Purification of recombinantly produced somatostatin-28 comparing hydrochloric acid and polyethyleneimine as E. coli extraction aids.

Peptides are used for diagnostics, therapeutics, and as antimicrobial agents. Most peptides are produced by chemical synthesis, but recombinant production has recently become an attractive alternative due to the advantages of high titers, less toxic waste and correct folding of tertiary structure. Somatostatin-28 is a peptide hormone that regulates the endocrine system, cell proliferation and inhibits the release of numerous secondary hormones in human body.

A production platform for disulfide-bonded peptides in the periplasm of Escherichia coli.

Background: Recombinant peptide production in Escherichia coli provides a sustainable alternative to environmentally harmful and size-limited chemical synthesis. However, in-vivo production of disulfide-bonded peptides at high yields remains challenging, due to degradation by host proteases/peptidases and the necessity of translocation into the periplasmic space for disulfide bond formation.

Results: In this study, we established an expression system for efficient and soluble production of disulfide-bonded peptides in the periplasm of E.

Understanding the mechanism of polyethyleneimine-mediated cell disintegration and protein extraction in E. coli: The role of floc network formation and PEI molecular weight.

Cell disintegration and protein extraction are crucial steps in downstream process development for biopharmaceuticals produced in E. coli. In this study, we explored the extraction mechanism of polyethyleneimine (PEI) at the cellular level and characterized the floc network that is formed upon PEI addition by Focused Beam Reflectance Measurement and Dispersion Analyzer.

Rapid purification of mAb using protein a membranes yielding high HCP clearance.

Protein A chromatography remains the crucial step in mAb purification because of the high binding specificity and impurity clearance. In recent years, highly productive membrane adsorbers emerged as an alternative to traditional resins allowing for rapid purification of biomolecules. In this study, we tested three commercially available protein A membranes (Sartobind® Rapid A, HiTrap Fibro™ PrismA and GORE™ Protein Capture Device) regarding flow distribution, permeability and binding performance.

Antibody fragments functionalized with non-canonical amino acids preserving structure and functionality - A door opener for new biological and therapeutic applications.

Functionalization of proteins by incorporating reactive non-canonical amino acids (ncAAs) has been widely applied for numerous biological and therapeutic applications. The requirement not to lose the intrinsic properties of these proteins is often underestimated and not considered. Main purpose of this study was to answer the question whether functionalization via residue-specific incorporation of the ncAA N-[(2-Azidoethoxy) carbonyl]-l-lysine (Azk) influences the properties of the anti-tumor-necrosis-factor-α-Fab (FTN2).

Resin structure impacts two-component protein adsorption and separation in anion exchange chromatography.

The influence of the resin structure, on the competitive binding and separation of a two-component protein mixture with anion exchange resins is evaluated using conalbumin and green fluorescent protein as a model system. Two macroporous resins, one with large open pores and one with smaller pores, are compared to a resin with grafted polymers. Investigations include measurements of single and two-component isotherms, batch uptake kinetics and two-component column breakthrough.

Polyethyleneimine efficiently extracts recombinant cytoplasmatic green fluorescent protein produced in Escherichia coli with high purity.

We used a polycationic polymer polyethyleneimine (PEI) to develop a method to extract recombinant proteins produced in the Escherichia coli (E. coli) cytosol. Compared to high pressure homogenization, commonly used to disrupt E.

Neutralising Effects of Different Antibodies on Toxins TcdA and TcdB in a Translational Approach.

Given the high prevalence of intestinal disease in humans and animals, there is a strong need for clinically relevant models recapitulating gastrointestinal systems, ideally replacing in vivo models in accordance with the principles of the 3R. We established a canine organoid system and analysed the neutralising effects of recombinant versus natural antibodies on toxins A and B in this in vitro system. Sulforhodamine B cytotoxicity assays in 2D and FITC-dextran barrier integrity assays on basal-out and apical-out organoids revealed that recombinant, but not natural antibodies, effectively neutralised toxins.

Desorption of plasmid DNA from anion exchangers: Salt concentration at elution is independent of plasmid size and load.

Detailed studies on the sorption behavior of plasmids on anion exchangers are rare compared to proteins. In this study, we systematically compare the elution behavior of plasmid DNA on three common anion exchange resins using linear gradient and isocratic elution experiments. Two plasmids of different lengths, 8 and 20 kbp, were studied and their elution characteristics were compared to a green fluorescent protein.

Computational fluid dynamics simulation improves the design and characterization of a plug-flow-type scale-down reactor for microbial cultivation processes.

The scale-up of bioprocesses remains one of the major obstacles in the biotechnology industry. Scale-down bioreactors have been identified as valuable tools to investigate the heterogeneities observed in large-scale tanks at the laboratory scale. Additionally, computational fluid dynamics (CFD) simulations can be used to gain information about fluid flow in tanks used for production.

Strain specific properties of Escherichia coli can prevent non-canonical amino acid misincorporation caused by scale-related process heterogeneities.

Background: Escherichia coli is one of the most important hosts for production of recombinant proteins in biopharmaceutical industry. However, when selecting a suitable production strain, it is often not considered that a lot of different sub-species exist, which can differ in their genotypes and phenotypes. Another important development step is the scale-up of bioprocesses with the particular challenge that heterogeneities and gradients occur at production scale.

Mass transfer of proteins in chromatographic media: Comparison of pure and crude feed solutions.

Elucidation of intraparticle mass transfer mechanisms in protein chromatography is essential for process design. This study investigates the differences of adsorption and diffusion parameters of basic human fibroblast factor 2 (hFGF2) in a simple (purified) and a complex (clarified homogenate) feed solution on the grafted agarose-based strong cation exchanger Capto S. Microscopic investigations using confocal laser scanning microscopy revealed slower intraparticle diffusion of hFGF2 in the clarified homogenate compared to purified hFGF2.

Alkaline treatment enhances mass transfer in Protein A affinity chromatography.

Cycle stability is important for preparative chromatography resins. Up to 200 cycles have been reported for Protein A affinity resins when used under optimized operating conditions. Through engineered ligands, alkaline resistant Protein A resins are available that can withstand repeated cleaning-in-place cycles with even 1 M NaOH.

Patterns of protein adsorption in ion-exchange particles and columns: Evolution of protein concentration profiles during load, hold, and wash steps predicted for pore and solid diffusion mechanisms.

Elucidation of protein transport mechanism in ion exchanges is essential to model separation performance. In this work we simulate intraparticle adsorption profiles during batch adsorption assuming typical process conditions for pore, solid and parallel diffusion. Artificial confocal laser scanning microscopy images are created to identify apparent differences between the different transport mechanisms.

A comprehensive antigen production and characterisation study for easy-to-implement, specific and quantitative SARS-CoV-2 serotests.

Background: Antibody tests are essential tools to investigate humoral immunity following SARS-CoV-2 infection or vaccination. While first-generation antibody tests have primarily provided qualitative results, accurate seroprevalence studies and tracking of antibody levels over time require highly specific, sensitive and quantitative test setups.

Methods: We have developed two quantitative, easy-to-implement SARS-CoV-2 antibody tests, based on the spike receptor binding domain and the nucleocapsid protein.

Efficient production of recombinant secretory IgA against Clostridium difficile toxins in CHO-K1 cells.

Despite the essential role secretory IgAs play in the defense against pathogenic invasion and the proposed value of recombinant secretory IgAs as novel therapeutics, currently there are no IgA-based therapies in clinics. Secretory IgAs are complex molecules and the major bottleneck limiting their therapeutic potential is a reliable recombinant production system. In this report, we addressed this issue and established a fast and robust production method for secretory IgAs in CHO-K1 cells using BAC-based expression vectors.

Integrated process development: The key to improve Fab production in E. coli.

Bioprocess development and optimization is a challenging, costly, and time-consuming effort. In this multidisciplinary task, upstream processing (USP) and downstream processing (DSP) are conventionally considered distinct disciplines. This consideration fosters "one-way" optimization disregarding interdependencies between unit operations; thus, the full potential of the process chain cannot be achieved.

Three-dimensional chromatography for purification and characterization of antibody fragments and related impurities from Escherichia coli crude extracts.

Antibody fragments (Fab) are often produced by recombinant methods in Escherichia coli as no glycosylation is needed. Besides the correctly expressed Fab molecule, a multitude of host cell impurities and product related impurities are present in the crude sample. The identification and characterization of the product-related impurities, such as modified Fab-molecules or free light chain, are of utmost importance.

Extraction of recombinant periplasmic proteins under industrially relevant process conditions: Selectivity and yield strongly depend on protein titer and methodology.

In this work, we attempted to identify a method for the selective extraction of periplasmic endogenously expressed proteins, which is applicable at an industrial scale. For this purpose, we used an expression model that allows coexpression of two fluorescent proteins, each of which is specifically targeted to either the cytoplasm or periplasm. We assessed a number of scalable lysis methods (high-pressure homogenization, osmotic shock procedures, extraction with ethylenediaminetetraacetic acid, and extraction with deoxycholate) for the ability to selectively extract periplasmic proteins rather than cytoplasmic proteins.

Influence of experimental constraints on micromechanical assessment of micromachined hard-tissue samples.

Continuing technological advancement of mechanical characterization at the microscale has enabled the isolation of micron-sized specimens and their direct mechanical characterization. Such techniques, initially developed for engineering materials and MEMS, can also be applied on hard biological materials. Bone is a material with a complex hierarchical structure ranging from the macro- all the way down to the nanoscale.

Scale up of a chromatographic capture step for a clarified bacterial homogenate - Influence of mass transport limitation and competitive adsorption of impurities.

A model-based approach for scaling up chromatographic capture step was developed. The purification of human basic fibroblast growth factor protein 2 (FGF2) from an E. coli homogenate on a cation exchange resin was selected as a case study.

Decoupling of recombinant protein production from Escherichia coli cell growth enhances functional expression of plant Leloir glycosyltransferases.

Sugar nucleotide-dependent (Leloir) glycosyltransferases from plants are important catalysts for the glycosylation of small molecules and natural products. Limitations on their applicability for biocatalytic synthesis arise because of low protein expression (≤10 mg/L culture) in standard microbial hosts. Here, we showed two representative glycosyltransferases: sucrose synthase from soybean and UGT71A15 from apple.

Hindered diffusion of proteins in mixture adsorption on porous anion exchangers and impact on flow-through purification of large proteins.

Complex adsorption kinetics behaviors of proteins in mixtures hampers chromatographic process development and complicates model-based prediction of separation. We investigated the adsorption characteristics of mixtures comprised of a larger protein (secretory immunoglobulins or thyroglobulin) and a smaller protein (serum albumin or green fluorescence protein) on the small-pore anion exchanger Q Sepharose FF. Confocal laser scanning microscopy measurements revealed that binding of the large protein was extremely slow and eventually stopped completely after the adsorption front penetrated just a few μm into the particle.

Hydrophobic interaction chromatography of proteins: Studies of unfolding upon adsorption by isothermal titration calorimetry.

Heat of adsorption is an excellent measure for adsorption strength and, therefore, very useful to study the influence of salt and temperature in hydrophobic interaction chromatography. The adsorption of bovine serum albumin and β-lactoglobulin to Toyopearl Butyl-650 M was studied with isothermal titration calorimetry to follow the unfolding of proteins on hydrophobic surfaces. Isothermal titration calorimetry is established as an experimental method to track conformational changes of proteins on stationary phases.

Secretory immunoglobulin purification from whey by chromatographic techniques.

Secretory immunoglobulins (SIg) are a major fraction of the mucosal immune system and represent potential drug candidates. So far, platform technologies for their purification do not exist. SIg from animal whey was used as a model to develop a simple, efficient and potentially generic chromatographic purification process.