Biophysical Determinants for the Viscosity of Concentrated Monoclonal Antibody Solutions.

Monoclonal antibodies (mAbs) are particularly relevant for therapeutics due to their high specificity and versatility, and mAb-based drugs are hence used to treat numerous diseases. The increased patient compliance of self-administration motivates the formulation of products for subcutaneous (SC) administration. The associated challenge is to formulate highly concentrated antibody solutions to achieve a significant therapeutic effect, while limiting their viscosity and preserving their physicochemical stability. Protein-protein interactions (PPIs) are in fact the root cause of several potential problems concerning the stability, manufacturability, and delivery of a drug product. The understanding of macroscopic viscosity requires an in-depth knowledge on protein diffusion, PPIs, and self-association/aggregation. Here, we study the self-diffusion of different mAbs of the IgG1 subtype in aqueous solution as a function of the concentration and temperature by quasi-elastic neutron scattering (QENS). QENS allows us to probe the short-time self-diffusion of the molecules and therefore to determine the hydrodynamic mAb cluster size and to gain information on the internal mAb dynamics. Small-angle neutron scattering (SANS) is jointly employed to probe structural details and to understand the nature and intensity of PPIs. Complementary information is provided by molecular dynamics (MD) simulations and viscometry, thus obtaining a comprehensive picture of mAb diffusion.

Predictive modeling of concentration-dependent viscosity behavior of monoclonal antibody solutions using artificial neural networks.

Solutions of monoclonal antibodies (mAbs) can show increased viscosity at high concentration, which can be a disadvantage during protein purification, filling, and administration. The viscosity is determined by protein-protein-interactions, which are influenced by the antibody's sequence as well as solution conditions, like pH, buffer type, or the presence of salts and other excipients. To predict viscosity, experimental parameters, like the diffusion interaction parameter (kD), or computational tools harnessing information derived from primary sequence, are often used, but a reliable predictive tool is still missing. We present a modeling approach employing artificial neural networks (ANNs) using experimental factors combined with simulation-derived parameters plus viscosity data from 27 highly concentrated (180 mg/mL) mAbs. These ANNs can be used to predict if mAbs exhibit problematic viscosity at distinct concentrations or to model viscosity-concentration-curves.

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., syringe, cartridge, or vial) that guarantees product integrity and enables transportation, storage, handling and clinical administration. The protein DP is exposed to several stress conditions during each of the unit operations in DP manufacturing, some of which can be detrimental to product quality. For example, particles, aggregates and chemically-modified proteins can form during manufacturing, and excessive amounts of these undesired variants might cause an impact on potency or immunogenicity. Therefore, DP manufacturing process development should include identification of critical quality attributes (CQAs) and comprehensive risk assessment of potential protein modifications in process steps, and the relevant steps must be characterized and controlled. In this commentary article we focus on the major unit operations in protein DP manufacturing, and critically evaluate each process step for stress factors involved and their potential effects on DP CQAs. Moreover, we discuss the current industry trends for risk mitigation, process control including analytical monitoring, and recommendations for formulation and process development studies, including scaled-down runs.

Adsorption of non-ionic surfactant and monoclonal antibody on siliconized surface studied by neutron reflectometry.

The adsorption of monoclonal antibodies (mAbs) on hydrophobic surfaces is known to cause protein aggregation and degradation. Therefore, surfactants, such as Poloxamer 188, are widely used in therapeutic formulations to stabilize mAbs and protect mAbs from interacting with liquid-solid interfaces. Here, the adsorption of Poloxamer 188, one mAb and their competitive adsorption on a model hydrophobic siliconized surface is investigated with neutron scattering coupled with contrast variation to determine the molecular structure of adsorbed layers for each case. Small angle neutron scattering measurements of the affinity of Poloxamer 188 to this mAb indicate that there is negligible binding at these solution conditions. Neutron reflectometry measurements of the mAb show irreversible adsorption on the siliconized surface, which cannot be washed off with neat buffer. Poloxamer 188 can be adsorbed on the surface already occupied by mAb, which enables partial removal of some adsorbed mAb by washing with buffer. The adsorption of the surfactant introduces significant conformational changes for mAb molecules that remain on the surface. In contrast, if the siliconized surface is first saturated with the surfactant, no adsorption of mAb is observed. Competitive adsorption of mAb and Poloxamer 188 from solution leads to a surface dominantly occupied with surfactant molecules, whereas only a minor amount of mAb absorbs. These findings clearly indicate that Poloxamer 188 can protect against mAb adsorption as well as modify the adsorbed conformation of previously adsorbed mAb.

Protein-Polydimethylsiloxane Particles in Liquid Vial Monoclonal Antibody Formulations Containing Poloxamer 188.

Surfactants play an important role in stabilizing proteins in liquid formulations against aggregate/particle formation during processing, handling, storage, and transportation. Only 3 surfactants are currently used in marketed therapeutic protein formulations: polysorbate 20, polysorbate 80, and poloxamer 188. While polysorbates are the most widely used surfactants, their intrinsic oxidative and hydrolytic degradation issues highlights the importance of alternative surfactants such as poloxamer 188. Here, we compare polysorbates and poloxamer 188 with regards to their stabilizing properties under various stress and storage conditions for several monoclonal antibody formulations. Our data shows that poloxamer 188 can provide suitable protection of monoclonal antibodies against interfacial stress in liquid formulations in vials. However, visible protein-polydimethylsiloxane (PDMS; silicone oil) particles were observed in vials after long-term storage at 2-8°C for some protein formulations using poloxamer 188, which were not observed in polysorbate formulations. The occurrence of these protein-PDMS particles in poloxamer 188 formulations is a protein-specific phenomenon that may correlate with protein physico-chemical properties. In this study, the primary source of the PDMS in particles found in vials was considered to be from the primary packaging stoppers used. Our findings highlight benefits, but also risks associated with using poloxamer 188 in liquid biotherapeutic formulations.

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. Therefore, the protein modifications potentially resulting from various process steps need to be characterized and controlled. This commentary brings together expertise and knowledge from biopharmaceutical scientists and discusses the various manufacturing process steps that could adversely impact the quality of drug substance (DS). The major process steps discussed here are commonly used in mAb production using mammalian cells. These include production cell culture, harvest, antibody capture by protein A, virus inactivation, polishing by ion-exchange chromatography, virus filtration, ultrafiltration-diafiltration, compounding followed by release testing, transportation and storage of final DS. Several of these process steps are relevant to protein DS production in general. The authors attempt to critically assess the level of risk in each of the DS processing steps, discuss strategies to control or mitigate protein modification in these steps, and recommend mitigation approaches including guidance on development studies that mimic the stress induced by the unit operations.

Synthetic Cargo Internalization Receptor System for Nanoparticle Tracking of Individual Cell Populations by Fluorine Magnetic Resonance Imaging.

Specific detection of target structures or cells lacking particular surface epitopes still poses a serious problem for all imaging modalities. Here, we demonstrate the capability of synthetic "cargo internalization receptors" (CIRs) for tracking of individual cell populations by 1H/19F magnetic resonance imaging (MRI). To this end, a nanobody for green fluorescent protein (GFP) was used to engineer cell-surface-expressed CIRs which undergo rapid internalization after GFP binding. For 19F MR visibility, the GFP carrier was equipped with "contrast cargo", in that GFP was coupled to perfluorocarbon nanoemulsions (PFCs). To explore the suitability of different uptake mechanisms for this approach, CIRs were constructed by combination of the GFP nanobody and three different cytoplasmic tails that contained individual internalization motifs for endocytosis of the contrast cargo (CIR1-3). Exposure of CIR+ cells to GFP-PFCs resulted in highly specific binding and internalization as confirmed by fluorescence microscopy as well as flow cytometry and enabled visualization by 1H/19F MRI. In particular, expression of CIR2/3 resulted in substantial incorporation of 19F cargo and readily enabled in vivo visualization of GFP-PFC recruitment to transplanted CIR+ cells by 1H/19F MRI in mice. Competition experiments with blood immune cells revealed that CIR+ cells are predominantly loaded with GFP-PFCs even in the presence of cells with strong phagocytotic capacity. Importantly, binding and internalization of GFP-PFCs did not result in the activation of signaling cascades and therefore does not alter cell physiology. Overall, this approach represents a versatile in vivo imaging platform for tracking of individual cell populations by making use of cell-type-specific CIR+ mice.

Epicardium-Derived Cells Formed After Myocardial Injury Display Phagocytic Activity Permitting In Vivo Labeling and Tracking.

UNLABELLED: Epicardium-derived cells (EPDCs) cover the heart surface and can function as a source of both progenitor cells and trophic factors for cardiac repair. Currently, EPDCs cannot be conveniently labeled in vivo to permit imaging and cell tracking. EPDCs formed after myocardial infarction (MI) preferentially take up a perfluorocarbon-containing nanoemulsion (PFC-NE; 130 ± 32 nm) injected 3 days after injury, as measured by (19)F-magnetic resonance imaging ((19)F-MRI). Flow cytometry, immune electron microscopy, and green fluorescent protein (GFP)-transgenic rats (only immune cells, but not epicardial cells, are GFP(+)) demonstrated that PFC-containing EPDCs are nonhematopoietic (CD45(-)/CD11b(-)) but stain positive for markers of mesenchymal stem cells such as platelet-derived growth factor receptor α (PDGFR-α) CD73, CD105, and CD90. When rhodamine-coupled PFC-NE was used, we found that ρ(+) vessel-like structures formed within the infarcted myocardium, comprising approximately 10% of all large vessels positive for smooth muscle actin (SM-actin). The epicardial cell layer, positive for Wilms' tumor 1 (WT-1), PDGFR-α, or KI-67, was shown to be well capillarized (293 ± 78 capillaries per mm(2)), including fenestrated endothelium. Freshly isolated EPDCs were positive for WT-1, GATA-4, KI-67, and FLK-1 (75%), PDGFR-α (50%), and SM-actin (28%) and also exhibited a high capacity for nanoparticle and cell debris uptake. This study demonstrates that EPDCs formed after MI display strong endocytic activity to take up i.v.-injected labeled nanoemulsions. This feature permitted in vivo labeling and tracking of EPDCs, demonstrating their role in myo- and vasculogenesis. The newly discovered endocytic activity permits in vivo imaging of EPDCs with (19)F-MRI and may be used for the liposomal delivery of substances to further study their reparative potential. SIGNIFICANCE: The present study reports that epicardium-derived cells (EPDCs) formed after myocardial infarction can specifically endocytose nanoparticles in vivo and in vitro. This novel feature permitted in vivo targeting of EPDCs with either a perfluorocarbon-containing or rhodamine-conjugated nanoemulsion to track migration and fate decision of EPDC with (19)F-magnetic resonance imaging and fluorescence microscopy. The liposomal nanoemulsions used in the present study may be useful in the future as a nanomedical device for the delivery of substances to direct cell fate of EPDCs.

Monitoring the Stability of Perfluorocarbon Nanoemulsions by Cryo-TEM Image Analysis and Dynamic Light Scattering.

Perfluorocarbon nanoemulsions (PFC-NE) are disperse systems consisting of nanoscale liquid perfluorocarbon droplets stabilized by an emulsifier, usually phospholipids. Perfluorocarbons are chemically inert and non-toxic substances that are exhaled after in vivo administration. The manufacture of PFC-NE can be done in large scales by means of high pressure homogenization or microfluidization. Originally investigated as oxygen carriers for cases of severe blood loss, their application nowadays is more focused on using them as marker agents in 19F Magnetic Resonance Imaging (19F MRI). 19F is scarce in organisms and thus PFC-NE are a promising tool for highly specific and non-invasive imaging of inflammation via 19F MRI. Neutrophils, monocytes and macrophages phagocytize PFC-NE and subsequently migrate to inflamed tissues. This technique has proven feasibility in numerous disease models in mice, rabbits and mini pigs. The translation to clinical trials in human needs the development of a stable nanoemulsion whose droplet size is well characterized over a long storage time. Usually dynamic light scattering (DLS) is applied as the standard method for determining particle sizes in the nanometer range. Our study uses a second method, analysis of transmission electron microscopy images of cryo-fixed samples (Cryo-TEM), to evaluate stability of PFC-NE in comparison to DLS. Four nanoemulsions of different composition are observed for one year. The results indicate that DLS alone cannot reveal the changes in particle size, but can even mislead to a positive estimation of stability. The combination with Cryo-TEM images gives more insight in the particulate evolution, both techniques supporting one another. The study is one further step in the development of analytical tools for the evaluation of a clinically applicable perfluorooctylbromide nanoemulsion.

Noninvasive Imaging of Early Venous Thrombosis by 19F Magnetic Resonance Imaging With Targeted Perfluorocarbon Nanoemulsions.

BACKGROUND: Noninvasive detection of deep venous thrombi and subsequent pulmonary thromboembolism is a serious medical challenge, since both incidences are difficult to identify by conventional ultrasound techniques. METHODS AND RESULTS: Here, we report a novel technique for the sensitive and specific identification of developing thrombi using background-free 19F magnetic resonance imaging, together with α2-antiplasmin peptide (α2AP)-targeted perfluorocarbon nanoemulsions (PFCs) as contrast agent, which is cross-linked to fibrin by active factor XIII. Ligand functionality was ensured by mild coupling conditions using the sterol-based postinsertion technique. Developing thrombi with a diameter<0.8 mm could be visualized unequivocally in the murine inferior vena cava as hot spots in vivo by simultaneous acquisition of anatomic matching 1H and 19F magnetic resonance images at 9.4 T with both excellent signal-to-noise and contrast-to-noise ratios (71±22 and 17±5, respectively). Furthermore, α2AP-PFCs could be successfully applied for the diagnosis of experimentally induced pulmonary thromboembolism. In line with the reported half-life of factor XIIIa, application of α2AP-PFCs>60 minutes after thrombus induction no longer resulted in detectable 19F magnetic resonance imaging signals. Corresponding results were obtained in ex vivo generated human clots. Thus, α2AP-PFCs can visualize freshly developed thrombi that might still be susceptible to pharmacological intervention. CONCLUSIONS: Our results demonstrate that 1H/19F magnetic resonance imaging, together with α2AP-PFCs, is a sensitive, noninvasive technique for the diagnosis of acute deep venous thrombi and pulmonary thromboemboli. Furthermore, ligand coupling by the sterol-based postinsertion technique represents a unique platform for the specific targeting of PFCs for in vivo 19F magnetic resonance imaging.