Modular and tunable alternative surfactants for biopharmaceuticals provide insights into Surfactant's Structure-Function relationship.

Surface-induced aggregation of protein therapeutics is opposed by employing surfactants, which are ubiquitously used in drug product development, with polysorbates being the gold standard. Since poloxamer 188 is currently the only generally accepted polysorbate alternative, but cannot be ubiquitously applied, there is a strong need to develop surfactant alternatives for protein biologics that would complement and possibly overcome known drawbacks of existing surfactants. Yet, a severe lack of structure-function relationship knowledge complicates the development of new surfactants. Herein, we perform a systematic analysis of the structure-function relationship of three classes of novel alternative surfactants. Firstly, the mode of action is thoroughly characterized through tensiometry, calorimetry and MD simulations. Secondly, the safety profiles are evaluated through cell-based in vitro assays. Ultimately, we could conclude that the alternative surfactants investigated possess a mode of action and safety profile comparable to polysorbates. Moreover, the biophysical patterns elucidated here can be exploited to precisely tune the features of future surfactant designs.

Not the Usual Suspects: Alternative Surfactants for Biopharmaceuticals.

Therapeutically relevant proteins naturally adsorb to interfaces, causing aggregation which in turn potentially leads to numerous adverse consequences such as loss of activity or unwanted immunogenic reactions. Surfactants are ubiquitously used in biotherapeutics drug development to oppose interfacial stress, yet, the choice of the surfactant is extremely limited: to date, only polysorbates (PS20/80) and poloxamer 188 are used in commercial products. However, both surfactant families suffer from severe degradation and impurities of the raw material, which frequently increases the risk of particle generation, chemical protein degradation, and potential adverse immune reactions. Herein, we assessed a total of 40 suitable alternative surfactant candidates and subsequently performed a selection through a three-gate screening process employing four protein modalities encompassing six different formulations. The screening is based on short-term agitation-induced aggregation studies coupled to particle analysis and surface tension characterization, followed by long-term quiescence stability studies connected to protein purity measurements and particle analysis. The study concludes by assessing the surfactant's chemical and enzymatic degradation propensity. The candidates emerging from the screening are de novo α-tocopherol-derivatives named VEDG-2.2 and VEDS, produced ad hoc for this study. They display protein stabilization potential comparable or better than polysorbates together with an increased resistance to chemical and enzymatic degradation, thus representing valuable alternative surfactants for biotherapeutics.

An Intra-Company Analysis of Inherent Particles in Biologicals Shapes the Protein Particle Mitigation Strategy Across Development Stages.

To better understand protein aggregation and inherent particle formation in the biologics pipeline at Novartis, a cross-functional team collected and analyzed historical protein particle issues. Inherent particle occurrences from the past 10 years were systematically captured in a protein particle database. Where the root cause was identified, a number of product attributes (such as development stage, process step, or protein format) were trended. Several key themes were revealed: 1) there was a higher propensity for inherent particle formation with non-mAbs than with mAbs; 2) the majority of particles were detected following manufacturing at scale, and were not predicted by the small-scale studies; 3) most issues were related to visible particles, followed by subvisible particles; 4) 50% of the issues were manufacturing related. These learnings became the foundation of a particle mitigation strategy across development and technical transfer, and resulted in a set of preventive actions. Overall, this study provides further insight into a recognized industry challenge and hopes to inspire the biopharmaceutical industry to transparently share their experiences with inherent particles formation.

Controlled release of therapeutic antibody formats.

The local administration of antibodies can represent in many cases a significant improvement for antibody-based therapies. The benefits of local delivery include high drug concentrations at the target site, the possibility of lower drug dosing and less systemic drug exposure. Currently, the most relevant delivery sites for therapeutic antibodies are the posterior segments of the eye, mucosal surfaces, the articular joints and the central nervous system (CNS). In addition, the oral and pulmonary route may enable non-invasive systemic antibody delivery. However, local antibody delivery to these sites is characterized by short drug residence times and a low compliance of administration. Controlled release (CR) systems can address these limitations and, thereby, enable and improve local delivery applications by achieving long lasting local drug concentrations, improved efficacy-dosing ratios and reduced treatment-associated side effects. The requirements for CR antibody formulations are more complex compared to conventional CR systems for small molecules, and their development poses an enormous technical challenge. Therefore, the review highlights experiences and challenges gathered in the development of the different CR systems for antibodies to date. Additionally, the unmet technological needs encountered in the field are described. This includes a critical evaluation of the limited capability of various CR systems to preserve antibody stability, delivery site specific considerations, as well as the processability of a CR system with a particular focus on drug loading and injectability. We believe that the success of CR and local delivery approaches could create an enormous added value for patients in the future.

Pharmacokinetics, biocompatibility and bioavailability of a controlled release monoclonal antibody formulation.

The sustained and localized delivery of monoclonal antibodies has become highly relevant, because of the increasing number of investigated local delivery applications in recent years. As the local delivery of antibodies is associated with high technological hurdles, very few successful approaches have been reported in the literature so far. Alginate-based delivery systems were previously described as promising sustained release formulations for monoclonal antibodies (mAbs). In order to further investigate their applicability, a single-dose animal study was conducted to compare the biocompatibility, the pharmacokinetics and the bioavailability of a human monoclonal antibody liquid formulation with two alginate-based sustained delivery systems after subcutaneous administration in rats. 28 days after injection, the depot systems were still found in the subcutis of the animals. A calcium cross-linked alginate formulation, which was injected as a hydrogel, was present as multiple compartments separated by subcutaneous tissue. An in situ forming alginate formulation was recovered as a single compact and cohesive structure. It can be assumed that the multiple compartments of the hydrogel formulation led to almost identical pharmacokinetic profiles for all tested animals, whereas the compact nature of the in situ forming system resulted in large interindividual variations in pharmacokinetics. As compared to the liquid formulation the hydrogel formulations led to lower mAb serum levels, and the in situ forming system to a shift in the time to reach the maximum mAb serum concentration (Tmax) from 2 to 4 days. Importantly, it was shown that after 28 days only marginal amounts of residual mAb were present in the alginate matrix and in the tissue at the injection site indicating nearly complete release. In line with this finding, systemic drug bioavailability was not affected by using the controlled release systems. This study successfully demonstrates the suitability and underlines the potential of polyanionic systems for local and controlled mAb delivery.

Understanding the freezing of biopharmaceuticals: first-principle modeling of the process and evaluation of its effect on product quality.

Freezing and thawing are important process steps in the manufacture of numerous biopharmaceuticals. It is well established that these process steps can significantly influence product quality attributes (PQA). Herein, we describe a physico-mathematical model to predict product temperature profiles based on the freezing program as input parameter in a commercial freeze-thaw module. Applying this model, the time from first nucleation until the last point to freeze (LPF) reaching -5°C and the time from -5°C at LPF to -30°C at LPF was varied to study the effect on PQA in a full factorial design. Effects of process parameter settings on a typical fully formulated, highly concentrated monoclonal antibody (mAb) solution as well as highly concentrated mAb solution formulated with buffer only were investigated. We found that both process phases affected PQA, such as aggregates by size-exclusion chromatography, polydispersity index by dynamic light scattering, and number of subvisible particles and turbidity in a complex way. In general, intermediate cooling and freezing times resulted in overall optimized PQA. Fully formulated mAb solution containing cryoprotectant and nonionic surfactant was significantly less affected by freezing-thawing than mAb solution formulated in buffer only.

The role of polysorbate 80 and HPßCD at the air-water interface of IgG solutions.

PURPOSE: To test the hypothesis of surface displacement as the underlying mechanism for IgG stabilization by polysorbates and HPßCD against surface-induced aggregation. METHODS: Adsorption/desorption-kinetics of IgG-polysorbate 80/-HPßCD were monitored. Maximum bubble pressure method was used for processes within seconds from surface formation. Profile analysis tensiometry was applied over long periods and to assess surface rheologic properties. Additionally, the kinetics of adsorption, desorption and surface displacement was followed by a double-capillary setup of the profile analysis tensiometer, allowing drop bulk exchange. RESULTS: Weak surface activity for HPßCD vs. much higher surface activity for polysorbate 80 was shown. Protein-displacement when exceeding a polysorbate 80 concentration close to the CMC and a lack of protein displacement for HPßCD was observed. The drop bulk exchange experiments show IgG displacement by polysorbate 80 independent of the adsorption order. In contrast, HPßCD coexists with IgG at the air-water interface when the surface layer is built from a mixed IgG-HPßCD-solution. Incorporation of HPßCD in a preformed IgG-surface-layer does not occur. CONCLUSIONS: The results confirm surface displacement as the stabilization mechanism of polysorbate 80, but refute the frequently held opinion, that HPßCD stabilizes proteins against aggregation at the air-water interface in a manner comparable to non-ionic surfactants.

Protein stabilization by cyclodextrins in the liquid and dried state.

Aggregation is arguably the biggest challenge for the development of stable formulations and robust manufacturing processes of therapeutic proteins. In search of novel excipients inhibiting protein aggregation, cyclodextrins and their derivatives have been under examination for use in parenteral protein products since more than 20 years and significant research work has been accomplished highlighting the great potential of cyclodextrins as stabilizers of therapeutic proteins. Oftentimes, the potential of cyclodextrins to inhibit protein aggregation has been attributed to their capability to incorporate hydrophobic residues on aggregation-prone proteins or on their partially unfolded intermediates into the hydrophobic cavity. In addition, also other mechanisms besides or even instead of complex formation play a role in the stabilization mechanism, e.g. non-ionic surfactant-like effects. In this review a comprehensive overview of the available research work on the beneficial use of cyclodextrins and their derivatives in protein formulations, liquid as well as dried, is provided. The mechanisms of stabilization against different kinds of stress conditions, such as thermal or surface-induced, are discussed in detail.

Inhibition of agitation-induced aggregation of an IgG-antibody by hydroxypropyl-beta-cyclodextrin.

In order to provide an alternative to nonionic surfactants as excipients for protein formulations, cyclodextrin-derivatives (CDs) were examined for their potential to inhibit agitation-induced aggregation of an IgG in aqueous solution. Loss of monomeric protein and protein aggregation were monitored throughout the agitation experiments by size exclusion chromatography. Hydroxypropyl-beta-cyclodextrin (HPbetaCD) completely suppressed IgG-aggregation at a remarkably low concentration (2.5 mM) and in contrast to other CDs it also did not negatively affect IgG-stability during storage in nonagitated solution. Further agitation experiments demonstrated the superiority of HPbetaCD to other common excipients in protein formulation, such as sugars and sugar alcohols or polysorbate 80 in low concentrations. Spectroscopic (fluorescence spectroscopy and Fourier transform infrared spectroscopy), thermodynamic (microcalorimetry), and physical investigations (surface tension measurements) were carried out to elucidate the mechanism of stabilization of the IgG. In contrast to other studies with HPbetaCD, protein stabilization could not be attributed to direct interaction between hydrophobic amino acids on the IgG and this excipient. Competition with the protein for the air-water interface appears to be the dominating mechanism of stabilization.