate of CtBP dehydrogenase activity and did not inhibit another dehydrogenase, lactase dehydrogenase. Finally, NSC95397 was able to disrupt CtBP-mediated transcriptional repression of a target gene. These studies present a new possibility for the development of a therapeutic agent targeting tumors through disrupting the CtBP transcriptional complex.

Allosteric inhibitors of the Eya2 phosphatase are selective and inhibit Eya2-mediated cell migration.

Eya proteins are essential co-activators of the Six family of transcription factors and contain a unique tyrosine phosphatase domain belonging to the haloacid dehalogenase family of phosphatases. The phosphatase activity of Eya is important for the transcription of a subset of Six1-target genes, and also directs cells to the repair rather than apoptosis pathway upon DNA damage. Furthermore, Eya phosphatase activity has been shown to mediate transformation, invasion, migration, and metastasis of breast cancer cells, making it a potential new drug target for breast cancer. We have previously identified a class of N-arylidenebenzohydrazide compounds that specifically inhibit the Eya2 phosphatase. Herein, we demonstrate that these compounds are reversible inhibitors that selectively inhibit the phosphatase activity of Eya2, but not Eya3. Our mutagenesis results suggest that this class of compounds does not bind to the active site and the binding does not require the coordination with Mg(2+). Moreover, these compounds likely bind within a site on the opposite face of the active site, and function as allosteric inhibitors. We also demonstrate that this class of compounds inhibits Eya2 phosphatase-mediated cell migration, setting the foundation for these molecules to be developed into chemical probes for understanding the specific function of the Eya2 phosphatase and to serve as a prototype for the development of Eya2 phosphatase specific anti-cancer drugs.

Identification of a selective small-molecule inhibitor series targeting the eyes absent 2 (Eya2) phosphatase activity.

Eya proteins are essential coactivators of the Six family of homeobox transcription factors and also contain a unique protein tyrosine phosphatase activity, belonging to the haloacid dehalogenase family of phosphatases. The phosphatase activity of Eya is important for a subset of Six1-mediated transcription, making this a unique type of transcriptional control. It is also responsible for directing cells to the repair instead of apoptosis pathway upon DNA damage. Furthermore, the phosphatase activity of Eya is critical for transformation, migration, invasion, and metastasis of breast cancer cells. Thus, inhibitors of the Eya phosphatase activity may be antitumorigenic and antimetastatic, as well as sensitize cancer cells to DNA damage-inducing therapies. In this article, we identified a previously unknown chemical series using high-throughput screening that inhibits the Eya2 phosphatase activity with IC(50)s ranging from 1.8 to 79 µM. Compound activity was confirmed using an alternative malachite green assay and H2AX, a known Eya substrate. Importantly, these Eya2 phosphatase inhibitors show specificity and do not significantly inhibit several other cellular phosphatases. Our studies identify the first selective Eya2 phosphatase inhibitors that can potentially be developed into chemical probes for functional studies of Eya phosphatase or into anticancer drugs in the future.

Matrix metalloproteinase-assisted triggered release of liposomal contents.

We offer a novel methodology for formulating liposomes by incorporating sequence-specific collagen-mimetic peptides such that they are specifically "uncorked" by a matrix metalloproteinase, MMP-9. By encapsulating carboxyfluorescein (as a self-quenching fluorescent dye), we demonstrate that the time-dependent release of the dye from liposomes is due to the specific enzymatic cleavage of the surface-exposed collagen-mimetic peptides. The specificity of such cleavage is attested by the fact that the liposomal "uncorking" and their content release occur only by MMP-9 and not by a general proteolytic enzyme, trypsin, despite the fact that the collagen mimetic peptides contain the trypsin cleavage site. The mechanistic details underlying the formulations of liposomes and their enzyme-selective "uncorking" and content release are discussed. Arguments are presented that such liposomes can be fine-tuned to serve as the drug delivery vehicles for the detection and treatment of various human diseases, which occur due to the overexpression of a variety of pathogenic matrix metalloproteinases.

"Uncorking" of liposomes by matrix metalloproteinase-9.

A triggered release methodology of liposomal contents via the enzyme MMP-9 is described.