Modulation of immune responses to liposomal vaccines by intrastructural help.

The encapsulation of HIV-unrelated T helper peptides into liposomal vaccines presenting trimers of the HIV-1 envelope glycoprotein (Env) on the surface (T helper liposomes) may recruit heterologous T cells to provide help for Env-specific B cells. This mechanism called intrastructural help can modulate the HIV-specific humoral immune response. In this study, we used cationic T helper liposomes to induce intrastructural help effects in a small animal model.

Impact of ergosterol content on acetic and lactic acids toxicity to Saccharomyces cerevisiae.

Organic acid stress often represents a major hurdle in industrial bio-based microbial processes. Organic acids can be released from lignocellulosic feedstocks pretreatment and can also be desirable products obtained by microbial fermentation with applications in different industrial sectors. Yeasts are prominent cell factories.

Design and Functional Characterization of HIV-1 Envelope Protein-Coupled T Helper Liposomes.

Functionalization of experimental HIV-1 virus-like particle vaccines with heterologous T helper epitopes (T helper VLPs) can modulate the humoral immune response via intrastructural help (ISH). Current advances in the conjugation of native-like HIV-1 envelope trimers (Env) onto liposomes and encapsulation of peptide epitopes into these nanoparticles renders this GMP-scalable liposomal platform a feasible alternative to VLP-based vaccines. In this study, we designed and analyzed customizable Env-conjugated T helper liposomes.

Conjugation of Native-Like HIV-1 Envelope Trimers onto Liposomes Using EDC/Sulfo-NHS Chemistry: Requirements and Limitations.

The display of native-like human immunodeficiency virus type 1 envelope (HIV-1 Env) trimers on liposomes has gained wide attention over the last few years. Currently, available methods have enabled the preparation of Env-liposome conjugates of unprecedented quality. However, these protocols require the Env trimer to be tagged and/or to carry a specific functional group.

Electrostatically Driven Encapsulation of Hydrophilic, Non-Conformational Peptide Epitopes into Liposomes.

Since the first use of liposomes as carriers for antigens, much work has been done to elucidate the mechanisms involved in the encapsulation of vaccine-relevant biomolecules. However, only a few studies have specifically investigated the encapsulation of hydrophilic, non-conformational peptide epitopes. We performed comprehensive and systematic screening studies, in order to identify conditions that favor the electrostatic interaction of such peptides with lipid membranes.

Lectin bio-layer interferometry for assessing product quality of Fc- glycosylated immunoglobulin G.

Glycosylation, as the most prominent posttranslational modification, is recognized as an important quality attribute of monoclonal antibodies affected by various bioprocess parameters and cellular physiology. A method of lectin-based bio-layer interferometry (LBLI) to relatively rank galactosylation and fucosylation levels was developed. For this purpose, Fc-glycosylated immunoglobulin G (IgG) was recombinantly produced with varying bioprocess conditions in 15 L bioreactor and accumulated IgG was harvested.

Impact of mammalian cell culture conditions on monoclonal antibody charge heterogeneity: an accessory monitoring tool for process development.

Recombinant monoclonal antibodies are predominantly produced in mammalian cell culture bioprocesses. Post-translational modifications affect the micro-heterogeneity of the product and thereby influence important quality attributes, such as stability, solubility, pharmacodynamics and pharmacokinetics. The analysis of the surface charge distribution of monoclonal antibodies provides aggregated information about these modifications.

Application of the Bradford Assay for Cell Lysis Quantification: Residual Protein Content in Cell Culture Supernatants.

Frequently measured mammalian cell culture process indicators include viability and total cell concentration (TCC). Cell lysis, an additional important process characteristic that substantially contributes to the overall product purity profiles, is often not addressed in detail. In the present study, an inexpensive and simple application of the Bradford assay is developed to determine the residual protein content (RPC) in cell culture supernatants.

Quantification of Recombinant Products in Yeast.

Quantification of various proteins expressed in yeast can be performed by different methods. In this respect, classical as well as advanced techniques can be applied, where the analysis of crude supernatants is of special interest in screening but also manufacturing.The following chapter addresses the analytical background of the introduced methods followed by specific recommendations for the quantification of different products of industrial interest.

Assessing the Quality of Recombinant Products Made in Yeast.

The product quality of recombinant proteins is of major importance for their intended purpose. The initial characterization of both simple and complex products should be performed as soon as practical. However, to comply with this high standard, appropriate selection of complementary methods is required.

Expression of full-length HER2 protein in Sf9 insect cells and its presentation on the surface of budded virus-like particles.

Biomarkers of cancer are often glycosylated membrane receptor proteins present on the cellular surface. In order to develop new antibodies for cancer diagnostics or treatment, it is a main pre-requisite that these target proteins are available in a native conformation. However, membrane receptor proteins are notoriously difficult to produce due to their hydrophobic nature and complex architecture.

An approach for liposome immobilization using sterically stabilized micelles (SSMs) as a precursor for bio-layer interferometry-based interaction studies.

Non-fluidic bio-layer interferometry (BLI) has rapidly become a standard tool for monitoring almost all biomolecular interactions in a label-free, real-time and high-throughput manner. High-efficiency screening methods which measure the kinetics of liposomes with a variety of compounds require the immobilization of liposomes. In this work, a method is described for immobilizing liposomes for interaction studies, based on the biophysical principles of this biosensor platform.

The vitamin-sensitive promoter P enables pre-defined autonomous induction of recombinant protein production in Pichia pastoris.

The methylotrophic yeast Pichia pastoris is widely used for production of recombinant proteins. Here we characterize a vitamin-sensitive regulatory sequence, which can be controlled independently of the main culture medium compounds such as carbon, nitrogen, or phosphor source. The THI11 promoter (P ) sequence derives from a gene involved in biosynthesis of thiamine.

A versatile, quantitative analytical method for pharmaceutical relevant lipids in drug delivery systems.

Over the past few years, liposomal formulations as drug carrier systems have markedly advanced in pharmaceutical research and development. Therefore, analytical methods to characterize liposome-based formulations are required. One particular issue in liposome analysis is the imbalance of lipid ratios within the vesicle formulations and the detectability of degradation products such as lysophospholipids and fatty acids caused by hydrolysis, especially in low molar ranges.

Generation of biologically active multi-sialylated recombinant human EPOFc in plants.

Hyperglycosylated proteins are more stable, show increased serum half-life and less sensitivity to proteolysis compared to non-sialylated forms. This applies particularly to recombinant human erythropoietin (rhEPO). Recent progress in N-glycoengineering of non-mammalian expression hosts resulted in in vivo protein sialylation at great homogeneity.

Expression of functionally active sialylated human erythropoietin in plants.

Recombinant human erythropoietin (rhEPO), a glycohormone, is one of the leading biopharmaceutical products. The production of rhEPO is currently restricted to mammalian cell expression systems because of rhEPO's highly complex glycosylation pattern, which is a major determinant for drug-efficacy. Here we evaluate the ability of plants to produce different glycoforms of rhEPO.

Application of Bio-Layer Interferometry for the analysis of protein/liposome interactions.

The development of biosensor technologies for the investigation of biomolecular interactions has markedly advanced over the last years. One promising biosensor platform, the Bio-Layer Interferometry (BLI), was developed by ForteBio with the main focus to qualify and quantify protein/protein interactions in research and routine applications. Here, a method to characterize protein/liposome binding interactions based on the biophysical principles of this platform is described.

Human pharmacokinetics of intravenous recombinant human Cu/Zn superoxide dismutase.

Objective: Oxidative stress plays an important role in human disease, but antioxidant therapies are limited. Under physiological conditions superoxide is controlled by the enzyme superoxide dismutase. A recombinant human Cu/Zn superoxide dismutase (rhSOD) might open new therapeutic possibilities.

Strategies for efficient transfection of CHO-cells with plasmid DNA.

Stable cell lines of Chinese hamster ovary (CHO) cells are the predominant source of commercial -biopharmaceutical proteins. Because making suitable CHO cell lines is time-consuming and costly, -preliminary experiments with transient expression are usually performed to optimize as many protein -production parameters as possible. Here, we describe protocols for optimizing expression in transient expression experiments and isolating stable CHO cell lines using two types of self-made reagents, namely, lipoplexes and polyplexes.

Rapid liposome quality assessment using a lab-on-a-chip.

Although liposomes have many outstanding features such as biocompatibility, biodegradability, low toxicity and structural diversity, and are successfully applied in many areas of chemistry and biotechnology, a lack of characterization standards and quality control tools are still inhibiting the translation of liposome technology into clinical routine. The greatest obstacle to clinical scale commercialization is the inability to ensure liposome formulation stability because small size variations or altered surface chemistries can significantly influence in vivo distribution and excretion kinetics that could in turn lead to unpredictable therapy outcomes. To enhance the product development process we have developed a microfluidic biochip containing embedded dielectric microsensors capable of providing quantitative results on formulation composition and stability based on the monitoring of the unique electric properties of liposomes.

Liposome technology for industrial purposes.

Liposomes, spherical vesicles consisting of one or more phospholipid bilayers, were first described in the mid 60s by Bangham and coworkers. Since then, liposomes have made their way to the market. Today, numerous lab scale but only a few large-scale techniques are available.

IgM characterization directly performed in crude culture supernatants by a new simple electrophoretic method.

A new electrophoretic technique for the qualitative and quantitative analyses of IgM isoforms and fragments has been developed. IgMs which are more complex than many other recombinantly expressed immunoglobulins are characterized by their high molecular weighted active forms and many additional isoforms and fragments in the molecular range between 25 and 1200kDa. To analyze the multimers, isoforms and fragments simultaneously a high-resolution method, which enables sufficient migration and separation is required.

Confocal microscopy of giant vesicles supports the absence of HIV-1 neutralizing 2F5 antibody reactivity to plasma membrane phospholipids.

The broadly neutralizing anti-HIV-1 2F5 monoclonal antibody recognizes a gp41 epitope proximal to the viral membrane. Potential phospholipid autoreactivity at cell surfaces has raised concerns about the use of this antibody for development of vaccines or immunotherapy. In this study, confocal microscopy of giant unilamellar vesicles (GUVs) was used to assess 2F5 reactivity with phospholipids assembled into bilayers with surface charge and curvature stress approximating those of the eukaryotic plasma membranes.