Challenges for the pharmaceutical technical development of protein coformulations.

OBJECTIVES: This review discusses challenges to stability, analytics and manufacturing of protein coformulations. Furthermore, general considerations to be taken into account for the pharmaceutical development of coformulated protein drug products are highlighted. KEY FINDINGS: Coformulation of two or more active substances in one single dosage form has recently seen increasing use offering several advantages, such as increased efficacy and/or the overall reduction of adverse event incidents in patients. Most marketed coformulated drug products are composed of small molecules. As proteins are not only comparatively large but also complex molecules, the maintenance of their physicochemical integrity within a formulation throughout pharmaceutical processing, storage, transport, handling and patient administration to ensure proper pharmacokinetics and pharmacodynamics in vivo already represents various challenges for single-entity products. Thus, nowadays, only sparse biologics-based coformulations can be found, as additional complexity during development is given for these products. SUMMARY: The complexity of the dosage form and the protein molecules results into additional challenges to formulation, manufacture, storage, transport, handling and patient administration, stability and analytics during the pharmaceutical development of protein coformulations. Various points have to be considered during different stages of development in order to obtain a safe and efficacious product.

If Euhydric and Isotonic Do Not Work, What Are Acceptable pH and Osmolality for Parenteral Drug Dosage Forms?

Parenteral products should aim toward being isotonic and euhydric (physiological pH). Yet, due to other considerations, this goal is often not reasonable or doable. There are no clear allowable ranges related to pH and osmolality, and thus, the objective of this review was to provide a better understanding of acceptable formulation pH, buffer strength, and osmolality taking into account the administration route (i.e., intramuscular, intravenous, subcutaneous) and administration technique (i.e., bolus, push, infusion). This evaluation was based on 3 different approaches: conventional, experimental, and parametric. The conventional way of defining formulation limits was based on standard pH and osmolality ranges. Experimental determination of titratable acidity or in vitro hemolysis testing provided additional drug product information. Finally, the parametric approach was based on the calculation of theoretical values such as (1) the maximal volume of injection which cannot shift the blood's pH or its molarity out of the physiological range and (b) a dilution ratio at the injection site and by verifying that threshold values are not exceeded. The combination of all 3 approaches can support the definition of acceptable pH, buffer strength, and osmolality of formulations and thus may reduce the risk of failure during preclinical and clinical development.

Prediction of intraocular antibody drug stability using ex-vivo ocular model.

Following intravitreal (IVT) injection, therapeutic proteins get exposed to physiological pH, temperature and components in the vitreous humor (VH) for a significantly long time. Therefore, it is of interest to study the stability of the proteins in the VH. However, the challenge posed by the isolated VH (such as pH shift upon isolation and incubation due to the formation of smaller molecular weight (MW) degradation products) can result in artefacts when investigating protein stability in relevance for the actual in vivo situation. In this current study, an ex-vivo intravitreal horizontal stability model (ExVit-HS) has been successfully developed and an assessment of long-term stability of a bi-specific monoclonal antibody (mAb) drug in the isolated VH for 3months at physiological conditions has been conducted. The stability assessment was performed using various analytical techniques such as microscopy, UV visible for protein content, target binding ELISA, Differential Scanning Calorimetry (DSC), Capillary-electrophoresis-SDS, Size Exclusion (SEC) and Ion-exchange chromatography (IEC) and SPR-Biacore. The results show that the ExVit-HS model was successful in maintaining the VH at physiological conditions and retained a majority of protein in the VH-compartment throughout the study period. The mAb exhibited significantly less fragmentation in the VH relative to the PBS control; however, chemical stability of the mAb was equally compromised in VH and PBS. Interestingly, in the PBS control, mAb showed a rapid linear loss in the binding affinity. The loss in binding was almost 20% higher compared to that in VH after 3months. The results clearly suggest that the mAb has different degradation kinetics in the VH compared to PBS. These results suggest that it is beneficial to investigate the stability in the VH for drugs intended for IVT injection and that are expected longer residence times in the VH. The studies show that the ExVit-HS model may become a valuable tool for evaluating stability of protein drugs and other molecules following IVT injection.

Evaluation of protein drug stability with vitreous humor in a novel ex-vivo intraocular model.

The stability of protein therapeutics during the residence time in the vitreous humor (VH) is an important consideration for intra ocular treatment and can possibly impact therapeutic efficacy and/or treatment intervals. Unavailability of the reliable Ex-vivo intravitreal (ExVit) model to estimate protein stability following IVT has driven the research focus to develop such model which can facilitate protein stability estimation before in-vivo experiments. In this manuscript, we have developed and evaluated three ExVit models, namely, ExVit static, semi-dynamic and dynamic. These models were utilized and compared when studying the in-vitro stability of model protein formulations under simulated intraocular conditions using porcine vitreous humor (VH). The ExVit static model exhibited significant precipitation and aggregation of proteins, most likely due to pH change occurred in the VH after isolation. The semi-dynamic model assessed was composed of two compartments i.e., VH- and buffer-compartment which has effectively stabilized the pH of the VH and facilitated the migration of VH degradation products. However, some limitations related to investigation of long-term protein stability were also observed with semi-dynamic model. The dynamic model developed, was comprised of three diffusion controlling barriers (two diffusion controlling membranes and a gel-matrix), which allowed modulation of the diffusion rate of macromolecules. The ability of dynamic model to modulate protein retention time in the VH will overcome the challenges faced by the semi-dynamic model such as long-term stability evaluation.