Evaluation of alternative surfactants as stabilizers for therapeutical protein formulations

Surfactants are potent stabilizers of proteins, preventing protein unfolding and aggregation presumably by competitive adsorption to interfaces. Due to limited data on e.g. performance and safety, only three surfactants are routinely found within marketed formulations of parenteral protein products: polysorbate 20 (PS20), polysorbate 80 (PS80), or poloxamer 188 (Px188). These molecules are well-established and safe for parenteral administration but possess liabilities such as degradation of the surfactant itself and/or chemical inhomogeneity (PS) or show decreased stabilizing effects at silicone oil-water interfaces (Px188). Thus, there is a need to evaluate alternative surfactants to expand the toolbox for product development and to ensure optimal drug product stability and quality. In our studies, potential alternative surfactants were evaluated and compared to PS20 and Px188 (e.g. during real-time and stress stability studies using a model mAb). Data from these efforts will be presented

Stability of Monoclonal Antibodies after Simulated Subcutaneous Administration

Changes in the environment from the drug product to the human physiology might lead to physical and/or chemical modifications of the protein drug, such as in vivo aggregation and fragmentation. Although subcutaneous (SC) injection is a common route of administration for therapeutic proteins, knowledge on in vivo stability in the SC tissue is limited. In this study, we developed a physiologic in vitro model simulating the SC environment in patients. We assessed the stability of two monoclonal antibodies (mAbs) in four different protein-free fluids under physiologic conditions. We monitored protein stability over two weeks using a range of analytical methods, in analogy to testing purposes of a drug product. Both mAbs showed an increase of protein aggregates, fragments, and acidic species. mAb1 was consistently more stable in this in vitro model than mAb2, highlighting the importance of comparing the stability of different mAbs under physiologic conditions. Throughout the study, both mAbs were substantially less stable in bicarbonate buffers as compared to phosphate-buffered saline. In summary, our developed model was able to differentiate stability between molecules. Bicarbonate buffers were more suitable compared to phosphate-buffered saline in regards to simulating the in vivo conditions and evaluating protein liabilities

Interaction of the antiviral drug telaprevir with renal and hepatic drug transporters

Telaprevir is a new, direct-acting antiviral drug that has been approved for the treatment of chronic hepatitis C viral infection. First data on drug-drug interactions with co-medications such as cyclosporine, tacrolimus and atorvastatin have been reported recently. Drug transporting proteins have been shown to play an important role in clinically observed drug-drug interactions. The aim of this study was therefore to systematically investigate the potential of telaprevir to inhibit drug transporting proteins. The effect of telaprevir on substrate uptake mediated by drug transporters located in human kidney and liver was investigated on a functional level in HEK293 cell lines that over-express single transporter. Telaprevir was shown to exhibit significant inhibition of the human renal drug transporters OCT2 and MATE1 with IC50 values of 6.4 µM and 23.0 µM, respectively, whereas no inhibitory effect on OAT1 and OAT3 mediated transport by telaprevir was demonstrated. Liver drug transporters were inhibited with IC50 of 2.2 µM for OATP1B1, 6.8 µM for OATP1B3 and 20.7 µM for OCT1. Our data show that telaprevir exhibited significant potential to inhibit human drug transporters. In view of the inhibitory potential of telaprevir, clinical co-administration of telaprevir together with drugs that are substrates of renal or hepatic transporters should be carefully monitored

The Effect of Formulation, Process, and Method Variables on the Reconstitution Time in Dual Chamber Syringes

Reconstitution time of dried products is influenced by various factors including formulation, process, and reconstitution method itself. This manuscript describes factors affecting reconstitution in a dual chamber syringe using highly concentrated human monoclonal antibody and bovine serum albumin model formulations. Freezing and drying conditions had only minor impact on the reconstitution time, whereas the primary container and thus the geometry of the lyophilization cake played a major role. Prewarmed diluent and agitation decreased reconstitution time. For effective agitation, short displacements and high agitation frequencies were found to be desirable conditions to minimize reconstitution time for a given lyophilization cake while foam formation was minimized. The article also provides general strategies (e.g., reduction of lyophilized cake density, use of an optimized fill finish process, and suitable method parameters) to reduce reconstitution time, especially for drug product presented in a dual chamber syringe configuration. LAY ABSTRACT: Dried drug products need to be reconstituted to a liquid form before being applied parenteral. Reconstitution time is an important attribute and needs to be as fast as possible in order to serve patients' compliance. Reconstitution time is influenced by various factors including formulation, process, and the reconstitution method itself. The article provides general strategies (e.g., reduction of dried drug product cake density, use of an optimized fill finish process, and suitable method parameters) to reduce reconstitution time, especially for drug product presented in a dual chamber syringe. Fast reconstitution of lyophilisates in dual chamber syringe can be achieved by a combination of optimized manufacturing procedures and clear instructions for the end-user (e.g., roll syringe between palms to warm and agitate it to accelerate reconstitution)

A Method to Determine the Kinetics of Sulute Mixing in Liquid/Liquid Formulation Dual-Chamber Syringes

Dual-chamber syringes were originally designed to separate a solid substance and its diluent. However, they can also be used to separate liquid formulations of two individual drug products, which cannot be co-formulated due to technical or regulatory issues. A liquid/liquid dual-chamber syringe can be designed to achieve homogenization and mixing of both solutions prior to administration, or it can be used to sequentially inject both solutions. While sequential injection can be easily achieved by a dual-chamber syringe with a bypass located at the needle end of the syringe barrel, mixing of the two fluids may provide more challenges. Within this study, the mixing behavior of surrogate solutions in different dual-chamber syringes is assessed. Furthermore, the influence of parameters such as injection angle, injection speed, agitation, and sample viscosity were studied. It was noted that mixing was poor for the commercial dual-chamber syringes (with a bypass designed as a longitudinal ridge) when the two liquids significantly differ in their physical properties (viscosity, density). However, an optimized dual-chamber syringe design with multiple bypass channels resulted in improved mixing of liquids. LAY ABSTRACT: Dual-chamber syringes were originally designed to separate a solid substance and its diluent. However, they can also be used to separate liquid formulations of two individual drug products. A liquid/liquid dual-chamber syringe can be designed to achieve homogenization and mixing of both solutions prior to administration, or it can be used to sequentially inject both solutions. While sequential injection can be easily achieved by a dual-chamber syringe with a bypass located at the needle end of the syringe barrel, mixing of the two fluids may provide more challenges. Within this study, the mixing behavior of surrogate solutions in different dual-chamber syringes is assessed. Furthermore, the influence of parameters such as injection angle, injection speed, agitation, and sample viscosity were studied. It was noted that mixing was poor for the commercially available dual-chamber syringes when the two liquids significantly differ in viscosity and density. However, an optimized dual-chamber syringe design resulted in improved mixing of liquids

Imaging Techniques to Characterize Cake Appearance of Freeze-Dried Products

Pharmaceutically elegant lyophilisates are highly desirable implying a stable and robust freeze-drying process. To ensure homogenous and intact cake appearance after process scale-up and transfer, characterization of lyophilisates during formulation and cycle development is required. The present study investigates different imaging techniques to characterize lyophilisates on different levels. Cake appearance of freeze-dried bovine serum albumin formulations with different dextran/sucrose ratios was studied by visual inspection, three-dimensional laser scanning, polydimethylsiloxane embedding, scanning electron microscopy, and microcomputed tomography (μ-CT). The set of techniques allowed a holistic evaluation of external cake appearance and internal structure providing complementary information at macroscopic and microscopic scale. In comparison to state of the art technologies like visual inspection or scanning electron microscopy, three-dimensional laser scanning and μ-CT provided quantitative information allowing comparison of visual cake appearance. In particular μ-CT enables a global, qualitative, and quantitative characterization of external and internal cake structure with a single measurement detecting heterogeneities of lyophilisates. We even demonstrated the use of noninvasive μ-CT for qualitative imaging of internal cake structure through the glass vial. Providing meaningful characterization of the entire lyophilisate, μ-CT can serve as a powerful tool during development of freeze-drying cycles, process scale-up, and transfer

Particle Analysis of Biotherapeutics in Human Serum Using Machine Learning

In recent years, an increasing number of studies assessed the stability of biotherapeutics in biological fluids. Such studies aim to simulate the conditions encountered in the human body and investigate the in vivo stability under in vitro conditions. However, on account of complexity of biological fluids, standard pharmaceutical methods are poorly suited to assess the stability of biotherapeutics. In this study, a fluorescent-labeled therapeutic immunoglobulin G (IgG) was analyzed for proteinaceous particles after mixing with human serum and after incubation at 37°C for 5 days. Samples were analyzed using standard pharmaceutical methods (light obscuration and dynamic imaging). Moreover, we developed a fluorescence microscopy method allowing to semiquantitatively detect IgG particles in serum. Several hundred IgG particles were detected after exposure to serum. Moreover, particle counts and particle size increased in serum over time. The results showed that an IgG may form particles on mixing with serum and novel methods such as fluorescence microscopy are required to gain insight on the stability of biotherapeutics in biological fluids. Furthermore, we showed distinct advantages of machine learning over traditional threshold-based methods by analyzing microscopy images. Machine learning allowed simplifying particles in regards to count, size, and shape

Tracking the physical stability of fluorescent-labeled mAbs under physiologic invitro conditions in human serum and PBS

In recent years, the stability of biotherapeutics in vivo has received increasing attention. Assessing the stability of biotherapeutics in serum may support the selection of adequate molecule candidates. In our study, we compared the physical stability of 8 different monoclonal antibodies (mAbs) in phosphate-buffered saline (PBS) and human serum. mAbs were Alexa Fluor 488-labeled and characterized with respect to fragmentation, aggregation, and proteinaceous particle formation. Samples were analyzed using size-exclusion chromatography, light obscuration, and flow imaging. In addition, novel methods such as flow cytometry and fluorescence microscopy were applied. mAbs were selected based on their hydrophobicity and isoelectric point. All mAbs studied were inherently less stable in human serum as compared to PBS. Particle size and particle counts increased in serum over time. Interestingly, certain mAbs showed significant levels of fragmentation in serum but not in PBS. We conclude that PBS cannot replicate the physical stability measured in serum. The stability of labeled mAbs in human serum did not correlate with their hydrophobicity and isoelectric point . Serum stability significantly differed amongst the tested mAbs

Stabilizing Polysorbate 20 and 80 Against Oxidative Degradation

Polysorbates are stabilizers typically required in therapeutic protein formulations. Due to their chemical structure, polysorbates are prone to degradation, which can render a pharmaceutical product instable or incompliant. The purpose of this study was to investigate if the addition of butylhydroxytoluene (BHT) and butylhydroxyanisole (BHA) protects Polysorbate 20 (PS20) and Polysorbate 80 (PS80) against oxidative degradation.; PS20 and PS80 solutions containing BHA, BHT - or as control without an antioxidant - were stressed by exposure to air at 40°C for seven weeks. The following assays were performed: ferrous oxidation-xylenol orange (FOX) assay, liquid chromatography coupled to ultraviolet (UV) and mass spectrometry (MS), pH-measurement, liquid chromatography fluorescence micelle assay (FMA), headspace-gas chromatography (HS-GC) coupled with MS.; PS20 and PS80 solutions containing an antioxidant were found to be more stable, demonstrated by lower peroxide levels, lower free fatty acid contents, stable pH, intact polysorbate micelle structure / composition, and less volatile degradants.; PS20 and PS80 solutions containing BHT or BHA are more stable against oxidative degradation compared to non-stabilized solutions. It might be beneficial to formulate bulk polysorbate with the antioxidant(s) to ensure stabilization during all process steps