Towards more tolerable subcutaneous administration: Review of contributing factors for improving combination product design.

Subcutaneous (SC) injections can be associated with local pain and discomfort that is subjective and may affect treatment adherence and overall patient experience. With innovations increasingly focused on finding ways to deliver higher doses and volumes (≥2 mL), there is a need to better understand the multiple intertwined factors that influence pain upon SC injection. As a priority for the SC Drug Development & Delivery Consortium, this manuscript provides a comprehensive review of known attributes from published literature that contribute to pain/discomfort upon SC injection from three perspectives: (1) device and delivery factors that cause physical pain, (2) formulation factors that trigger pain responses, and (3) human factors impacting pain perception. Leveraging the Consortium's collective expertise, we provide an assessment of the comparative and interdependent factors likely to impact SC injection pain. In addition, we offer expert insights and future perspectives to fill identified gaps in knowledge to help advance the development of patient-centric and well tolerated high-dose/high-volume SC drug delivery solutions.

Poly(glutamic acid)-Based Viscosity Reducers for Concentrated Formulations of a Monoclonal IgG Antibody.

Above a concentration threshold, the viscosity of solutions of proteins increases abruptly, which hampers the injectability of therapeutic formulations. Concentrations above 200 g/L are an ideal goal for subcutaneous application of antibodies. Molecular additives, such as amino acids (e.g., arginine) help decrease the viscosity, but they are used at concentrations as high as about 200 mmol/L. We addressed the question of whether poly(amino acids) could be more efficient than small molecular additives. We observed marked fluidification of a model therapeutic monoclonal antibody (mAb) solution by poly(d,l-glutamic acid) and poly(l-glutamic acid) derivatives added at concentrations of <6.5 g/L (i.e., a mAb/polymer chain molar ratio between 4:1 and 1:1 mol/mol). The bare poly(glutamate) parent chains were compared with polyethylene glycol-grafted chains as PEGylation is a common way to enhance stability. Viscosity could be decreased to ∼20 mPa s as compared to values of ∼100 mPa s in the absence of polymers at 200 g/L mAb. Formation of complexes between the mAb and the polyglutamates was characterized by capillary electrophoresis analysis in dilute solutions (1 g/L mAb) and by observation of phase separation at higher concentrations, suggesting tight association at about 2:1 mol/mol mAb/polymer. Altogether, these results show that polyglutamate derivatives hold an untapped potential as an excipient for fluidification of concentrated protein solutions.

A biomimetic chip to assess subcutaneous bioavailability of monoclonal antibodies in humans.

Subcutaneous (subQ) injection is a common route for delivering biotherapeutics, wherein pharmacokinetics is largely influenced by drug transport in a complex subQ tissue microenvironment. The selection of good drug candidates with beneficial pharmacokinetics for subQ injections is currently limited by a lack of reliable testing models. To address this limitation, we report here a Subcutaneous Co-Culture Tissue-on-a-chip for Injection Simulation (SubCuTIS). SubCuTIS possesses a 3D coculture tissue architecture, and it allows facile quantitative determination of relevant scale independent drug transport rate constants. SubCuTIS captures key in vivo physiological characteristics of the subQ tissues, and it differentiates the transport behavior of various chemically distinct molecules. We supplemented the transport measurements with theoretical modeling, which identified subtle differences in the local absorption rate constants of seven clinically available mAbs. Accounting for first-order proteolytic catabolism, we established a mathematical framework to assess clinical bioavailability using the local absorption rate constants obtained from SubCuTIS. Taken together, the technology described here broadens the applicability of organs-on-chips as a standardized and easy-to-use device for quantitative analysis of subQ drug transport.

Inhaled IgG1 antibodies: The buffering system is an important driver of stability during mesh-nebulization.

In the past decade, oral inhalation has been a thriving focus of research to administer antibody directly to the lungs as an aerosol, for local treatment of respiratory diseases. Formulation of inhaled antibodies is central for the stability of antibody, lung safety and to ensure inhaler performances. Surfactants have already been shown to prevent antibody degradation during aerosolization, but little is known about the impact of other components of liquid formulations on the structural stability of antibodies. Here, we report for the first time to the best of our knowledge, a significant effect of the buffering system on monoclonal antibodies stability, during mesh-nebulization. While the monoclonal antibody extensively aggregated in citrate buffer after nebulization and required high concentration of polysorbate 80 (PS80) to maintain protein integrity, acetate and histidine buffers resulted in a slight to moderate aggregation without PS80 and low concentration of PS80 was sufficient to stabilize antibody during mesh-nebulization.

Industry Perspective on the Use and Characterization of Polysorbates for Biopharmaceutical Products Part 2: Survey Report on Control Strategy Preparing for the Future.

Polysorbate (PS) 20 and 80 are the main surfactants used to stabilize biopharmaceutical products. Industry practices on various aspects of PS based on a confidential survey and following discussions by 16 globally acting major biotechnology companies is presented in two publications. Part 1 summarizes the current practice and use of PS during manufacture in addition to aspects like current understanding of the (in)stability of PS, the routine QC testing and control of PS, and selected regulatory aspects of PS.1 The current part 2 of the survey focusses on understanding, monitoring, prediction, and mitigation of PS degradation pathways in order to propose an effective control strategy. The results of the survey and extensive cross-company discussions are put into relation with currently available scientific literature.

Industry Perspective on the use and Characterization of Polysorbates for Biopharmaceutical Products Part 1: Survey Report on Current State and Common Practices for Handling and Control of Polysorbates.

Polysorbates (PS) are widely used as a stabilizer in biopharmaceutical products. Industry practices on various aspects of PS are presented in this part 1 survey report based on a confidential survey and following discussions by 16 globally acting major biotechnology companies. The current practice and use of PS during manufacture across their global manufacturing sites are covered in addition to aspects like current understanding of the (in)stability of PS, the routine QC testing and control of PS, and selected regulatory aspects of PS. The results of the survey and extensive cross-company discussions are put into relation with currently available scientific literature. Part 2 of the survey report (upcoming) will focus on understanding, monitoring, prediction, and mitigation of PS degradation pathways to develop an effective control strategy.

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. However, their protection efficacy related to the nature of the plastic material is still poorly investigated. METHODS: We use real-time surface-sensitive techniques and immunosorbent assays, to quantify surfactant and monoclonal antibody adsorption on hydrophobic model surfaces and different plastic polymers to analyse the effect of material surface properties on the level of surfactant protection. RESULTS: We show that Polysorbate 80 protects monoclonal antibodies significantly better from adsorption on a polystyrene surface than on a hexadecane self-assembled monolayer, used as a model surface with similar hydrophobicity. This enhanced protective effect on polystyrene is observed for different antibodies and also other surfactants, and its extent depends on the surfactant concentration for a given antibody concentration. A comparative adsorption study allows ranking different in-use plastics and highlights the dependence of Polysorbate 80 protection efficacy on the nature of the plastic material. CONCLUSION: This study demonstrates that, beyond hydrophobicity, the nature of plastic polymer surfaces affects surfactant adsorption and thereby impacts their protection efficacy in therapeutic antibody formulations.

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. The latter are difficult to measure accurately because a depletion layer forms rapidly in the bulk solution above a bare surface, slowing down adsorption. Adapting flow conditions and local surface chemistry, we are able to minimize depletion limitations and measure apparent adsorption rate constants of three monoclonal antibodies, other proteins and surfactants with a hydrophobic surface. We show that surface adsorption rates scale with the molecular mass of the molecule, with polysorbates therefore showing thousand times slower rates than antibodies. Moreover, we observed that the desorption dynamic of polysorbates from a given hydrophobic surface depends on surface coverage, whereas this is not the case for Poloxamer 188. These novel contributions to surface adsorption dynamics enable a new perspective on the evaluation of drug surface compatibility and can, together with diffusion rates, be used to predict the protective potential of surfactants in given conditions.

Inhaled antibodies: formulations require specific development to overcome instability due to nebulization.

Respiratory infections are life-threatening and therapeutic antibodies (Ab) have a tremendous opportunity to benefit to patients with pneumonia due to multidrug resistance bacteria or emergent virus, before a vaccine is manufactured. In respiratory infections, inhalation of anti-infectious Ab may be more relevant than intravenous (IV) injection-the standard route-to target the site of infection and improve Ab therapeutic index. One major challenge associated to Ab inhalation is to prevent protein instability during the aerosolization process. Ab drug development for IV injection aims to design a high-quality product, stable to different environment stress. In this study, we evaluated the suitability of Ab formulations developed for IV injection to be extended for inhalation delivery. We studied the aerosol characteristics and the aggregation profile of three Ab formulations developed for IV injection after nebulization, with two mesh nebulizers. Although the formulations for IV injection were compatible with mesh nebulization and deposition into the respiratory tract, the Ab were more unstable during nebulization than exposition to a vigorous shaking. Overall, our findings indicate that Ab formulations developed for IV delivery may not easily be repurposed for inhalation delivery and point to the requirement of a specific formulation development for inhaled Ab.

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. In this work, we propose an innovative approach based on optimised design of microfluidic converging-diverging channels coupled with a microscope-based tracking method to characterise the dynamic behaviour of individual bio-particles under homogeneous straining flow. The tracking algorithm, combining a motorised stage and a microscopy imaging system controlled by external signals, allows us to follow individual bio-particles transported over long-distances with high-quality images. We demonstrate experimentally the ability of the numerically optimised microchannels to provide linear velocity streamwise gradients along the centreline of the device, allowing for extended consecutive regions of homogeneous elongation and compression. We selected three test cases (DNA, actin filaments and protein aggregates) to highlight the ability of our approach for investigating dynamics of objects with a wide range of sizes, characteristics and behaviours of relevance in the biological world.

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. Here we investigate a microfluidic model system to gain fundamental understanding of the clogging of constrictions by monoclonal antibody aggregates. Highly concentrated solutions of monoclonal antibodies were used to create protein aggregates (larger than 50 microns) using mechanical or heat stress. We show that clogging occurs when aggregates reach the size of the constriction and that clogs can in some cases be released by increasing the applied pressure. This indicates the important role of protein aggregate deformability. We perform systematic experiments for different relative aggregate sizes and applied pressures, and measure the resulting flow-rate. This allows us to present first in situ estimates of an effective Young's modulus. Despite their different shapes and densities, we can predict the number of clogging events for a given constriction size from the aggregate size distribution measured by Flow Imaging Microscopy (MFI). In addition our device can detect the occurrence of very rare big aggregates often overlooked by other detection methods.

Distinctive Low-Resolution Structural Features of Dimers of Antibody-Drug Conjugates and Parent Antibody Determined by Small-Angle X-ray Scattering.

Structural features of lysine-conjugated antibody-drug conjugate (ADC) from humanized IgG1 were studied by small-angle X-ray scattering (SAXS). As the physicochemical properties of the cytotoxic drug (payload) and linker may impact the conformational and colloidal stabilities of the conjugated monoclonal antibody (mAb), it is essential to characterize how the conjugation may affect the overall higher order structure and therefore the physical stability and integrity of the ADCs upon storage conditions. Here, the ADC monomer and aggregates generated upon thermal stress were analyzed by high performance liquid chromatography coupled to SAXS with a particular focus on the fraction of dimers (3-10% depending on the storage conditions at 25 and 40 °C). In addition to average parameters such as radius of gyration, molecular weight, and maximal end-to-end distance, the structural information obtained from SAXS patterns were visualized as a low-resolution average envelope of both monomers and dimers (implementation of two methods: ab initio reconstruction and modeling Fab and Fc as rigid bodies with a flexible hinge). We showed that the monomer envelope of the ADC was similar to the corresponding (nonconjugated) parent monoclonal antibody (mAb). ADC dimers appeared more compact and less polydisperse than the dimers of mAb, which was also confirmed by atomic force microscopy. The generated envelopes of the mAb dimers suggest elongated structures with one or few inter-mAb contacts at the outermost region of Fab or Fc domains. The structural features of ADC dimers are independent of the tested pH buffering system (pH 5.0/acetate and pH 6.0/histidine with or without NaCl) and characterized by multiple, tighter contacts between the Fab and Fc domains and distortion of the monomer native shape. Results from the SAXS structural study show in the present case that conjugation has favored innermost inter-ADC contacts in the dimer, which differ from the inter-mAb ones. In general, it is likely that many parameters affect inter-ADC association, including the chemical nature of linkers and drugs, degree of conjugation, conjugation sites, etc. Making a qualitative difference between mAb and ADC dimers as a function of these parameters can help point to the presence of tight associations that must be abolished in protein drug formulations.

Exploring the fate of inhaled monoclonal antibody in the lung parenchyma by microdialysis.

Therapeutic antibodies (Abs) are emerging as major drugs to treat respiratory diseases, and inhalation may provide substantial benefits for their delivery. Understanding the behavior of Abs after pulmonary deposition is critical for their development. We investigated the pharmacokinetics of a nebulized Ab by continuous sampling in lung parenchyma using microdialysis in non-human primates. We defined the optimal conditions for microdialysis of Ab and demonstrated that lung microdialysis of Ab is feasible over a period of several days. The concentration-profile indicated a two-phase non-linear elimination and/or distribution of inhaled mAbX. Lung exposition was higher than the systemic one over a period of 33 hours and above MabX affinity for its target. The microdialysis results were supported by an excellent relationship with dosages from lung extracts.

[Developability assessment]. / Développabilité.

The therapeutic antibodies and their by-products (antibody fragments, conjugated, etc.) establish one of the most dynamic biopharmaceutical market segments today. Due to their intrinsic properties of specificity towards their target, towards their flexible affinity and due to their stability, antibodies became therapeutic agents of the very first choice. One of the challenges of this sector is to create antibodies of very good quality, more and more quickly, while having less and less consequent development costs in fine.

TITLE: Développabilité. ABSTRACT: Les anticorps thérapeutiques et leurs dérivés (fragments d'anticorps, conjugués, etc.) constituent aujourd'hui l'un des segments du marché biopharmaceutique les plus dynamiques. De par leurs propriétés intrinsèques de spécificité vis-à-vis de leur cible, de leur affinité modulable et de par leur stabilité, les anticorps sont devenus des agents thérapeutiques de tout premier choix. Un des challenges de ce secteur est de créer des anticorps de très bonne qualité, de plus en plus rapidement, tout en ayant des coûts de développement de moins en moins conséquents in fine.

[Developability assessment with case studies highlighting the decision taking]. / Exemples d'études de développabilité apportant un éclairage à la prise de décision.

Therapeutic antibodies generation has to be faster with less development costs. This requires combination of in silico predictions associated with cutting edge screening and characterization technologies. Here, non-exhaustive examples illustrate this simultaneity need.

TITLE: Exemples d'études de développabilité apportant un éclairage à la prise de décision. ABSTRACT: De nos jours, la génération d'anticorps thérapeutiques doit être plus rapide avec des coûts de développement moins importants. Pour cela, des prédictions in silico sont associées à des technologies de criblage et de caractérisation de pointe. Les exemples choisis ici sont non-exhaustifs mais illustrent ce besoin de travailler en parallèle.