How spray drying processing and solution composition can affect the mAbs stability in reconstituted solutions for subcutaneous injections. Part I: Contribution of processing stresses against composition.

Spray drying is increasingly being applied to process biopharmaceuticals, particularly monoclonal antibodies (mAbs). However, due to their protein nature, mAbs are susceptible to degradation when subjected to various stresses during a drying process. Despite extensive research in this domain, identifying the appropriate formulation composition and spray drying conditions remains a complex challenge, requiring further studies to enhance the understanding on how process and formulation parameters impact mAb stability in reconstituted solutions.

How spray drying processing and solution composition can affect the mAbs stability in reconstituted solutions for subcutaneous injections. Part II: Exploring each protein stabilizer effect.

Despite extensive research in spray drying of biopharmaceuticals, identifying the optimal formulation composition and process conditions to minimize the various stresses a biopharmaceutical undergoes during this drying process. The current study extends previous research on investigating how spray drying processing and solution composition can affect the stability of monoclonal antibodies (mAbs) in reconstituted solutions for subcutaneous injections. The decoupling process stresses on a model mAb (mAb-A) compared to the effect of coupled spray-drying stresses revealed that excipients and protein concentration had a more pronounced effect on stabilizing mAb-A against shear and thermal/dehydration stresses than spray drying operating conditions.

Native and Non-Native aggregation pathways of antibodies anticipated by cold-accelerated studies.

Assessment of cold stability is essential for manufacture and commercialization of biotherapeutics. Storage stability is often estimated by measuring accelerated rates at elevated temperature and using mathematical models (as the Arrhenius equation). Although, this strategy often leads to an underestimation of protein aggregation during storage.

Mechanistic understanding of metal-catalyzed oxidation of polysorbate 80 and monoclonal antibody in biotherapeutic formulations.

Surfactants are commonly used in biotherapeutic formulations to prevent the formation of aggregates and protect proteins from denaturation. Among them polysorbates are the most widely used. However, they are known to be prone to degradation, mainly via enzymatic hydrolysis and oxidation.

A Systematic Approach to Evaluating Closed System Drug-Transfer Devices During Drug Product Development.

The use of Closed System Drug-Transfer Devices (CSTDs) has increased significantly in recent years due to NIOSH and USP recommendations to use them during preparation of hazardous drugs. Mechanistic and material differences between CSTDs and traditional in-use components warrant an assessment of their impact on product quality and dosing accuracy. Using a combination of prevalent CSTDs with biologic molecules, we performed an extensive assessment of the effect of using CSTDs for dose preparation and observed no negative impact on product quality attributes.

Surfactant Protection Efficacy at Surfaces Varies with the Nature of Hydrophobic Materials.

Objective: Monoclonal antibodies are in contact with many different materials throughout their life cycle from production to patient administration. Plastic surfaces are commonly found in single use bags, syringes, perfusion bags and tubing and their hydrophobic nature makes them particularly prone for adsorption of therapeutic proteins. The addition of surfactants in therapeutic formulations aims at minimizing surface and interface adsorption of the active molecules.

Stress Factors in Protein Drug Product Manufacturing and Their Impact on Product Quality.

Injectable protein-based medicinal products (drug products, or DPs) must be produced by using sterile manufacturing processes to ensure product safety. In DP manufacturing the protein drug substance, in a suitable final formulation, is combined with the desired primary packaging (e.g.

Adsorption rate constants of therapeutic proteins and surfactants for material surfaces.

Adsorption of therapeutic proteins to material surfaces can be a pivotal issue in drug development, especially for low concentration products. Surfactants are used to limit adsorption losses. For each formulation component, surface adsorption is the result of a combination of its diffusion and surface adsorption rates.

Optimised hyperbolic microchannels for the mechanical characterisation of bio-particles.

The transport of bio-particles in viscous flows exhibits a rich variety of dynamical behaviour, such as morphological transitions, complex orientation dynamics or deformations. Characterising such complex behaviour under well controlled flows is key to understanding the microscopic mechanical properties of biological particles as well as the rheological properties of their suspensions. While generating regions of simple shear flow in microfluidic devices is relatively straightforward, generating straining flows in which the strain rate is maintained constant for a sufficiently long time to observe the objects' morphologic evolution is far from trivial.

Clogging of microfluidic constrictions by monoclonal antibody aggregates: role of aggregate shape and deformability.

The formation of aggregates in solutions of monoclonal antibodies is difficult to prevent. Even if the occurrence of large aggregates is rare, their existence can lead to partial or total clogging of constrictions in injection devices, with drastic effects on drug delivery. Little is known on the origin and characteristics of such clogging events.

Stress Factors in mAb Drug Substance Production Processes: Critical Assessment of Impact on Product Quality and Control Strategy.

The success of biotherapeutic development heavily relies on establishing robust production platforms. During the manufacturing process, the protein is exposed to multiple stress conditions that can result in physical and chemical modifications. The modified proteins may raise safety and quality concerns depending on the nature of the modification.

Nanoparticulate Impurities in Pharmaceutical-Grade Sugars and their Interference with Light Scattering-Based Analysis of Protein Formulations.

Purpose: In the present study we investigated the root-cause of an interference signal (100-200 nm) of sugar-containing solutions in dynamic light scattering (DLS) and nanoparticle tracking analysis (NTA) and its consequences for the analysis of particles in biopharmaceutical drug products.

Methods: Different sugars as well as sucrose of various purity grades, suppliers and lots were analyzed by DLS and NTA before and (only for sucrose) after treatment by ultrafiltration and diafiltration. Furthermore, Fourier transform infrared (FTIR) microscopy, scanning electron microscopy coupled energy-dispersive X-ray spectroscopy (SEM-EDX), and fluorescence spectroscopy were employed.

Small amounts of sub-visible aggregates enhance the immunogenic potential of monoclonal antibody therapeutics.

Purpose: Determine the effect of minute quantities of sub-visible aggregates on the in vitro immunogenicity of clinically relevant protein therapeutics.

Methods: Monoclonal chimeric (rituximab) and humanized (trastuzumab) antibodies were subjected to fine-tuned stress conditions to achieve low levels (<3% of total protein) of sub-visible aggregates. The effect of stimulating human dendritic cells (DC) and CD4(+) T cells with the aggregates was measured in vitro using cytokine secretion, proliferation and confocal microscopy.

Photodynamic therapy with conventional and PEGylated liposomal formulations of mTHPC (temoporfin): comparison of treatment efficacy and distribution characteristics in vivo.

A major challenge in the application of a nanoparticle-based drug delivery system for anticancer agents is the knowledge of the critical properties that influence their in vivo behavior and the therapeutic performance of the drug. The effect of a liposomal formulation, as an example of a widely-used delivery system, on all aspects of the drug delivery process, including the drug's behavior in blood and in the tumor, has to be considered when optimizing treatment with liposomal drugs, but that is rarely done. This article presents a comparison of conventional (Foslip®) and polyethylene glycosylated (Fospeg®) liposomal formulations of temoporfin (meta-tetra[hydroxyphenyl]chlorin) in tumor-grafted mice, with a set of comparison parameters not reported before in one model.

In vivo fluorescence imaging of IgG1 aggregates after subcutaneous and intravenous injection in mice.

Purpose: To monitor the biodistribution of IgG1 aggregates upon subcutaneous (SC) and intravenous (IV) administration in mice and measure their propensity to stimulate an early immune response.

Methods: A human mAb (IgG1) was fluorescently labeled, aggregated by agitation stress and injected in SKH1 mice through SC and IV routes. The biodistribution of monomeric and aggregated formulations was monitored over 47 days by fluorescence imaging and the early immune response was measured by quantifying the level of relevant cytokines in serum using a Bio-plex assay.

Stability of rituximab in freeze-dried formulations containing trehalose or melibiose under different relative humidity atmospheres.

The objective of the study was to compare the effectiveness of trehalose with that of melibiose in protecting a monoclonal antibody (rituximab) from aggregation, fragmentation, and secondary structure alterations during processing and subsequent storage. Because reducing disaccharides such as melibiose participate in Maillard reaction with proteins, especially in the presence of water, the lyophilizates were stored under different relative humidity (RH 5%, 11%, and 23%) atmospheres. Freeze drying was shown to cause clear alterations in rituximab secondary structure, an increase in noncovalent protein aggregation, and in some cases fragmentation.

Immunogenicity of different stressed IgG monoclonal antibody formulations in immune tolerant transgenic mice.

The presence of protein aggregates in biopharmaceutical formulations is of great concern for safety and efficacy reasons. The aim of this study was to correlate the type and amount of IgG monoclonal antibody aggregates with their immunogenic potential. IgG degradation was obtained by freeze-thawing cycles, pH-shift cycles, heating, shaking and metal-catalyzed oxidation.

Asymmetrical flow field-flow fractionation method for the analysis of submicron protein aggregates.

For the analysis of protein aggregates in the submicron size range, there is still a need for reliable, quantitative methods that can assist the development of therapeutic protein formulations. The aim of our study was to develop an asymmetrical flow field-flow fractionation (AF4) method for the analysis of protein aggregates in the size range of up to approximately 1000 nm. Method development was performed with polystyrene standard beads (60, 200, and 1000 nm) and a heat-stressed IgG formulation containing a substantial amount of submicron aggregates.

Transient molten globules and metastable aggregates induced by brief exposure of a monoclonal IgG to low pH.

The presence of aggregates in therapeutic protein formulations is of great concern due to quality, safety, and efficacy issues. Nonetheless, the mechanisms and kinetics of protein aggregation are only partly understood. In this study, metastable immunoglobulin G (IgG) aggregates induced by a brief exposure to pH 1 were kept at 4°C and analyzed over time by size-exclusion chromatography (SEC), nanoparticle tracking analysis, light obscuration, dynamic light scattering, fluorescence spectroscopy, and circular dichroism.

Detection and characterization of subvisible aggregates of monoclonal IgG in serum.

Purpose: To detect and characterize the aggregation of therapeutic monoclonal antibodies in undiluted biological fluids.

Methods: Fluorescently labeled subvisible IgG aggregates formed by applying either heat stress or by pH-shift were investigated immediately after addition to human serum, and after 24 h. Unstressed and stressed IgG formulations were analyzed by fluorescence single particle tracking, confocal laser scanning microscopy and flow cytometry.

Fluorescence single particle tracking for the characterization of submicron protein aggregates in biological fluids and complex formulations.

Purpose: To evaluate the potential of fluorescence single particle tracking (fSPT) for the characterization of submicron protein aggregates in human serum, plasma and formulations containing human serum albumin (HSA).

Methods: A monoclonal IgG was covalently labeled with a fluorescent dye and cross-linked with glutaraldehyde. IgG aggregates and fluorescent beads of 0.

Mass spectrometric analysis of intact human monoclonal antibody aggregates fractionated by size-exclusion chromatography.

Purpose: The aim of this study was to develop a method to characterize intact soluble monoclonal IgG1 antibody (IgG) oligomers by mass spectrometry.

Methods: IgG aggregates (dimers, trimers, tetramers and high-molecular-weight oligomers) were created by subjecting an IgG formulation to several pH jumps. Protein oligomer fractions were isolated by high performance size exclusion chromatography (HP-SEC), dialyzed against ammonium acetate pH 6.

Critical evaluation of Nanoparticle Tracking Analysis (NTA) by NanoSight for the measurement of nanoparticles and protein aggregates.

Purpose: To evaluate the nanoparticle tracking analysis (NTA) technique, compare it with dynamic light scattering (DLS) and test its performance in characterizing drug delivery nanoparticles and protein aggregates.

Methods: Standard polystyrene beads of sizes ranging from 60 to 1,000 nm and physical mixtures thereof were analyzed with NTA and DLS. The influence of different ratios of particle populations was tested.

Fluorescent molecular rotors as dyes to characterize polysorbate-containing IgG formulations.

Purpose: The aim was to evaluate fluorescent molecular rotors (DCVJ and CCVJ), which are mainly sensitive to viscosity, for the characterization of polysorbate-containing IgG formulations and compare them to the polarity-sensitive dyes ANS, Bis-ANS and Nile Red.

Methods: IgG formulations with polysorbate 20 or 80 were stressed below the aggregation temperature and analyzed by steady-state and time-resolved fluorescence and by HP-SEC with UV and fluorescent dye detection (Bis-ANS and CCVJ). Furthermore, commercial protein preparations of therapeutic proteins (Enbrel 50 mg, Humira 40 mg and MabThera 100 mg) were aggregated accordingly and analyzed with CCVJ fluorescence and HP-SEC.