b-tagging in DELPHI at LEP

Abstract: The standard method used for tagging b-hadrons in the DELPHI experiment at the CERN LEP Collider is discussed in detail. The main ingredient of b-tagging is the impact parameters of tracks, which relies mostly on the vertex detector. Additional information, such as the mass of particles associated to a secondary vertex, significantly improves the selection efficiency and the background suppression. The paper describes various discriminating variables used for the tagging and the procedure of their combination. In addition, applications of b-tagging to some physics analyses, which depend crucially on the performance and reliability of b-tagging, are described briefly

Measurement of the Tau Lepton Polarisation at LEP2

A first measurement of the average polarisation P_tau of tau leptons produced in e+e- annihilation at energies significantly above the Z resonance is presented. The polarisation is determined from the kinematic spectra of tau hadronic decays. The measured value P_tau = -0.164 +/- 0.125 is consistent with the Standard Model prediction for the mean LEP energy of 197 GeV.A first measurement of the average polarisation Pτ of tau leptons produced in e + e − annihilation at energies significantly above the Z resonance is presented. The polarisation is determined from the kinematic spectra of tau hadronic decays. The measured value Pτ=−0.164±0.125 is consistent with the Standard Model prediction for the mean LEP energy of 197 GeV.A first measurement of the average polarisation P_tau of tau leptons produced in e+e- annihilation at energies significantly above the Z resonance is presented. The polarisation is determined from the kinematic spectra of tau hadronic decays. The measured value P_tau = -0.164 +/- 0.125 is consistent with the Standard Model prediction for the mean LEP energy of 197 GeV

Rheological investigation of manufacturability and injectability of highly concentrated monoclonal antibody formulations

Highly concentrated protein therapeutics offer a convenient way for subcutaneous (sc) drug administration by the patient him-/herself or a healthcare professional. As the therapy e.g. with monoclonal antibodies requires quite high doses in the range of mg per kg body weight, the development of highly concentrated protein formulations is needed due to the limited injection volume, generally considered being 1 - 2 mL for sc administration. The development of highly concentrated formulations exceeding 50 - 100 mg/mL poses several challenges including chemical and physical stability (e.g. aggregation) as well as solution viscosity. Thereby, the increase in viscosity observed with higher protein concentration may cause severe limitations during product development as well as processing and drug administration. These limitations are defined by the flow rate/injection rate depending on the applied pressure which is needed during manufacture (fill-finish), in particular during filtration, and drug administration. The focus of this work was to investigate the rheological behavior of protein solutions at high protein concentrations. The main objective was to obtain a profound understanding of two critical, hydrodynamic processes for highly concentrated protein solutions, which were drug administration and filtration, and to elucidate the role of viscosity with regard to potential limitations. The current work provides a detailed overview on product characteristics of ten commercially available, highly concentrated protein therapeutics (Chapter 1). This technical overview summarizes formulation properties like viscosity and number of visible and sub-visible particles, physico-chemical properties like pH and osmolality as well as injection device characteristics, such as device dimensions. The analysis of marketed products revealed significant differences between the products. The current benchmark for maximum protein concentration and of viscosity was identified as a liquid formulation at a protein concentration of 200 mg/mL with a dynamic viscosity of 102 mPas (20? C). This product, which is provided in a pre-filled syringe, also exhibits the largest inner needle diameter of 25 G compared to other commercial products using 27 G needle for the injection device. In the following (Chapter 2), advantages and limitations of different methods for viscosity determination of protein formulations are discussed. Moreover, a high-throughput method to measure viscosity was established. This method uses a capillary electrophoresis instrumentation without operation of the electrical field. The established method has the advantage of being automated offering the possibility for high-throughput by use of low sample amounts in the microliter range at the same time. (Allmendinger et al., J Pharm Biomed Anal, 99 (2014) 51-58) Based on these studies, the present work investigated and characterized the subcutaneous drug administration process of highly concentrated protein formulations providing quantitative in vitro (Chapter 3) and in vivo data (G¨ottingen minipigs) of injection forces (Chapter 4). Chapter 3 describes in detail the establishment of an in silico model to predict injection forces depending on syringe and needle dimensions, solution viscosity, and injection rate. Importantly, this model accounts for shear thinning behavior (non-Newtonian flow behavior) of highly concentrated protein olutions, which leads to lower effective injection forces than expected from current literature models. (Allmendinger et al., Eu J Pharm Biopharm, 87 (2014) 318-328) To address the in vivo situation, Chapter 4 investigates and quantifies the contribution of the subcutaneous tissue backpressure and specifically reports the additional influence of body temperature on injection forces, which was found to compensate the tissue backpressure to some parts. Overall, an extended model, which addresses the injection force as a function of viscosity, volumetric flow/injection rate, needle/device dimensions, shear-thinning behavior, sc backpressure, and body temperature, was developed to predict injection forces representative for the in vivo situation. This knowledge is of key importance for the development of combination products (e.g. autoinjectors or pre-filled syringes) as a detailed understanding of injection forces depending on various parameters is required. It may be also supportive for the definition of limits during the evaluation, planning, and design phase during the development of injection devices. (Allmendinger et al., submitted to Pharm Research, 2014) Besides drug administration, filtration was investigated as another critical hydrodynamic process for highly concentrated protein formulations, depending on formulation composition and filter material (Chapter 5). For both processes, filtration and drug administration, shear thinning behavior was found for some of the products depending on viscosity and protein concentration, shear rate, and formulation composition. Within the present work it was shown, that the two investigated hydrodynamic processes, filtration and drug administration by injection, are two highly complex processes which are influenced by various factors. Thereby, the final limiting parameter for the injection process is given by the user capability of the patient population. However, the needle inner diameter was shown to have major influence on injection forces. It is related to injection forces by the power of four compared to other parameters like viscosity, injection rate, and contribution of sc backpressure being directly proportional. For the filtration process, the final limiting parameter may be discussed controversially. The study showed that the filtration pressure is mainly defined by the pore size distribution of the filter material, which was furthermore found to trigger the rheological behavior at high protein concentrations dependent on filtration rate. Moreover, literature data reported that the influence of filtration pressure on product quality might not be the limiting parameter during filtration. For the formulations previously tested, the shear stress exposure during manufacture was not considered important for final product quality, however only tested up to a protein concentration of 100 mg/mL. More important causes of aggregation were suggested to be the presence of air-bubbles, adsorption to solid surfaces, or contamination by particulates. Nevertheless, the stability of formulations showing pronounced shear-thinning behavior at high shear rates, which is most likely only the case for higher protein concentrations than previously tested, needs further experiments and has to be evaluated on a case-by-case basis dependent on product and process characteristics. (Allmendinger et al., submitted to J Pharm Sci, 2014) With respect to viscosity, the current work has demonstrated for both processes, drug administration and filtration, that the potential limitation defined by the proportional increase in pressure based on Newtonian flow behavior was overestimated due to the presence of shear-thinning behavior which was shown for highly concentrated protein formulations

Impact of formulation and process parameters on near-infrared spectra: Application for water determination in biopharmaceuticals

Traditionally, the water content of freeze-dried biopharmaceuticals is determined by time-consuming methods such as Karl Fischer titration. As a fast and non-destructive method, many studies demonstrated the efficiency of Near-Infrared (NIR) spectroscopy for that purpose [1]. In this study, NIR was applied to different freeze-dried monoclonal antibody. The aim was to evaluate the robustness of a NIR model depending on formulation composition and process parameters of the lyophilization parameters, and the benefits of NIR when developing a freeze-drying cycle for a new pharmaceutical product. A full Design of experiments (DoE) was established in order to produce materials with various formulations and various process parameters. As a first step, a calibration model was created and validated. The model creation was based on 4 target lyophilized cycles which were manufactured to obtain samples with different water content concentration. Then, 20 lyophilized cycles were produced according to the DoE. Two levels of protein and sucrose concentration, and two levels of pressure / primary drying temperature and process time were investigated. Furthermore, several samples of each experiment stored at different temperature and relative humidity conditions were evaluated. Chemometrics using Principal Component Analysis (PCA) and Partial Least Squares (PLS) were used to evaluate the process variations and to determine the water content, respectively. NIR is capable to differentiate between different lyophilization process conditions, based on chemometrics. Robust calibration NIR model for water determination was generated against KF independent on lyophilization process parameters and formulation composition. NIR is suitable and robust method for drug product development of freeze-dried formulation

Comparison of Techniques to Control Ice Nucleation during Lyophilization

Controlling ice nucleation during lyophilization of parenteral drug products increases the homogeneity of critical quality attributes, such as residual moisture, across drug product batches and shortens lyophilization cycle time. In the present study, we compare three mechanistically different techniques to control ice nucleation during the freezing step of lyophilization, which are referred to as “depressurization”, “partial vacuum”, and “ice fog” techniques. The techniques are compared with respect to their operational limitations and challenges. Installation considerations are also discussed. Using the aforementioned nucleation techniques, we investigated a monoclonal antibody formulation and an enzyme formulation at different protein concentrations using feasible nucleation temperatures and different vial formats and fill volumes. Samples were compared for solid state properties and other critical quality attributes on stability. When nucleated at the same temperature, the three techniques produced products with the same quality attributes and stability behavior. Under conditions resulting in micro-collapse, stability behavior can be different. We found that each technology had considerations for achieving robust nucleation. The present comparison may serve as guidance in selecting a nucleation method

Excipients for Room Temperature Stable Freeze-Dried Monoclonal Antibody Formulations

Sucrose is a commoncryoprotectant and lyoprotectant to stabilize labile biopharmaceuticals during freezedrying and storage. Sucrose-based formulations require low primary drying temperatures to avoid collapse and monoclonal antibody (mAb) containing products need to be stored refrigerated. The objective of this study is to investigate different excipients enabling storage at room temperature and aggressive, shorter lyophilization cycles. We studied combinations of 2-hydroxypropyl-beta-cyclodextrin (CD), recombinant human albumin, polyvinylpyrroldione (PVP), dextran 40 kDa (Dex), and sucrose (Suc) using 2 mAbs. Samples were characterized for collapse temperature (T c ), glass transition temperature of the liquid (T g 1 ) and freeze-dried formulation (T g ), cake appearance, residual moisture, and reconstitution time. Freezedried formulations were stored at 5C, 25C, and 40C for up to 9 months and mAb stability was analyzed for color, turbidity, visible and sub-visible particles, andmonomer content. Formulations with CD/Suc or CD/PVP/Suc were superior to pure Suc formulations for long-term storage at 40C. When using aggressive freeze-drying cycles, these formulations were characterized by pharmaceutically elegant cakes, short reconstitution times, higher T g 1 , T c , and T g . We conclude that the addition of CD allows for shorter freeze-drying cycles with improved cake appearance and enables storage at room temperature, which might reduce costs of goods substantially

Impact of dextran on thermal properties, product quality attributes, and monoclonal antibody stability in freeze-dried formulations

Freeze-drying is commonly used to improve stability of liquid formulations of labile biopharmaceuticals. Lyo- and cryoprotectants such as sucrose are traditionally utilized as excipients, but have low glass transition (Tg') and collapse temperatures (Tc). Consequently, these formulations require low primary drying temperatures making the lyophilization cycle time-consuming and costly. We investigated different dextrans (1, 40, 150, and 500 kDa) and mixtures of dextran with sucrose as alternative excipients. The influence of dextran on thermal properties, cake appearance, and other quality attributes in the solid state was studied using bovine serum albumin as model protein. Especially at higher weight ratios of dextran to sucrose, dextrans of medium to high molecular weight (MW) of 40-500 kDa showed up to 20°C higher Tc compared to sucrose, which was reflected in elegant lyophilisates. However, this resulted in slower reconstitution times. Addition of dextran led to lower residual moisture levels and higher Tg values compared to sucrose. We confirmed the thermal properties for two monoclonal antibodies (mAb) at two weight ratios of sucrose and dextran with different MW, and tested for stability at 40°C for 14 days. While no loss in relative potency of the antibodies was observed after storage, size exclusion chromatography and isoelectric focusing revealed a strong increase in high molecular weight species (HMWs) and acidic species, which were dependent on the MW of the dextrans. With further characterization of selected formulations (dextran 1 kDa) by boronate affinity chromatography and mass spectrometry analysis, we demonstrated that HMWs were a result of glycation by free terminal glucose of the dextran. This chemical modification was strongly reduced when adding sucrose, which protects the protein possibly by shielding its surface. Our results demonstrate that formulation scientists need to use dextrans as excipients in freeze-dried mAb formulations with caution. A binary mixture of sucrose and dextran in adequate ratio however might potentially be superior to pure sucrose formulations allowing for faster freeze-drying cycles resulting in elegant lyophilisates and good protein stability

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

Be Aggressive! Amorphous Excipients Enabling Single-Step Freeze-Drying of Monoclonal Antibody Formulations

Short freeze-drying cycles for biopharmaceuticals are desirable. Formulations containing an amorphous disaccharide, such as sucrose, are prone to collapse upon aggressive primary drying at higher shelf temperature. We used 2-hydroxypropyl-betacyclodextrin (HPBCD) in combination with sucrose and polyvinylpyrrolidone (PVP) to develop an aggressive lyophilization cycle for low concentration monoclonal antibody (mAb) formulations. Glass transition temperature and collapse temperature of the formulations were determined, and increasingly aggressive cycle parameters were applied. Using a shelf temperature of +30 °C during primary drying, the concept of combining sublimation and desorption of water in a single drying step was investigated. Cake appearance was evaluated visually and by micro-computed tomography. Lyophilisates were further analyzed for reconstitution time, specific surface area, residual moisture, and glass transition temperature. We demonstrated the applicability of single-step freeze-drying, shortening the total cycle time by 50% and providing elegant lyophilisates for pure HPBCD and HPBCD/sucrose formulations. HPBCD/PVP/sucrose showed minor dents, while good mAb stability at 10 mg/mL was obtained for HPBCD/sucrose and HPBCD/PVP/sucrose when stored at 40 °C for 3 months. We conclude that HPBCD-based formulations in combination with sucrose are highly attractive, enabling aggressive, single-step freeze-drying of low concentration mAb formulations, while maintaining elegant lyophilisates and ensuring protein stability at the same time