Assessing Physicochemical Stability of Monoclonal Antibodies in a Simulated Subcutaneous Environment.

Monoclonal antibodies (mAbs) are being increasingly administered by the subcutaneous (SC) route compared to the traditional intravenous route. Despite the growing popularity of the subcutaneous route, our current knowledge regarding the intricate mechanistic changes happening in the formulation after injection in the subcutaneous space, as well as the in vivo stability of administered mAbs, remains quite limited. Changes in the protein environment as it transitions from a stabilized, formulated drug product in an appropriate container closure to the SC tissue environment can drastically impact the structural stability and integrity of the injected protein. Interactions of the protein with components of the extracellular matrix can lead to changes in its structure, potentially impacting both safety and efficacy. Investigating protein stability in the SC space can enable early assessment of risk and performance of subcutaneously administered proteins influencing clinical decisions and formulation development strategies. The Subcutaneous Injection Site Simulator (SCISSOR) is a novel in vitro system that mimics the subcutaneous injection site and models the events that a protein goes through as it transitions from a stabilized formulation environment to the dynamic physiological space. In this paper, we utilize the SCISSOR to probe for biophysical and chemical changes in seven mAbs post SC injection using a variety of analytical techniques. After 24 h, all mAbs demonstrated a relative decrease in conformational stability, an increase in fragmentation, and elevated acidic species. Higher order structure analysis revealed a deviation in the secondary structure from the standard and an increase in the number of unordered species. Our findings suggest an overall reduced stability of mAbs after subcutaneous administration. This reduced stability could have a potential impact on safety and efficacy. In vitro systems such as the SCISSOR combined with downstream analyses have potential to provide valuable information for assessing the suitability of lead molecules and aid in formulation design optimized for administration in the intended body compartment, thus improving chances of clinical success.

Utility of High Resolution 2D NMR Fingerprinting in Assessing Viscosity of Therapeutic Monoclonal Antibodies.

PURPOSE: The viscosity of highly concentrated therapeutic monoclonal antibody (mAb) formulations at concentrations ≥ 100 mg/mL can significantly affect the stability, processing, and drug product development for subcutaneous delivery. An early identification of a viscosity prone mAb during candidate selection stages are often beneficial for downstream processes. Higher order structure of mAbs may often dictate their viscosity behavior at high concentration. Thus it is beneficial to gauge or rank-order their viscosity behavior using noninvasive structural fingerprinting methods and to potentially screen for suitable viscosity lowering excipients. METHODS: In this study, Dynamic Light Scattering (DLS) and 2D NMR based methyl fingerprinting were used to correlate viscosity behavior of a set of Pfizer mAbs. The viscosities of mAbs were determined. Respective Fab and Fc domains were generated for studies. RESULT: Methyl fingerprinting of intact mAbs allows for differentiation of viscosity prone mAbs from well behaved ones even at 30-40 mg/ml, where bulk viscosity of the solutions are near identical. For viscosity prone mAbs, peak broadening and or distinct chemical shift changes were noted in intact and fragment fingerprints, unlike the well-behaved mAbs, indicative of protein protein interactions (PPI). CONCLUSION: Fab-Fab or Fab-Fc interactions may lead to formation of protein networks at high concentration. The early transients to these network formation may be manifested through peak broadening or peak shift in the 2D NMR spectrum of mAb/mAb fragments. Such insights go beyond rank ordering mAbs based on viscosity behavior, which can be obtained by other methods as well..

Correction to "Water-Soluble Blue Fluorescent Nonconjugated Polymer Dots from Hyaluronic Acid and Hydrophobic Amino Acids".

[This corrects the article DOI: 10.1021/acsomega.1c01343.].

Water-Soluble Blue Fluorescent Nonconjugated Polymer Dots from Hyaluronic Acid and Hydrophobic Amino Acids.

Fluorescent polymers have been increasingly investigated to improve their water solubility and biocompatibility to enhance their performance in drug delivery and theranostic applications. However, the environmentally friendly synthesis and dual functionality of such systems remain a challenge due to the complicated synthesis of conventional fluorescent materials. Herein, we generated a novel blue fluorescent polymer dot through chemical conjugation of hydrophobic amino acids to hyaluronic acid (HA) under one-pot green chemistry conditions. These nonconjugated fluorescent polymer dots (NCPDs) are water soluble, nontoxic to cells, have high fluorescence quantum yield, and can be used for in vitro bioimaging. HA-derived NCPDs exhibit excitation wavelength-dependent fluorescent properties. In addition, the NCPDs also show enhanced doxorubicin loading and delivery in naive and drug-resistant breast cancer cells in 2D and 3D tumor cellular systems. These results demonstrate the potential for successful synthetic scale-up and applications for HA-derived NCPDs.

Sulfation modulates the targeting properties of hyaluronic acid to P-selectin and CD44.

Many targeting strategies can be employed to direct nanoparticles to tumors for imaging and therapy. However, tumors display a dynamic, heterogeneous microenvironment that undergoes spatiotemporal changes, including the expression of targetable cell-surface biomarkers. Here, we develop a nanoparticle system to effectively target two receptors overexpressed in the microenvironment of aggressive tumors. Hyaluronic acid (HA) was regioselectivity modified using a multi-step synthetic approach to alter binding specificities for CD44 and P-selectin to tumor cell interaction. The dual-targeting strategy utilizes sulfate modifications on HA that targets P-selectin, in addition to native targeting of CD44, which exploits spatiotemporal alterations in the expression patterns of these two receptors in cancer sites. Using biophysical characterization and in vitro studies, we demonstrate that modified HA nanoparticles effectively targets both P-selectin+ and CD44+ cells, which lays the groundwork for future in vivo biomedical applications.

ω-Hydroxy isoprenoid bisphosphonates as linkable GGDPS inhibitors.

The enzyme geranylgeranyl diphosphate synthase (GGDPS) is a potential therapeutic target for multiple myeloma. Malignant plasma cells produce and secrete large amounts of monoclonal protein, and inhibition of GGDPS results in disruption of protein geranylgeranylation which in turn impairs intracellular protein trafficking. Our previous work has demonstrated that some isoprenoid triazole bisphosphonates are potent and selective inhibitors of GGDPS. To explore the possibility of selective delivery of such compounds to plasma cells, new analogues with an ω-hydroxy group have been synthesized and examined for their enzymatic and cellular activity. These studies demonstrate that incorporation of the ω-hydroxy group minimally impairs GGDPS inhibitory activity. Furthermore conjugation of one of the novel ω-hydroxy GGDPS inhibitors to hyaluronic acid resulted in enhanced cellular activity. These results will allow future studies to focus on the in vivo biodistribution of HA-conjugated GGDPS inhibitors.