Comprehensive Impurity Profiling of mRNA: Evaluating Current Technologies and Advanced Analytical Techniques.

In vitro transcription (IVT) of mRNA is a versatile platform for a broad range of biotechnological applications. Its rapid, scalable, and cost-effective production makes it a compelling choice for the development of mRNA-based cancer therapies and vaccines against infectious diseases. The impurities generated during mRNA production can potentially impact the safety and efficacy of mRNA therapeutics, but their structural complexity has not been investigated in detail yet. This study pioneers a comprehensive profiling of IVT mRNA impurities, integrating current technologies with innovative analytical tools. We have developed highly reproducible, efficient, and stability-indicating ion-pair reversed-phase liquid chromatography and capillary gel electrophoresis methods to determine the purity of mRNA from different suppliers. Furthermore, we introduced the applicability of microcapillary electrophoresis for high-throughput (<1.5 min analysis time per sample) mRNA impurity profiling. Our findings revealed that impurities are mainly attributed to mRNA variants with different poly(A) tail lengths due to aborted additions or partial hydrolysis and the presence of double-stranded mRNA (dsRNA) byproducts, particularly the dsRNA 3'-loop back form. We also implemented mass photometry and native mass spectrometry for the characterization of mRNA and its related product impurities. Mass photometry enabled the determination of the number of nucleotides of different mRNAs with high accuracy as well as the detection of their size variants [i.e., aggregates and partial and/or total absence of the poly(A) tail], thus providing valuable information on mRNA identity and integrity. In addition, native mass spectrometry provided insights into mRNA intact mass, heterogeneity, and important sequence features such as poly(A) tail length and distribution. This study highlights the existing bottlenecks and opportunities for improvement in the analytical characterization of IVT mRNA, thus contributing to the refinement and streamlining of mRNA production, paving the way for continued advancements in biotechnological applications.

C-Terminal Lysine Processing of IgG in Human Suction Blister Fluid: Implications for Subcutaneous Administration.

C-terminal lysine (CTK) is often classified as a potential critical quality attribute for therapeutic antibodies being developed for subcutaneous (SC) administration because of its potential to impact antibody SC bioavailability/pharmacokinetics (PK). This classification both inflates development costs and increases comparability risks for SC administration of antibodies. However, prior risk assessments of CTK have not fully considered biotransformation of CTK in the SC space, which may play an important role given that circulating carboxypeptidases in humans rapidly process CTK on intravenously administered antibodies. Here, CTK biotransformation in biofluid derived from human SC space was investigated. The representative fluid from the human SC space was sampled from 10 healthy human subjects using the suction blister method. Glycosylated antibody containing high levels of CTK (expressed using a carboxypeptidase D CRISPR/Cas9 knockout CHO cell line) was incubated in the collected suction blister fluids (SBFs), recovered using cognate antigen pulldowns, and characterized for remaining CTK levels using intact and reduced liquid chromatography-mass spectrometry (LC-MS) analysis. CTK processing (i.e., carboxypeptidase activity) was evident in all SBF and exhibited first-order kinetics with rate constants of 2.18 ± 0.57 d-1 (at 37 °C). PK simulations that integrated CTK processing pathways and their associated rate constants were subsequently performed using a range of clinically observed PK parameters for therapeutic antibodies, including atezolizumab- and pertuzumab-specific parameters. The impact of CTK content (even up to 100%) on SC PK outcomes such as bioavailability and Ctrough were modest (<14%) for all combinations of PK parameters tested in the sensitivity analysis. This study forms the cornerstone data package for derisking CTK as a PK liability for antibody SC programs and highlights the usefulness of fully considering biotransformation during product quality criticality assessments.

Hybrid peptides as platform for synchronized combination therapy.

Combination therapy, where two or more therapeutic agents are combined to target different cellular pathways, is an effective tool in cancer treatment but often difficult to execute. Here we present the collagen peptide-based platform that allows for synchronous and colocalized cellular delivery of three different agents. The peptide is a hybrid between collagen and cell penetrating peptide (CPP) that assembles into a heterotrimer helix and forms fully organic, high aspect ratio nanoparticles. The validity of the approach was tested with three chemically different agents (Paclitaxel, Doxorubicin, and 5-Fluorouracil; a combination used in clinical treatment of (ER)-positive and (PR)-positive breast cancer) conjugated to N-terminus of the peptide. The design of this peptide-based drug delivery system provides several advantages: it avoids drug loading problems; removes the need for orthogonal synthesis; and allows for colocalized delivery of up to three drugs (which leads to the same biodistribution for each drug). In addition, hybrid collagen/CPP peptides are known to enhance cellular uptake and improve solubility of drugs. The synergistic effect, in terms of enhanced efficacy, of the Paclitaxel-Doxorubicin-5-Fluorouracil combination was also calculated. We envision self-assembling peptides as a platform for drug codelivery that can be expanded into a library of personalized combinations that may also include other functionalities like targeting or imaging.

Identification and quantitation of hinge cysteinylation on endogenous IgG2 antibodies from human serum.

Cysteinylation is a post-translational modification (PTM) that occurs when a cysteine residue on a protein forms a disulfide bond with a terminal cysteine molecule. This PTM has been found in the hinge region of several recombinant therapeutic IgG2 antibodies, but the impact of cysteinylation on the safety and immunogenicity of therapeutics remains unclear. In this study, we characterized recombinant and endogenous IgG2 antibodies to quantify their levels of hinge cysteinylation, if present. To the best of our knowledge, this is the first study to identify and quantify hinge cysteinylation in endogenous IgG2 antibodies from healthy human serum. We used anti-IgG2 immunopurification of human serum to specifically enrich for endogenous IgG2 antibodies, and then subjected the resulting samples to Lys-C peptide mapping coupled with targeted mass spectrometry techniques. Using this analytical workflow, we found that all healthy human serum samples tested (N = 10) contained quantifiable levels of hinge cysteinylation (0.8 ± 0.3%) in their endogenous human IgG2s (IgG2-A isoform). These findings demonstrate that hinge cysteinylation in therapeutic IgG2s, at least up to a certain level, is well tolerated in humans and pose minimal safety or immunogenicity risks.

Higher Order Architecture of Designer Peptides Forms Bioinspired 10 nm siRNA Delivery System.

The higher-order architecture observed in biological systems, like viruses, is very effective in nucleic acid transport. The replications of this system has been attempted with both synthetic and naturally occurring polymers with mixed results. Here we describe a peptide/siRNA quaternary complex that functions as an siRNA delivery system. The rational design of a peptide assembly is inspired by the viral capsids, but not derived from them. We selected the collagen peptide (COL) to provide the structural stability and the folding framework, and hybridize it with the cell penetrating peptide (CPP) that allows for effective penetration of biological barriers. The peptide/siRNA quaternary complex forms stoichiometric, 10 nm nanoparticles, that show fast cellular uptake (<30 min), effective siRNA release, and gene silencing. The complex provides capsid-like protection for siRNA against nucleases without being immunostimulatory, or cytotoxic. Our data suggests that delivery vehicles based on synthetic quaternary structures that exhibit higher-order architecture may be effective in improving delivery and release of nucleic acid cargo.

Conjugation of Paclitaxel to Hybrid Peptide Carrier and Biological Evaluation in Jurkat and A549 Cancer Cell Lines.

Paclitaxel (PTX) is one of the most potent cancer drugs; however, its low solubility and strong systemic side effects limit its clinical applications. To overcome these issues, new drug formulations and chemical modifications have been proposed. In this study, we present conjugation of PTX to hybrid collagen-cell penetrating peptide (COL-CPP) carriers. The peptide carrier is highly soluble and utilizes a unique stabilization strategy: folding into a triple helix. Here, we report the formation of PTX-COL-CPP prodrug that has similar drug potency as free PTX when tested in Jurkat (human T lymphocyte of acute T cell leukemia) cells but not in A549 (human epithelial of lung carcinoma) cells. Confocal images and flow cytometry show that this behavior originates from lower cellular uptake of COL-CPP and endosomal entrapment of the prodrug in A549, but not in Jurkat cells.