Integration of continuous precipitation, crystallization and flocculation of recombinant proteins

Increased titer in biopharmaceutical production requires new strategies for economical processing. Precipitation, crystallization and flocculation are unit operation which overcomes productivity limits of chromatography and membrane technology. General engineering principles how to set up a precipitation, crystallization, or flocculation process for purification of recombinant proteins have shown in the past. The biophysical principles of precipitation by salt, organic solvent and non-ionic polymers will be explained and commonality with crystallization and flocculation discussed and it will be shown how they can be integrated into a continuous process for recovery proteins. Thermodynamic (phase diagrams)and engineering models, and kinetics of precipitation, crystallization, and flocculation (orthokinetic and perikinetic phase, induction time) have been developed for several proteins such as antibodies and interferon gamma. Scale up rules will be explained and how a process can be transferred into a continuous operation; in particular, the concept of fractal dimension (Figure 1) and the Camp number discussed. Please click Additional Files below to see the full abstract

An Endophytic Fungus as a Source of New Antifungal Compounds

Medical advances in society such as organ transplants, prolonged chemotherapy, and those that lengthen the lives of AIDS patients and the elderly increase the number of immunocompromised individuals(1). When the immune system is compromised, opportunistic fungi can flourish and become fatal. Current antifungal treatments are limited and often toxic(2,3). In addition, strains of fungi resistant to available antifungals are emerging(4,5). Fungi were selected as the source of potential new antifungal agents because fungal antagonism has been reported in most fungal ecosystems(6). Endophytic fungi, which inhabit the spaces between plant cells, are known producers of natural products and that assist plants in fending off plant fungal pathogens(7). Therefore, endophytic fungi should produce compounds with antifungal activity. The endophytic fungus, KG146A, a basidiomycete found in the wild rosemary tree/shrub Ceratiola ericoides, demonstrated antifungal activity in plug assays against Candida, a human pathogen. KG146A was cultured in liquid Sabouraud\u27s Dextrose broth and extracted with ethyl acetate. The organic extracts (~5mg/ diffusion disk) revealed antifungal activity in disc diffusion assays against Candida albicans 406, Candida albicans wisconsin, Candida albicans MEN, and Saccharomyces cerevisiae. Purification of the active component involving LH-20 gel chromatography followed by reverse-phase HPLC was developed. Final purification will be completed through the use of HPLC and structure elucidation will be achieved through NMR spectroscopy, IR spectroscopy, and mass spectrometry

HPV Vaccination, Sociodemographic Variables, and Physician Recommendation in Select U.S. Areas

Human papillomavirus (HPV) is the most common sexually transmitted virus, and is causally related to several cancers. HPV vaccination rates are far below HealthyPeople 2020 targets and vary across geographic, socioeconomic, and demographic populations. The purpose of this research was to test the relationships among socioeconomic and demographic variables, HPV vaccination, social vulnerability, and physician recommendation within select local areas in the United States. Fundamental cause theory and behavioral economics informed this quantitative secondary analysis of National Immunization Survey-Teen and Social Vulnerability Index data (n = 43,271). Statistical analyses included chi-square and binomial logistic regression. Teens whose mothers had less than a college degree were more likely to initiate the HPV vaccine series (p \u3c .01), while teens living in Hidalgo County and Houston were less likely to initiate the series (p \u3c .001). Younger teens (p \u3c .001), males (p \u3c .001) and teens whose mothers had some college (p \u3c .01) were less likely to complete the series, while older teens (p \u3c .001) and teens living in Philadelphia and Houston (p \u3c .01) were more likely to complete the series. Fewer teens in Bexar County received a physician recommendation (p \u3c .01); there was no difference between vaccine initiation and select local area. These findings highlight the need to consider local sociodemographic influences on underlying disparities in health and physician behavior. Informed interventions may produce positive social change by reducing variance in health care quality, tailoring public health efforts to local needs, and moving persons experiencing disparities in health outcomes toward a healthy future

Representation of Death in Cristopher Marlowe’s Tamburlaine the Great, Parts One and Two

In 1587, Christopher Marlowe introduced a new approach to theatre in Ta mburlaine the Great and reformed the reigning theatrical practices. The popularity of the plays’ main motif, Death, can be traced back to ancient Greek and Roman texts, but Marlowe’s portrayal of the Death-topos differs from the traditional depictions on many levels. My main aim in this essay is to point out the Marlovian innovations concerning the theatrical representation of death by relying on contemporary popular culture. I also intend to emphasise the complementary relationship of rhetoric and imagery in order to prove the shared importance of verbality and imagery

Continuous desalting of refolding solution by ion exchange chromatography

Recombinant proteins expressed in E. coli as insoluble inclusion bodies need to be resolubilized and refolded to obtain its native structure. These steps require certain salts, which lead to buffers with elevated conductivity. When loading such a refolding solution on an ion exchange column for capturing only relatively low binding capacities can be achieved. In order to overcome this problem, an additional process step has to be introduced. The traditional approach is dilution, diafiltration or dialysis. Here we present a novel alternative process for salt removal of protein solutions. We applied anion and cation exchangers of a micro-pore type, where only salts can penetrate into the pores, but no proteins, in order to desalt the solution. The columns were connected together to run in a serial setup. In order to increase operation performance, a continuous process was developed comprising of four columns, two anion and two cation exchangers. Continuous mode was achieved by staggered cycling operation, where one set of columns was loaded while the other set was regenerated. Proof of concept using a scFv as model protein was performed. The refolding solution could be successfully deionized resulting in constantly low conductivity below 0.5 mS/cm. By running the process continuously process time could be reduced by 38.5% and at the same time productivity was increased to 163% compared to batch operation. Desalting of the protein solution resulted in 5-7 fold higher binding capacities in subsequent ion exchange capture step by conventional protein binding resins

Modeling the residence time distribution of an end to end integrated biomanufacturing process

With the advancements in continuous manufacturing focused mainly on the development of individual unit operations, only a few end-to-end integrated continuous bioprocesses (ICB) have been reported. As the scope starts shifting also towards commercial applications, detailed process understanding is required for quality process design, process optimization and developing process control strategies. Please click Additional Files below to see the full abstract

Sleep Disturbances in Mental Health Care: A Review of Literature on Nursing Interventions

Sleep is essential in the repair and renewal of cells and tissues. It allows the body to recover from the wear and tear of life’s daily activities, slows metabolism, decreases heart workload, relaxes body muscles, and preserves energy. Sleep deprivation caused by psychological disorder can cause anxiety, euphoria, irritability, and memory impairment. Insomnia is associated with depression, anxiety disorders, deficient work performance, drug abuse, and reduction in productivity. The purpose of this study is to determine what nursing interventions are available to improve the sleep quality of mental health care patients

Representation of Death in Cristopher Marlowe’s Tamburlaine the Great, Parts One and Two

In 1587, Christopher Marlowe introduced a new approach to theatre in Ta mburlaine the Great and reformed the reigning theatrical practices. The popularity of the plays’ main motif, Death, can be traced back to ancient Greek and Roman texts, but Marlowe’s portrayal of the Death-topos differs from the traditional depictions on many levels. My main aim in this essay is to point out the Marlovian innovations concerning the theatrical representation of death by relying on contemporary popular culture. I also intend to emphasise the complementary relationship of rhetoric and imagery in order to prove the shared importance of verbality and imagery

Integration of continuous ethanol precipitation and flocculation into manufacturing of antibodies

Precipitation and flocculation are optimal unit operations for continuous capture of proteins from culture supernatant. Precipitation and flocculation can be operated in a real continuous manner. The methods can be combined and after this capture step the precipitate can be stored as a concentrated solution or even as a precipitate. We have developed several precipitation/ flocculation protocols for capture of antibodies from culture supernatant. These include the combinations of CaCl2 flocculation with either cold ethanol precipitation or PEG precipitation and octanoic acid precipitation with PEG precipitation. The precipitation conditions have been screened in microtiter plates or in case of cold ethanol precipitation in small scale reactors. For cold ethanol precipitation a combination with CaCl2 flocculation is best suited. In the first step the high molecular mass impurities can be removed by flocculation with CaCl2 while in the second step the antibody is precipitated by addition of ethanol and low molecular mass impurities are removed. The whole procedure can be repeated and then final polishing can be performed by an anion exchange step in flow through mode. Octanoic acid precipitation is also a very efficient step but an additional phase can be formed which is difficult to remove. In Figure 1 a bench top reactor for cold ethanol precipitation is depicted. The rector consists of two sections (Figure 1). First a concurrent cooling is installed to ensure a constant cooling rate while ethanol is added to the culture supernatant or pre-treated supernatant. In the second section a countercurrent cooling is installed to keep the reactors at the requested temperature. The industry standard for antibody capture is protein A affinity chromatography. Thus the properties of the antibody after cold ethanol precipitation have been compared to protein A purified material. No significant difference could be observed (Figure 2) in either composition or structure. Further purification has been tested by anion-exchange monoliths in order to remove further host cell proteins. The different protocols will be compared to other standard platforms for antibody purification. The possibilities to integrate in-process control and maintenance of steady state will be discussed. The economics of such a process will be discussed for the different scenarios of clinical phase manufacturing and how strategies can be developed when antibodies become commodities or when oral delivery becomes reality. Next steps will be shown how to scale up such a process