Subcutaneous Injection Site Pain of Formulation Matrices.

Purpose: The objective of this work was to systematically evaluate the effects of formulation composition on subcutaneous injection site pain (ISP) using matrices comprising of common pharmaceutical excipients.

Methods: Two randomized, blinded, crossover studies in healthy subjects were conducted at a single site, where subjects received 1 mL SC injections of the buffer matrices. ISP intensity was measured using a 100 mm visual analogue scale (VAS), which was then analyzed via heatmap, categorical grouping, subgroup analysis, and paired delta analysis.

A Novel Sample Preparation for Shotgun Proteomics Characterization of HCPs in Antibodies.

Residual host cell proteins (HCPs) in biopharmaceuticals derived from recombinant DNA technology can present potential safety risks to patients or compromise product stability. Thus, the downstream purification process is designed to demonstrate robust removal of these impurities. ELISA using polyclonal anti-HCP antibodies as reagents for capture, detection, and quantitation purposes is most commonly used to monitor HCP removal during process development, but this technique has limitations.

Endotoxin recovery using limulus amebocyte lysate (LAL) assay.

A phenomenon initially reported by Chen and Vinther in 2013 [1], and now commonly referred to as low endotoxin recovery (LER), has prompted the Food and Drug Administration (FDA) to request specific data demonstrating the capability of the LAL BET method (i.e., USP <85>) to recover endotoxin from spiked samples over time.

Subvisible (2-100 μm) particle analysis during biotherapeutic drug product development: Part 2, experience with the application of subvisible particle analysis.

Measurement and characterization of subvisible particles (including proteinaceous and non-proteinaceous particulate matter) is an important aspect of the pharmaceutical development process for biotherapeutics. Health authorities have increased expectations for subvisible particle data beyond criteria specified in the pharmacopeia and covering a wider size range. In addition, subvisible particle data is being requested for samples exposed to various stress conditions and to support process/product changes.

Method for characterization of PEGylated bioproducts in biological matrixes.

PEGylation of peptides and proteins has been widely used to enhance stability and reduce immunogenicity of biotherapeutics. Characterizing the degradation of these PEGylated products in biological fluids can yield essential information to support pharmacokinetic evaluations and provide clues about their in vivo properties useful for further molecular optimization. In this paper, we describe a novel and uncomplicated approach to characterize PEGylated peptides or proteins and their related degradation products in biological matrixes.

An automated robotic platform for rapid profiling oligosaccharide analysis of monoclonal antibodies directly from cell culture.

Oligosaccharides attached to Asn297 in each of the CH2 domains of monoclonal antibodies play an important role in antibody effector functions by modulating the affinity of interaction with Fc receptors displayed on cells of the innate immune system. Rapid, detailed, and quantitative N-glycan analysis is required at all stages of bioprocess development to ensure the safety and efficacy of the therapeutic. The high sample numbers generated during quality by design (QbD) and process analytical technology (PAT) create a demand for high-performance, high-throughput analytical technologies for comprehensive oligosaccharide analysis.

Identification of racemization sites using deuterium labeling and tandem mass spectrometry.

Racemization of amino acids is a common chemical degradation pathway observed in biopharmaceuticals and is particularly prevalent in synthetic peptides. The identification of racemized amino acid residue(s) by mass spectrometry is particularly challenging due to isobaric mass between the isomeric forms. In this paper, we present a novel methodology combining stable deuterium labeling with collisionally induced dissociation-tandem mass spectrometry (CID-MS/MS) to elucidate racemized amino acid residues in immunoglobulin samples.

Elucidation of PEGylation site with a combined approach of in-source fragmentation and CID MS/MS.

Poly(ethylene glycol) (PEG)ylation of peptides and proteins creates significant challenges for detailed structural characterization, such as PEG heterogeneity, site of addition and number of attached PEGylated moieties. Recently, we published a novel LC/MS methodology with a post-column addition of amines to obtain accurate masses of PEGylated peptides and proteins. The accurate masses can be used to assign the structures and number of attached PEGs [15], but the PEGylation site remains unclear in situations where multiple potential attachments are involved.

Linear epitope mapping by native mass spectrometry.

The identification of antigenic epitopes is important for the optimization of monoclonal antibodies (mAbs) intended as therapeutic agents. MS has proven to be a powerful tool for the study of noncovalent molecular interactions such as those involved in antibody-antigen (Ab-Ag) binding. In this work, we described a novel methodology for mapping a linear epitope based on direct mass spectrometric measurement of Ab-Ag complexes.

Analysis of 3-(acetylamino)-6-aminoacridine-derivatized oligosaccharides from recombinant monoclonal antibodies by liquid chromatography-mass spectrometry.

Glycosylation has been established as playing a pivotal role in various aspects of recombinant monoclonal antibodies (MAbs), ranging from pharmacokinetics to enhancement of effector function. Consequently, characterization of these oligosaccharides is of great importance and requires sensitive analytical techniques. Here we present a method for the rapid elucidation of 3-(acetylamino)-6-aminoacridine-labeled N-glycans present on MAbs using liquid chromatography-mass spectrometry.

In vitro stability of insulin lispro in continuous subcutaneous insulin infusion.

Background: The stability of insulin lispro for use in continuous subcutaneous insulin infusion (CSII) therapy was evaluated using a stress test incorporating high temperature and mechanical agitation combined with simulated basal/bolus administration.

Methods: Insulin lispro formulation contained in MiniMed 507c (Medtronic MiniMed, Sylmar, CA), H-TRONplus (Disetronic Medical Systems, St. Paul, MN), and D-TRON CSII (Disetronic Medtronic Systems) devices was subjected to a stress test involving exposure to elevated temperature (37 degrees C) and mechanical agitation (shaking at 100 strokes/min) for 7 days.

Amyloid fibrils of glucagon characterized by high-resolution atomic force microscopy.

Glucagon solutions at pH 2.0 were subjected to mechanical agitation at 37 degrees C in the presence of a hydrophobic surface to explore the details of aggregation and fiber formation. High-resolution intermittent-contact atomic force microscopy performed in solution revealed the presence of aggregates after 0.

Non-native intermediate conformational states of human growth hormone in the presence of organic solvents.

Purpose: Manufacturing processes expose protein pharmaceuticals to organic solvents that may perturb the native folded state, increasing the potential for irreversible aggregation or surface adsorption. The aim of this study was to characterize the conformational states of human growth hormone (hGH) in aqueous ethanolic solutions.

Methods: The higher order structure of hGH was investigated using far- and near-UV circular dichroism (CD) and fluorescence spectroscopy as orthogonal techniques, and the hydrodynamic size was monitored using dynamic light scattering.