The Strengths of Total Holographic Video Microscopy in Detecting Sub-Visible Protein Particles in Biopharmaceuticals: A Comparison to Flow Imaging and Resonant Mass Measurement.

Determination of subvisible particle (SVP) content in biopharmaceuticals is a prerequisite to ensure the quality of liquid biopharmaceutical products. Here, we present a comparison of the recently introduced holographic video microscopy (total holographic characterization, THC) with two orthogonal and well-established analytical technologies: micro flow imaging (MFI) and resonant mass measurement (RMM). The capabilities of the THC were investigated under conditions commonly applied in drug product development. Three different antibody products were used at different concentrations and formulations to cover a wide range of realistic use-cases. The comparison was particularly focused on protein aggregates to investigate the applicability of THC to this critical class of particles in drug product development. Protein concentrations up to 100 mg/ml were investigated covering a broad range of viscosity and refractive indices, both important parameters in particle detection. The comparison reveals that THC is highly sensitive to detect protein aggregates in a size range from 0.5 µm to 10 µm. THC shows a significant superiority to FI and RMM in detecting heterogenous protein aggregates which often appear as transparent and porous particles. Additionally, THC needs very small sample amount of about 30 µl and short measurement times, making it applicable for early development stages and high-throughput approaches. These results show that THC is a valuable supplement to the existing particle characterization method portfolio in drug product development.

Comparisons of electrophysiological and psychophysical fitting methods for cochlear implants.

OBJECTIVE: This study compared two different versions of an electrophysiology-based software-guided cochlear implant fitting method with a procedure employing standard clinical software. The two versions used electrically evoked compound action potential (ECAP) thresholds for either five or all twenty-two electrodes to determine sound processor stimulation level profiles. Objective and subjective performance results were compared between software-guided and clinical fittings. DESIGN: Prospective, double-blind, single-subject repeated-measures with permuted ABCA sequences. STUDY SAMPLE: 48 post linguistically deafened adults with ≤15 years of severe-to-profound deafness who were newly unilaterally implanted with a Nucleus device. RESULTS: Speech recognition in noise and quiet was not significantly different between software- guided and standard methods, but there was a visit/learning-effect. However, the 5-electrode method gave scores on the SSQ speech subscale 0.5 points lower than the standard method. Clinicians judged usability for all methods as acceptable, as did subjects for comfort. Analysis of stimulation levels and ECAP thresholds suggested that the 5-electrode method could be refined. CONCLUSIONS: Speech recognition was not inferior using either version of the electrophysiology-based software-guided fitting method compared with the standard method. Subject-reported speech perception was slightly inferior with the five-electrode method. Software-guided methods saved about 10 min of clinician's time versus standard fittings.

Industry Perspective on the Use and Characterization of Polysorbates for Biopharmaceutical Products Part 2: Survey Report on Control Strategy Preparing for the Future.

Polysorbate (PS) 20 and 80 are the main surfactants used to stabilize biopharmaceutical products. Industry practices on various aspects of PS based on a confidential survey and following discussions by 16 globally acting major biotechnology companies is presented in two publications. Part 1 summarizes the current practice and use of PS during manufacture in addition to aspects like current understanding of the (in)stability of PS, the routine QC testing and control of PS, and selected regulatory aspects of PS.1 The current part 2 of the survey focusses on understanding, monitoring, prediction, and mitigation of PS degradation pathways in order to propose an effective control strategy. The results of the survey and extensive cross-company discussions are put into relation with currently available scientific literature.

Industry Perspective on the use and Characterization of Polysorbates for Biopharmaceutical Products Part 1: Survey Report on Current State and Common Practices for Handling and Control of Polysorbates.

Polysorbates (PS) are widely used as a stabilizer in biopharmaceutical products. Industry practices on various aspects of PS are presented in this part 1 survey report based on a confidential survey and following discussions by 16 globally acting major biotechnology companies. The current practice and use of PS during manufacture across their global manufacturing sites are covered in addition to aspects like current understanding of the (in)stability of PS, the routine QC testing and control of PS, and selected regulatory aspects of PS. The results of the survey and extensive cross-company discussions are put into relation with currently available scientific literature. Part 2 of the survey report (upcoming) will focus on understanding, monitoring, prediction, and mitigation of PS degradation pathways to develop an effective control strategy.

A Multicompany Assessment of Submicron Particle Levels by NTA and RMM in a Wide Range of Late-Phase Clinical and Commercial Biotechnology-Derived Protein Products.

One of the major product quality challenges for injectable biologics is controlling the amount of protein aggregates and particles present in the final drug product. This article focuses on particles in the submicron range (<2 µm). A cross-industry collaboration was undertaken to address some of the analytical gaps in measuring submicron particles (SMPs), developing best practices, and surveying the concentration of these particles present in 52 unique clinical and commercial protein therapeutics covering 62 dosage forms. Measured particle concentrations spanned a range of 4 orders of magnitude for nanoparticle tracking analysis and 3 orders of magnitude for resonant mass measurement. The particle concentrations determined by the 2 techniques differed significantly for both control and actual product. In addition, results suggest that these techniques exhibit higher variability compared to well-established subvisible particle characterization techniques (e.g., flow-imaging or light obscuration). Therefore, in their current states, nanoparticle tracking analysis and resonant mass measurement-based techniques can be used during product and process characterization, contributing information on the nature and propensity for formation of submicron particles and what is normal for the product, but may not be suitable for release or quality control testing. Evaluating the level of SMPs to which humans have been routinely exposed during the administration of several commercial and late-phase clinical products adds critical knowledge to our understanding of SMP levels that may be considered acceptable from a safety point of view. This article also discusses dependence of submicron particle size and concentration on the dosage form attributes such as physical state, primary packaging, dose strength, etc. To the best of our knowledge, this is the largest study ever conducted to characterize SMPs in late-phase and commercial products.

Systems for the cell-free synthesis of proteins.

We describe a system for the cell-free expression of proteins based on extracts from Escherichia coli. Two reaction configurations, batch and continuous exchange, are discussed and analytical scale as well as preparative scale setups are documented. Guidelines for the systematic development and optimization of cell-free expression protocols are given in detail. We further provide specific protocols and parameters for the cell-free production of membrane proteins. High-throughput screening applications of CF expression systems are exemplified as new tools for genomics and proteomics studies.

Advances in cell-free protein synthesis for the functional and structural analysis of membrane proteins.

Cell-free expression has emerged as a powerful technique to overcome major restrictions of classical in vivo membrane protein production, with sample yields of mgms of protein per ml reaction volume possible in less than a day. The open nature and high versatility of cell-free expression allows a variety of completely new ways to rationally design and optimise expression environments as well as to modulate folding kinetics for membrane proteins independent of their origin, size, topology and function. This article summarises the array of currently available options to modify and develop cell-free expression protocols adapted to the specific requirements of individual membrane proteins. We give further an overview of the recent advances of cell-free production of membrane proteins for structural and functional analysis.

Remote fitting in Nucleus cochlear implant recipients.

CONCLUSION: Remote programming is a viable alternative to face-to-face programming. The procedure can be regarded as safe, time and cost saving, and clinically feasible. OBJECTIVES: The aim of this study was to determine the suitability of commercially available video conferencing technology and remote control software for remote programming of sound processors in Nucleus cochlear implant recipients by assessing the feasibility, efficiency, risks, and benefits of remote programming compared to face-to-face programming. METHODS: This was a randomized, prospective study. Seventy Nucleus implant recipients were recruited for a random sequence comparison of one remote and one local programming session each. The time required for local or remote programming was measured and resulting MAP T and C levels were compared. The recipient provided feedback on the local and remote programming session. The audiologist and monitoring clinician were asked for their feedback on remote programming. RESULTS: Remote programming sessions were successfully finished for 69 recipients. No significant differences between T and C levels obtained by local and remote programming were found. The audiologists and monitoring clinicians agreed that the remote programming system provided an acceptable level of performance after most sessions. More than 50 participating recipients considered remote programming an efficient alternative to face-to-face-programming.

Modulation of G-protein coupled receptor sample quality by modified cell-free expression protocols: a case study of the human endothelin A receptor.

G-protein coupled receptors still represent one of the most challenging targets in membrane protein research. Here we present a strategic approach for the cell-free synthesis of these complex membrane proteins exemplified by the preparative scale production of the human endothelin A receptor. The versatility of the cell-free expression system was used to modulate sample quality by alteration of detergents hence presenting different solubilization environments to the synthesized protein at different stages of the production process. Sample properties after co-translational and post-translational solubilization have been analysed by evaluation of homogeneity, protein stability and receptor ligand binding competence. This is a first quality evaluation of a membrane protein obtained in two different cell-free expression modes and we demonstrate that both can be used for the production of ligand-binding competent endothelin A receptor in quantities sufficient for structural approaches. The presented strategy of cell-free expression protocol development could serve as basic guideline for the production of related receptors in similar systems.

Membrane protein expression in cell-free systems.

Cell-free expression has emerged as a promising tool for the fast and efficient production of membrane proteins. The rapidly growing number of successfully produced targets in combination with the continuous development of new applications significantly promotes the distribution of this technology. Membrane protein synthesis by cell-free expression does not appear to be restricted by origin, size or topology of the target, and its global application is therefore a highly valuable characteristic. The technology is relatively fast to establish in standard biochemical labs, and it does not require expensive equipment. Moreover, it enables the production of membrane proteins in completely new modes, like the direct translation into detergent micelles, which is not possible with any other expression system. In this protocol, we focus on the currently most efficient cell-free expression system for membrane proteins based on Escherichia coli extracts.

Cell-free expression and stable isotope labelling strategies for membrane proteins.

Membrane proteins are highly underrepresented in the structural data-base and remain one of the most challenging targets for functional and structural elucidation. Their roles in transport and cellular communication, furthermore, often make over-expression toxic to their host, and their hydrophobicity and structural complexity make isolation and reconstitution a complicated task, especially in cases where proteins are targeted to inclusion bodies. The development of cell-free expression systems provides a very interesting alternative to cell-based systems, since it circumvents many problems such as toxicity or necessity for the transportation of the synthesized protein to the membrane, and constitutes the only system that allows for direct production of membrane proteins in membrane-mimetic environments which may be suitable for liquid state NMR measurements. The unique advantages of the cell-free expression system, including strong expression yields as well as the direct incorporation of almost any combination of amino acids with very little metabolic scrambling, has allowed for the development of a wide-array of isotope labelling techniques which facilitate structural investigations of proteins whose spectral congestion and broad line-widths may have earlier rendered them beyond the scope of NMR. Here we explore various labelling strategies in conjunction with cell-free developments, with a particular focus on alpha-helical transmembrane proteins which benefit most from such methods.

Transmembrane segment enhanced labeling as a tool for the backbone assignment of alpha-helical membrane proteins.

Recent advances in cell-free expression protocols have opened a new avenue toward high-resolution structural investigations of membrane proteins by x-ray crystallography and NMR spectroscopy. One of the biggest challenges for liquid-state NMR-based structural investigations of membrane proteins is the significant peak overlap in the spectra caused by large line widths and limited chemical shift dispersion of alpha-helical proteins. Contributing to the limited chemical shift dispersion is the fact that approximately 60% of the amino acids in transmembrane regions consist of only six different amino acid types. This principle disadvantage, however, can be exploited to aid in the assignment of the backbone resonances of membrane proteins; by (15)N/(13)C-double-labeling of these six amino acid types, sequential connectivities can be obtained for large stretches of the transmembrane segments where number and length of stretches consisting exclusively of these six amino acid types are enhanced compared with the remainder of the protein. We show by experiment as well as by statistical analysis that this labeling scheme provides a large number of sequential connectivities in transmembrane regions and thus constitutes a tool for the efficient assignment of membrane protein backbone resonances.

Preparative scale expression of membrane proteins in Escherichia coli-based continuous exchange cell-free systems.

Cell-free expression is emerging as a prime method for the rapid production of preparative quantities of high-quality membrane protein samples. The technology facilitates easy access to large numbers of proteins that have been extremely difficult to obtain. Most frequently used are cell-free systems based on extracts of Escherichia coli cells, and the reaction procedures are reliable and efficient. This protocol describes the preparation of all essential reaction components such as the E. coli cell extract, T7 RNA polymerase, DNA templates as well as the individual stock solutions. The setups of expression reactions in analytical and preparative scales, including a variety of reaction designs, are illustrated. We provide detailed reaction schemes that allow the preparation of milligram amounts of functionally folded membrane proteins of prokaryotic and eukaryotic origin in less than 24 h.

Functional analysis of cell-free-produced human endothelin B receptor reveals transmembrane segment 1 as an essential area for ET-1 binding and homodimer formation.

The functional and structural characterization of G-protein-coupled receptors (GPCRs) still suffers from tremendous difficulties during sample preparation. Cell-free expression has recently emerged as a promising alternative approach for the synthesis of polytopic integral membrane proteins and, in particular, for the production of G-protein-coupled receptors. We have now analyzed the quality and functional folding of cell-free produced human endothelin type B receptor samples as an example of the rhodopsin-type family of G-protein-coupled receptors in correlation with different cell-free expression modes. Human endothelin B receptor was cell-free produced as a precipitate and subsequently solubilized in detergent, or was directly synthesized in micelles of various supplied mild detergents. Purified cell-free-produced human endothelin B receptor samples were evaluated by single-particle analysis and by ligand-binding assays. The soluble human endothelin B receptor produced is predominantly present as dimeric complexes without detectable aggregation, and the quality of the sample is very similar to that of the related rhodopsin isolated from natural sources. The binding of human endothelin B receptor to its natural peptide ligand endothelin-1 is demonstrated by coelution, pull-down assays, and surface plasmon resonance assays. Systematic functional analysis of truncated human endothelin B receptor derivatives confined two key receptor functions to the membrane-localized part of human endothelin B receptor. A 39 amino acid fragment spanning residues 93-131 and including the proposed transmembrane segment 1 was identified as a central area involved in endothelin-1 binding as well as in human endothelin B receptor homo-oligomer formation. Our approach represents an efficient expression technique for G-protein-coupled receptors such as human endothelin B receptor, and might provide a valuable tool for fast structural and functional characterizations.