Biophysical characterization approaches to aid the selection of protein formulations by predicting their physical stability during long-term storage

The formulation of therapeutic proteins is a critical process which aims at finding the most suitable conditions that impede protein degradation during long-term storage. One degradation path of high interest is the non-native aggregation1. The latter can be greatly suppressed by the selection of suitable solution conditions2. Over the years, various biophysical techniques have been explored as tools to quickly select the most promising formulations for long-term storage. In this talk, we share the experience in our lab how some of these techniques can be integrated into protein formulation studies. We discuss the application of differential scanning calorimetry and the usefulness of determining the apparent protein melting temperatures to select protein formulations with high physical stability3. In the next part, we show how state-of-the-art equipment that employs fluorimetric and light scattering measurements during heating allows the sample saving characterization of proteins to select stable formulations. We then discuss the limitations of the existing non-isothermal techniques and show how the results from formulation studies can be biased when some excipient properties change during heating. In this context, we explore whether isothermal chemical denaturation is a useful complementary tool to overcome the limitations of non-isothermal techniques4. Finally, we show how the assessment of the aggregation of partially folded species during refolding can provide additional information for the selection of protein formulations with high physical stability during storage4. The talk will be concluded with few general suggestions how to select solution conditions that impede aggregation during long-term storage of liquid protein formulations. References 1. Roberts, C. J. Non-Native Protein Aggregation Kinetics. 98, (Springer New York, 2007). 2. Shire, S. J. Formulation and manufacturability of biologics. Curr. Opin. Biotechnol. 20, 708–714 (2009). 3. Youssef, A. M. K. & Winter, G. A critical evaluation of microcalorimetry as a predictive tool for long term stability of liquid protein formulations: Granulocyte Colony Stimulating Factor (GCSF). Eur. J. Pharm. Biopharm. 84, 145–155 (2013). 4. Svilenov, H., Markoja, U. & Winter, G. Isothermal chemical denaturation as a complementary tool to overcome limitations of thermal differential scanning fluorimetry in predicting physical stability of protein formulations. Eur. J. Pharm. Biopharm. 125, 106–113 (2018)

The ReFOLD assay for protein formulation studies and prediction of protein aggregation during long-term storage

The formulation of novel therapeutic proteins is a challenging task which aims at finding formulation conditions that will minimize protein degradation during long-term storage. One particularly important and difficult-to-predict protein degradation pathway is the so-called non-native aggregation. The qualitative and quantitative prediction of the latter has been a subject of extensive research over the past two decades. An increasing body of evidence shows that the widely-used short-term biophysical techniques cannot accurately rank formulation conditions in order of their effect on the aggregation during long-term storage of some therapeutic proteins, e.g. monoclonal antibodies. Here we suggest a novel approach for the selection of formulation conditions that will suppress the formation of protein aggregates during long-term storage. We postulate that conditions (i.e. pH, buffer type, ionic strength) that reduce the isothermal aggregation of various denaturant-induced partially folded protein species will be conditions that impede protein aggregation during long-term storage. To test our hypothesis, we developed an isothermal microdialysis-based unfolding/refolding assay, named ReFOLD, which we use to induce moderate aggregation of partially folded proteins. Next, we assessed the relative monomer yield after isothermal unfolding/refolding of two monoclonal antibodies, each formulated in 12 different conditions. Using the proposed approach, we were able to accurately rank the formulations in order of their effect on the amount of protein aggregates detected after storage for 12 months at 4 °C and 25 °C, while widely-used stability-indicating parameters like protein melting and aggregation onset temperatures failed to provide accurate predictive formulation rankings

Formulations that suppress aggregation during long-term storage of a bispecific antibody are characterized by high refoldability and colloidal stability

Understanding the formulation features that ensure sufficient stability during long-term storage is critical for developing next-generation therapeutic proteins. In this work, we investigate the physical stability of a bispecific antibody (Bis-mAb) in 12 different formulation conditions. Isothermal chemical denaturation with urea indicates a higher resistance to denaturant-induced unfolding when pH is increased from 5.0 to 6.5 but shows minor influence from the buffer type and ionic strength. Dynamic and static light scattering are used to derive the interaction parameter (kD) and second virial coefficient (A2), respectively. These two parameters indicate that Bis-mAb exhibits highest colloidal stability in formulations containing 10 mM histidine buffer without added sodium chloride. Further, we observe that the highest relative monomer yield (RMY) after isothermal refolding, i.e. the highest refoldability, from urea is measured for the low ionic strength histidine formulations. Finally, we show long-term stability data on all 12 Bis-mAb formulations after storage at 4 °C and 25 °C for 12 months. The least amount of soluble aggregates and subvisible particles were detected in the Bis-mAb formulations with the highest colloidal stability and refoldability from urea. We suggest that the optimization of these two features is crucial for obtaining physically stable formulations of Bis-mAb

Exploring Chemical Space for new Substances to stabilize a therapeutic Monoclonal Antibody

The physical stability of therapeutic proteins is a major concern in the development of liquid protein formulations. The number of degrees of freedom to tweak a given protein’s stability is limited to pH, ionic strength and type and concentration of excipient. There are only very few, mostly similar excipients currently in use, limited to the short list of substances generally recognized as safe for human use by the FDA. Opposed to the limited number of molecules the formulation scientist has at hand to stabilize a protein, there is the vastness of chemical space which is hypothesized to consist of 1060 compounds. Its potential to stabilize proteins has never been explored systematically in the context of stabilization of therapeutic proteins. Here we present a screening strategy to discover new excipients to further stabilize an already stable formulation of a therapeutic antibody. We use our data to build a predictive model to evaluate the stabilizing potential of small molecules. We argue that prior to worrying about the hurdles of toxicity and approval of novel excipient candidates, it is mandatory to assess the actual potential hidden in the chemical space

Plectin isoform 1b mediates mitochondrion–intermediate filament network linkage and controls organelle shape

Plectin is a versatile intermediate filament (IF)–bound cytolinker protein with a variety of differentially spliced isoforms accounting for its multiple functions. One particular isoform, plectin 1b (P1b), remains associated with mitochondria after biochemical fractionation of fibroblasts and cells expressing exogenous P1b. Here, we determined that P1b is inserted into the outer mitochondrial membrane with the exon 1b–encoded N-terminal sequence serving as a mitochondrial targeting and anchoring signal. To study P1b-related mitochondrial functions, we generated mice that selectively lack this isoform but express all others. In primary fibroblasts and myoblasts derived from these mice, we observe a substantial elongation of mitochondrial networks, whereas other mitochondrial properties remain largely unaffected. Normal morphology of mitochondria could be restored by isoform-specific overexpression of P1b in P1b-deficient as well as plectin-null cells. We propose a model where P1b both forms a mitochondrial signaling platform and affects organelle shape and network formation by tethering mitochondria to IFs

Comparison of the Effects of Early Pregnancy with Human Interferon, Alpha 2 (IFNA2), on Gene Expression in Bovine Endometrium

Interferon tau (IFNT), a type I IFN similar to alpha IFNs (IFNA), is the pregnancy recognition signal produced by the ruminant conceptus. To elucidate specific effects of bovine IFNT and of other conceptus-derived factors, endometrial gene expression changes during early pregnancy were compared to gene expression changes after intrauterine application of human IFNA2. In experiment 1, endometrial tissue samples were obtained on Day (D) 12, D15, and D18 postmating from nonpregnant or pregnant heifers. In experiment 2, heifers were treated from D14 to D16 of the estrous cycle with an intrauterine device releasing IFNA2 or, as controls, placebo lipid extrudates or PBS only. Endometrial biopsies were performed after flushing the uterus. All samples from both experiments were analyzed with an Affymetrix Bovine Genome Array. Experiment 1 revealed differential gene expression between pregnant and nonpregnant endometria on D15 and D18. In experiment 2, IFNA2 treatment resulted in differential gene expression in the bovine endometrium. Comparison of the data sets from both studies identified genes that were differentially expressed in response to IFNA2 but not in response to pregnancy on D15 or D18. In addition, genes were found that were differentially expressed during pregnancy but not after IFNA2 treatment. In experiment 3, spatiotemporal alterations in expression of selected genes were determined in uteri from nonpregnant and early pregnant heifers using in situ hybridization. The overall findings of this study suggest differential effects of bovine IFNT compared to human IFNA2 and that some pregnancy-specific changes in the endometrium are elicited by conceptus-derived factors other than IFNT

Inhalative Nanoparticulate CpG Immunotherapy in Severe Equine Asthma: An Innovative Therapeutic Concept and Potential Animal Model for Human Asthma Treatment

Severe equine asthma is the most common globally widespread non-infectious equine respiratory disease (together with its mild and moderate form), which is associated with exposure to hay dust and mold spores, has certain similarities to human asthma, and continues to represent a therapeutic problem. Immunomodulatory CpG-ODN, bound to gelatin nanoparticles as a drug delivery system, were successfully administered by inhalation to severe equine asthmatic patients in several studies. It was possible to demonstrate a significant, sustained, and allergen-independent one-to-eight-week improvement in key clinical parameters: the arterial partial pressure of oxygen, the quantity and viscosity of tracheal mucus, and neutrophilic inflammatory cells in the respiratory tracts of the severe equine asthmatic subjects. At the immunological level, an upregulation of the regulatory antiallergic and anti-inflammatory cytokine IL-10 as well as a downregulation of the proallergic IL-4 and proinflammatory IFN-γ in the respiratory tracts of the severe equine asthmatic patients were identified in the treatment groups. CD4+ T lymphocytes in the respiratory tracts of the asthmatic horses were demonstrated to downregulate the mRNA expression of Tbet and IL-8. Concentrations of matrix metalloproteinase-2 and -9 and tissue inhibitors of metalloproteinase-2 were significantly decreased directly after the treatment as well as six weeks post-treatment. This innovative therapeutic concept thus opens new perspectives in the treatment of severe equine asthma and possibly also that of human asthma

Homogeneous Heat Transfer During Freeze-Drying Using Cyclic Olefin Polymer Vials

In pharmaceutical freeze-drying processes, batch homogeneity is an important quality attribute. In this context, the edge-vial-effect is a challenging phenomenon. Shortly, this effect describes that vials at the edges of the shelf dry faster and at a higher temperature compared to vials in the middle of the shelf. Studies by Ehlers et al. revealed that this effect mainly origins from the number of neighbor vials cooling each other, which is reduced for vials in corners and edges compared to vials in the middle. Due to the reduced heat transfer in cyclic olefin polymer (COP) vials, the adverse edge-vial-effect should be greatly reduced allowing a better batch uniformity. In this focused study, glass and COP vials are compared regarding this effect on a fully loaded shelf. A reference experiment with vials placed at distance using a specially designed frame is presented as well