Molecular basis of high viscosity in concentrated antibody solutions: Strategies for high concentration drug product development

Effective translation of breakthrough discoveries into innovative products in the clinic requires proactive mitigation or elimination of several drug development challenges. These challenges can vary depending upon the type of drug molecule. In the case of therapeutic antibody candidates, a commonly encountered challenge is high viscosity of the concentrated antibody solutions. Concentration-dependent viscosity behaviors of mAbs and other biologic entities may depend on pairwise and higher-order intermolecular interactions, non-native aggregation, and concentration-dependent fluctuations of various antibody regions. This article reviews our current understanding of molecular origins of viscosity behaviors of antibody solutions. We discuss general strategies and guidelines to select low viscosity candidates or optimize lead candidates for lower viscosity at early drug discovery stages. Moreover, strategies for formulation optimization and excipient design are also presented for candidates already in advanced product development stages. Potential future directions for research in this field are also explored.     

Strategies for selecting Recombinant CHO cell lines for cGMP manufacturing: Realizing the potential in bioreactors

Manufacture of recombinant proteins from mammalian cell lines requires the use of bioreactor systems at scales of up to 20,000 L. The cost and complexity of such systems can prohibit their extensive use during the process to construct and select the manufacturing cell line. It is therefore common practice to develop a model of the production process in a small scale vessel, such as a shake‐flask, where lower costs, ease of handling, and higher throughput are possible. This model can then be used to select a small number of cell lines for further evaluation in bioreactor culture. Here, we extend our previous work investigating cell line construction strategies to assess how well the behavior of cell lines in such a shake‐flask assessment predicts behavior in the associated bioreactor production process. A panel of 29 GS‐CHO cell lines, all producing the same antibody, were selected to include a mixture of high and low producers from a pool of 175 transfectants. Assessment of this panel in 10 L bioreactor culture revealed wide variation in parameters including growth, productivity, and metabolite utilization. In general, those cell lines which were high producing in the bioreactor cultures had also been higher producing in an earlier shake‐flask assessment. However, some changes in rank position of the evaluated cell lines were seen between the two systems. A potential explanation of these observations is discussed and approaches to improve the predictability of assessments used for cell line selection are considered. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Strategies for selecting recombinant CHO cell lines for cGMP manufacturing: Improving the efficiency of cell line generation

Transfectants with a wide range of cellular phenotypes are obtained during the process of cell line generation. For the successful manufacture of a therapeutic protein, a means is required to identify a cell line with desirable growth and productivity characteristics from this phenotypically wide‐ranging transfectant population. This identification process is on the critical path for first‐in‐human studies. We have stringently examined a typical selection strategy used to isolate cell lines suitable for cGMP manufacturing. One‐hundred and seventy‐five transfectants were evaluated as they progressed through the different assessment stages of the selection strategy. High producing cell lines, suitable for cGMP manufacturing, were identified. However, our analyses showed that the frequency of isolation of the highest producing cell lines was low and that ranking positions were not consistent between each assessment stage, suggesting that there is potential to improve upon the strategy. Attempts to increase the frequency of isolation of the 10 highest producing cell lines, by in silico analysis of alternative selection strategies, were unsuccessful. We identified alternative strategies with similar predictive capabilities to the typical selection strategy. One alternate strategy required fewer cell lines to be progressed at the assessment stages but the stochastic nature of the models means that cell line numbers are likely to change between programs. In summary, our studies illuminate the potential for improvement to this and future selection strategies, based around use of assessments that are more informative or that reduce variance, paving the way to improved efficiency of generation of manufacturing cell lines. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Stopping azithromycin mass drug administration for trachoma: A systematic review

The World Health Organization (WHO) recommends continuing azithromycin mass drug administration (MDA) for trachoma until endemic regions drop below 5% prevalence of active trachoma in children aged 1–9 years. Azithromycin targets the ocular strains of Chlamydia trachomatis that cause trachoma. Regions with low prevalence of active trachoma may have little if any ocular chlamydia, and, thus, may not benefit from azithromycin treatment. Understanding what happens to active trachoma and ocular chlamydia prevalence after stopping azithromycin MDA may improve future treatment decisions. We systematically reviewed published evidence for community prevalence of both active trachoma and ocular chlamydia after cessation of azithromycin distribution. We searched electronic databases for all peer-reviewed studies published before May 2020 that included at least 2 post-MDA surveillance surveys of ocular chlamydia and/or the active trachoma marker, trachomatous inflammation–follicular (TF) prevalence. We assessed trends in the prevalence of both indicators over time after stopping azithromycin MDA. Of 140 identified studies, 21 met inclusion criteria and were used for qualitative synthesis. Post-MDA, we found a gradual increase in ocular chlamydia infection prevalence over time, while TF prevalence generally gradually declined. Ocular chlamydia infection may be a better measurement tool compared to TF for detecting trachoma recrudescence in communities after stopping azithromycin MDA. These findings may guide future trachoma treatment and surveillance efforts.     

Brown drug substance color investigation in cell culture manufacturing using chemically defined media: A case study

Drug substance (DS) color is an important quality attribute for release, stability and comparability studies of biologics. With the increase of DS concentrations and biologics pipelines made in chemically defined media, atypical DS color other than colorless or pale yellow has been recently reported in the biopharmaceutical industry. We recently observed a brown DS color in manufacturing. Although analytical characterization data indicated that the brown color DS had no major quality issue, it is necessary to find the root cause and reduce DS color to ease placebo design for clinical use. It was demonstrated that the brown color was caused by the chemically defined basal medium containing high levels of iron and vitamin B12 (VB12) regardless of cell lines. Iron caused tryptophan oxidation in the protein to form N-formylkynurenine and kynurenine products, which likely contributed to a yellow DS color. A pink DS color was caused by the residual VB12 bound to DS. The brown color was the result of the combinatory effect of yellow and pink colors. Finally a modified basal medium was developed to produce a pale yellow DS in manufacturing.     

Hydrogen/deuterium exchange-mass spectrometry analysis of high concentration biotherapeutics: application to phase-separated antibody formulations

High concentration biotherapeutic formulations are often required to deliver large doses of drugs to achieve a desired degree of efficacy and less frequent dose. However, highly concentrated protein-containing solutions may exhibit undesirable therapeutic properties, such as increased viscosity, aggregation, and phase separation that can affect drug efficacy and raise safety issues. The characterization of high concentration protein formulations is a critical yet challenging analytical task for therapeutic development efforts, due to the lack of technologies capable of making accurate measurements under such conditions. To address this issue, we developed a novel dilution-free hydrogen/deuterium exchange (HDX) mass spectrometry (MS) method for the direct conformational analysis of high concentration biotherapeutics. Here, we particularly focused on studying phase separation phenomenon that can occur at high protein concentrations. First, two aliquots of monoclonal antibodies (mAbs) were dialyzed in either hydrogen- or deuterium-containing buffers at low salt and pH. Phases that separated were then discretely sampled and subjected to dilution-free HDX-MS analysis through mixing the non-deuterated and deuterated protein aliquots. Our HDX-MS results analyzed at a global protein level reveal less deuterium incorporation for the protein-enriched phase compared to the protein-depleted phase present in high concentration formulations. A peptide level analysis further confirmed these observed differences, and a detailed statistical analysis provided direct information surrounding the details of the conformational changes observed. Based on our HDX-MS results, we propose possible structures for the self-associated mAbs present at high concentrations. Our new method can potentially provide useful insights into the unusual behavior of therapeutic proteins in high concentration formulations, aiding their development.     

A rapid capillary gel electrophoresis method for the quantitative determination of RuBisCo in spinach

A capillary gel electrophoretic (CGE) method for the quantitative analysis of RuBisCo in spinach leaves was developed. RuBisCo was resolved into large and small subunits in the presence of sodium dodecyl sulphate (SDS) by the CGE procedure which enabled the determination of the molecular weight of each unit accurately; the values so determined were in close agreement with those reported using other methods. Advantages of CGE over SDS-polyacrylamide gel electrophoresis and high-pressure gel filtration include decreased sample preparation and analysis time, superior resolution and greater sensitivity permitting reduced sample size and trace analysis. In addition, CGE provided precise quantification of RuBisCo and was demonstrated to be a viable alternative to other available methods of protein analysis.     

Strategies for enhancing fermentative production of acetoin: A review

Acetoin is a volatile compound widely used in foods, cigarettes, cosmetics, detergents, chemical synthesis, plant growth promoters and biological pest controls. It works largely as flavour and fragrance. Since some bacteria were found to be capable of vigorous acetoin biosynthesis from versatile renewable biomass, acetoin, like its reduced form 2,3-butanediol, was also classified as a promising bio-based platform chemical. In spite of several reviews on the biological production of 2,3-butanediol, little has concentrated on acetoin. The two analogous compounds are present in the same acetoin (or 2,3-butanediol) pathway, but their production processes including optimal strains, substrates, derivatives, process controls and product recovery methods are quite different. In this review, the usages of acetoin are reviewed firstly to demonstrate its importance. The biosynthesis pathway and molecular regulation mechanisms are then outlined to depict the principal network of functioning in typical species. A phylogenetic tree is constructed and the relationship between taxonomy and acetoin producing ability is revealed for the first time, which will serve as a useful guide for the screening of competitive acetoin producers. Genetic engineering, medium optimization, and process control are effective strategies to improve productivity as well. Currently, downstream processing is one of the main barriers in efficient and economical industrial acetoin fermentation. The future prospects of microbial acetoin production are discussed in light of the current progress, challenges, and trends in this field.     

Natural product derived promising anti-MRSA drug leads: A review

Multi-drug resistant Staphylococcus aureus infections have created a critical need for the development of new classes of antibacterials. Discovery of new naturally derived antibacterial agents with new mechanism of action remains a high priority globally. Several of the available antibacterial agents like β-lactams, polyketides, phenylpropanoids, aminoglycosides, macrolides, glycopeptides, streptogramins and lipopeptides are natural products or their semisynthetic variations. In the current scenario of alarming rise in antibacterial resistance, revisiting natural products with modern chemistry and biology tools has fascinated many medicinal chemists for discovery and development of natural products or derived semisynthetic derivatives as effective antibacterial agents. This review underlines the structures and anti-MRSA activity of various natural product derivatives covering recent reports, in vivo activities and brief Structure Activity Relationships (SARs).     

Key factors influencing ADME properties of therapeutic proteins: A need for ADME characterization in drug discovery and development

Protein therapeutics represent a diverse array of biologics including antibodies, fusion proteins, and therapeutic replacement enzymes. Since their inception, they have revolutionized the treatment of a wide range of diseases including respiratory, vascular, autoimmune, inflammatory, infectious, and neurodegenerative diseases, as well as cancer. While in vivo pharmacokinetic, pharmacodynamic, and efficacy studies are routinely carried out for protein therapeutics, studies that identify key factors governing their absorption, distribution, metabolism, and excretion (ADME) properties have not been fully investigated. Thorough characterization and in-depth study of their ADME properties are critical in order to support drug discovery and development processes for the production of safer and more effective biotherapeutics. In this review, we discuss the main factors affecting the ADME characteristics of these large macromolecular therapies. We also give an overview of the current tools, technologies, and approaches available to investigate key factors that influence the ADME of recombinant biotherapeutic drugs, and demonstrate how ADME studies will facilitate their future development.     

A Novel Solution for the High Defibrillation Threshold in Patients with a DF-4 Lead: Adding a High Voltage Adaptor/Splitter

A high defibrillation threshold occurs in approximately 6% of implants. The defibrillation threshold can be improved by addition of a defibrillation lead. However, the DF-4 high energy ICD header precludes the addition of a defibrillation lead. Here we report on use of a new high voltage adaptor/splitter that enables the addition of an extra defibrillation lead.     

Protein and solute distribution in drug substance containers during frozen storage and post-thawing: a tool to understand and define freezing-thawing parameters in biotechnology process development

Active pharmaceutical ingredient for biotechnology-based drugs, commonly known as drug substance (DS), is often stored frozen for longer shelf-life. Freezing DS enhances stability by slowing down reaction rates that lead to protein instability, minimizes the risk of microbial growth, and eliminates the risk of transport-related stress. High density polyethylene bottles are commonly used for storing monoclonal antibody DS due to good mechanical stress/strain resistant properties even at low temperatures. Despite the aforementioned advantages for frozen storage of DS, this is not devoid of risks. Proteins are known to undergo ice-water surface denaturation, cryoconcentration, and cold denaturation during freezing. A systematic investigation was performed to better understand the protein and solute distribution along with potential of aggregate formation during freeze and thaw process. A significant solute and protein concentration gradient was observed for both frozen and thawed DS bottles. In case of thawed DS, cryoconcentration was localized in the bottom layer and a linear increase in concentration as a function of liquid depth was observed. On the other hand, for frozen DS, a "bell shaped" cryoconcentration distribution was observed between the bottom layers and centre position. A cryoconcentration of almost three-fold was observed for frozen DS in the most concentrated part when freezing was conducted at -20 and -40 °C and 2.5-fold cryoconcentration was observed in the thawed DS before mixing. The information obtained in this study is critical to design freeze thaw experiments, storage condition determination, and process improvement in manufacturing environment.     

Lecithin organogels as a potential phospholipid-structured system for topical drug delivery: A review

The purpose of this review is to give an insight into the considerable potential of lecithin organogels (LOs) in the applications meant for topical drug delivery. LOs are clear, thermodynamically stable, viscoelastic, and biocompatible jelly-like phases, chiefly composed of hydrated phospholipids and appropriate organic liquid. These systems are currently of interest to the pharmaceutical scientist because of their structural and functional benefits. Several therapeutic agents have been formulated as LOs for their facilitated transport through topical route (for dermal or transdermal effect), with some very encouraging results. The improved topical drug delivery has mainly been attributed to the biphasic drug solubility, the desired drug partitioning, and the modification of skin barrier function by the organogel components. Being thermodynamically stable, LOs are prepared by spontaneous emulsification and therefore posses prolonged shelf life. The utility of this novel matrix as a topical vehicle has further increased owing to its very low skin irritancy potential. Varied aspects of LOs viz formation, composition, phase behavior, and characterization have been elaborated, including a general discussion on the developmental background. Besides a comprehensive update on the topical applications of lecithin organogels, the review also includes a detailed account on the mechanistics of organogelling. Published: October 6, 2005     

Application of a PEG precipitation method for solubility screening: A tool for developing high protein concentration formulations

Previous publications demonstrated that the extrapolated solubility by polyethylene glycol (PEG) precipitation method (Middaugh et al., J Biol Chem 1979; 254:367–370; Juckes, Biochim Biophys Acta 1971; 229:535–546; Foster et al., Biochim Biophys Acta 1973; 317:505; Mahadevan and Hall, AIChE J 1990; 36:1517–1528; Stevenson and Hageman, Pharm Res 1995; 12:1671–1676) has a strong correlation to experimentally measured solubility of proteins. Here, we explored the utility of extrapolated solubility as a method to compare multiple protein drug candidates when nonideality of a highly soluble protein prohibits accurate quantitative solubility prediction. To achieve high efficiency and reduce the amount of protein required, the method is miniaturized to microwell plate format for high‐throughput screening application. In this simplified version of the method, comparative solubility of proteins can be obtained without the need of concentration measurement of the supernatant following the precipitation step in the conventional method. The monoclonal antibodies with the lowest apparent solubilities determined by this method are the most difficult to be concentrated, indicating a good correlation between the prediction and empirical observations. This study also shows that the PEG precipitation method gives results for opalescence prediction that favorably compares to experimentally determined opalescence levels at high concentration. This approach may be useful in detecting proteins with potential solubility and opalescence problems prior to the time‐consuming and expensive development process of high concentration formulation.     

Community perceptions and acceptability of mass drug administration for the control of neglected tropical diseases in Asia-Pacific countries: A systematic scoping review of qualitative research

BackgroundPreventative chemotherapy and mass drug administration have been identified as effective strategies for the prevention, treatment, control and elimination of several NTDs in the Asia-Pacific region. Qualitative research can provide in-depth insight into the social dynamics and processes underlying effective implementation of and adherence to mass drug administration programs. This scoping review examines published qualitative literature to examine factors influencing community perceptions and acceptability of mass drug administration approaches to control NTDs in the Asia-Pacific region.MethodologyTwenty-four peer reviewed published papers reporting qualitative data from community members and stakeholders engaged in the implementation of mass drug administration programs were identified as eligible for inclusion.FindingsThis systematic scoping review presents available data from studies focussing on lymphatic filariasis, soil-transmitted helminths and scabies in eight national settings (India, Indonesia, Philippines, Bangladesh, Laos, American Samoa, Papua New Guinea, Fiji). The review highlights the profoundly social nature of individual, interpersonal and institutional influences on community perceptions of willingness to participate in mass drug administration programs for control of neglected tropical diseases (NTD). Future NTD research and control efforts would benefit from a stronger qualitative social science lens to mass drug administration implementation, a commitment to understanding and addressing the social and structural determinants of NTDs and NTD control in complex settings, and efforts to engage local communities as equal partners and experts in the co-design of mass drug administration and other efforts to prevent, treat, control and eliminate NTDs.ConclusionFor many countries in the Asia-Pacific region, the “low hanging fruit has been picked” in terms of where mass drug administration has worked and transmission has been stopped. The settings that remain–such as remote areas of Fiji and Papua New Guinea, or large, highly populated, multi-cultural urban settings in India and Indonesia–present huge challenges going forward.     

Metabolite induction via microorganism co-culture: A potential way to enhance chemical diversity for drug discovery

Microorganisms have a long track record as important sources of novel bioactive natural products, particularly in the field of drug discovery. While microbes have been shown to biosynthesize a wide array of molecules, recent advances in genome sequencing have revealed that such organisms have the potential to yield even more structurally diverse secondary metabolites. Thus, many microbial gene clusters may be silent under standard laboratory growth conditions. In the last ten years, several methods have been developed to aid in the activation of these cryptic biosynthetic pathways. In addition to the techniques that demand prior knowledge of the genome sequences of the studied microorganisms, several genome sequence-independent tools have been developed. One of these approaches is microorganism co-culture, involving the cultivation of two or more microorganisms in the same confined environment. Microorganism co-culture is inspired by the natural microbe communities that are omnipresent in nature. Within these communities, microbes interact through signaling or defense molecules. Such compounds, produced dynamically, are of potential interest as new leads for drug discovery. Microorganism co-culture can be achieved in either solid or liquid media and has recently been used increasingly extensively to study natural interactions and discover new bioactive metabolites. Because of the complexity of microbial extracts, advanced analytical methods (e.g., mass spectrometry methods and metabolomics) are key for the successful detection and identification of co-culture-induced metabolites.     

Different ways to die in a changing world: Consequences of climate change for tree species performance and survival through an ecophysiological perspective

Anthropogenic activities such as uncontrolled deforestation and increasing greenhouse gas emissions are responsible for triggering a series of environmental imbalances that affect the Earth's complex climate dynamics. As a consequence of these changes, several climate models forecast an intensification of extreme weather events over the upcoming decades, including heat waves and increasingly severe drought and flood episodes. The occurrence of such extreme weather will prompt profound changes in several plant communities, resulting in massive forest dieback events that can trigger a massive loss of biodiversity in several biomes worldwide. Despite the gravity of the situation, our knowledge regarding how extreme weather events can undermine the performance, survival, and distribution of forest species remains very fragmented. Therefore, the present review aimed to provide a broad and integrated perspective of the main biochemical, physiological, and morpho‐anatomical disorders that may compromise the performance and survival of forest species exposed to climate change factors, particularly drought, flooding, and global warming. In addition, we also discuss the controversial effects of high CO₂ concentrations in enhancing plant growth and reducing the deleterious effects of some extreme climatic events. We conclude with a discussion about the possible effects that the factors associated with the climate change might have on species distribution and forest composition.