Analysis of the Structure of 14 Therapeutic Antibodies Using Circular Dichroism Spectroscopy.

Understanding the impact of the manufacturing environment on therapeutic monoclonal antibody (mAb) structures requires new process analytical technology. Here, we describe the creation of a new reference set for the circular dichroism (CD) spectra of mAbs. Data sets of the highest quality were collected by synchrotron radiation CD for 14 different mAbs in both native and acid-stressed states.

Integrated system for temperature-controlled fast protein liquid chromatography. IV. Continuous 'one-column' 'low-salt' hydrophobic interaction chromatography.

Systematic development of a temperature-controlled isocratic process for one-column low-salt hydrophobic interaction chromatography (HIC) of proteins employing a travelling cooling zone reactor (TCZR) system, is described. Batch binding and confocal scanning microscopy were employed to define process conditions for temperature-reversible binding of bovine serum albumin (BSA) which were validated in pulse-response temperature switching HIC experiments, before transferring to TCZR-HIC. A thin-walled stainless-steel column mounted with a movable assembly of copper blocks and Peltier elements (travelling cooling zone, TCZ) was used for TCZR-HIC.

Probing fibronectin adsorption on chemically defined surfaces by means of single molecule force microscopy.

Atomic force microscope (AFM) based single molecule force spectroscopy (SMFS) and a quartz crystal microbalance (QCM) were respectively employed to probe interfacial characteristics of fibronectin fragment FNIII and full-length fibronectin (FN) on CH-, OH-, COOH-, and NH-terminated alkane-thiol self-assembled monolayers (SAMs). Force-distance curves acquired between hexahistidine-tagged FNIII immobilised on trisNTA-Ni functionalized AFM cantilevers and the OH and COOH SAM surfaces were predominantly 'loop-like' (76% and 94% respectively), suggesting domain unfolding and preference for 'end-on' oriented binding, while those generated with NH and CH SAMs were largely 'mixed type' (81% and 86%, respectively) commensurate with unravelling and desorption, and 'side-on' binding. Time-dependent binding of FN to SAM-coated QCM crystals occurred in at least two phases: initial rapid coverage over the first 5 min; and variably diminishing adsorption thereafter (5-70 min).

Tribocorrosion behaviour of pure titanium in bovine serum albumin solution: A multiscale study.

Tribocorrosion behaviour of pure titanium in phosphate buffer saline (PBS) solution has been investigated systematically as a function of surface chemistry and bovine serum albumin (BSA) content in the solution. A ball-on-disk tribometer coupled with an electrochemical cell was used to study the effect of electrochemical conditions (i.e.

Effect of the electrochemical characteristics of titanium on the adsorption kinetics of albumin.

An electrochemical quartz crystal microbalance (EQCM) was used to examine the electrochemical behaviour of pure titanium in phosphate buffered saline (PBS) and PBS-containing bovine serum albumin (BSA) solutions, and the associated adsorption characteristics of BSA under cathodic and anodic applied potentials. It was found that the electrochemical behaviours of bulk titanium substrate and titanium-coated QCM sensors are slightly different in PBS buffer solution, which is attributed to the difference in their surface roughness. The oxide film formed on the surface of the QCM sensor during potentiostatic tests was found to affect its electrochemical behaviour, while cathodic cleaning is not sufficient to have it removed.

Automated High-Throughput Capillary Circular Dichroism and Intrinsic Fluorescence Spectroscopy for Rapid Determination of Protein Structure.

Assessing the physical stability of proteins is one of the most important challenges in the development, manufacture, and formulation of biotherapeutics. Here, we describe a method for combining and automating circular dichroism and intrinsic protein fluorescence spectroscopy. By robotically injecting samples from a 96-well plate into an optically compliant capillary flow cell, complementary information about the secondary and tertiary structural state of a protein can be collected in an unattended manner from considerably reduced volumes of sample compared to conventional techniques.

Integrated system for temperature-controlled fast protein liquid chromatography. III. Continuous downstream processing of monoclonal antibodies.

Three different applications of travelling heating zone reactor (THZR) chromatography for the downstream processing of monoclonal antibodies (mAbs) are described. mAb containing feedstocks were applied to a fixed bed of the thermoresponsive rProtein A matrix, Byzen Pro™, contained in a bespoke column (held at 15 °C) fitted with a travelling heating (42 °C) device encircling a narrow section of the column. For the demonstration of continuous concentration, uninterrupted loading of 1.

Use of a design of experiments approach to optimise production of a recombinant antibody fragment in the periplasm of Escherichia coli: selection of signal peptide and optimal growth conditions.

Production of recombinant proteins such as antibody fragments in the periplasm of the bacterium Escherichia coli has a number of advantages, including the ability to form disulphide bonds, aiding correct folding, and the relative ease of release and subsequent capture and purification. In this study, we employed two N-terminal signal peptides, PelB and DsbA, to direct a recombinant scFv antibody (single-chain variable fragment), 13R4, to the periplasm via the Sec and SRP pathways respectively. A design of experiments (DoE) approach was used to optimise process conditions (temperature, inducer concentration and induction point) influencing bacterial physiology and the productivity, solubility and location of scFv.

Adsorption of Fibronectin Fragment on Surfaces Using Fully Atomistic Molecular Dynamics Simulations.

The effect of surface chemistry on the adsorption characteristics of a fibronectin fragment (FNIII) was investigated using fully atomistic molecular dynamics simulations. Model surfaces were constructed to replicate self-assembled monolayers terminated with methyl, hydroxyl, amine, and carboxyl moieties. It was found that adsorption of FNIII on charged surfaces is rapid, specific, and driven by electrostatic interactions, and that the anchoring residues are either polar uncharged or of opposing charge to that of the targeted surfaces.

Development of a simple intensified fermentation strategy for growth of Magnetospirillum gryphiswaldense MSR-1: Physiological responses to changing environmental conditions.

The development of a simple pH-stat fed-batch fermentation strategy for the production of Magnetospirillum gryphiswaldense MSR-1 and magnetosomes (nanoscale magnetic organelles with biotechnological applications) is described. Flow cytometry was exploited as a powerful analytical tool for process development, enabling rapid monitoring of cell morphology, physiology and polyhydroxyalkanoate production. The pH-stat fed-batch growth strategy was developed by varying the concentrations of the carbon source (lactic acid) and the alternative electron acceptor (sodium nitrate) in the feed.

Flow cytometry as a rapid analytical tool to determine physiological responses to changing O and iron concentration by Magnetospirillum gryphiswaldense strain MSR-1.

Magnetotactic bacteria (MTB) are a diverse group of bacteria that synthesise magnetosomes, magnetic membrane-bound nanoparticles that have a variety of diagnostic, clinical and biotechnological applications. We present the development of rapid methods using flow cytometry to characterize several aspects of the physiology of the commonly-used MTB Magnetospirillum gryphiswaldense MSR-1. Flow cytometry is an optical technique that rapidly measures characteristics of individual bacteria within a culture, thereby allowing determination of population heterogeneity and also permitting direct analysis of bacteria.

Encapsulated membrane proteins: A simplified system for molecular simulation.

Over the past 50years there has been considerable progress in our understanding of biomolecular interactions at an atomic level. This in turn has allowed molecular simulation methods employing full atomistic modelling at ever larger scales to develop. However, some challenging areas still remain where there is either a lack of atomic resolution structures or where the simulation system is inherently complex.

Integrated system for temperature-controlled fast protein liquid chromatography. II. Optimized adsorbents and 'single column continuous operation'.

Continued advance of a new temperature-controlled chromatography system, comprising a column filled with thermoresponsive stationary phase and a travelling cooling zone reactor (TCZR), is described. Nine copolymer grafted thermoresponsive cation exchangers (thermoCEX) with different balances of thermoresponsive (N-isopropylacrylamide), hydrophobic (N-tert-butylacrylamide) and negatively charged (acrylic acid) units were fashioned from three cross-linked agarose media differing in particle size and pore dimensions. Marked differences in grafted copolymer composition on finished supports were sourced to base matrix hydrophobicity.

Continuous protein purification using functionalized magnetic nanoparticles in aqueous micellar two-phase systems.

A novel technique for technical-scale continuous purification of proteins is presented. It is based on the combined use of functionalized magnetic nano-particles and an Aqueous Micellar Two-Phase System featuring the non-ionic surfactant, Eumulgin ES, which undergoes temperature induced phase separation at ∼25°C. In the first step, conducted below the transition temperature (i.

Integrated system for temperature-controlled fast protein liquid chromatography comprising improved copolymer modified beaded agarose adsorbents and a travelling cooling zone reactor arrangement.

An integrated approach to temperature-controlled chromatography, involving copolymer modified agarose adsorbents and a novel travelling cooling zone reactor (TCZR) arrangement, is described. Sepharose CL6B was transformed into a thermoresponsive cation exchange adsorbent (thermoCEX) in four synthetic steps: (i) epichlorohydrin activation; (ii) amine capping; (iii) 4,4'-azobis(4-cyanovaleric acid) immobilization; and 'graft from' polymerization of poly(N-isopropylacrylamide-co-N-tert-butylacrylamide-co-acrylic acid-co-N,N'-methylenebisacrylamide). FT-IR, (1)H NMR, gravimetry and chemical assays allowed precise determination of the adsorbent's copolymer composition and loading, and identified the initial epoxy activation step as a critical determinant of 'on-support' copolymer loading, and in turn, protein binding performance.

Multi-cycle recovery of lactoferrin and lactoperoxidase from crude whey using fimbriated high-capacity magnetic cation exchangers and a novel "rotor-stator" high-gradient magnetic separator.

Cerium (IV) initiated "graft-from" polymerization reactions were employed to convert M-PVA magnetic particles into polyacrylic acid-fimbriated magnetic cation exchange supports displaying ultra-high binding capacity for basic target proteins. The modifications, which were performed at 25 mg and 2.5 g scales, delivered maximum binding capacities (Qmax ) for hen egg white lysozyme in excess of 320 mg g(-1) , combined with sub-micromolar dissociation constants (0.

Surfactant-free purification of membrane proteins with intact native membrane environment.

In order to study the structure and function of a protein, it is generally required that the protein in question is purified away from all others. For soluble proteins, this process is greatly aided by the lack of any restriction on the free and independent diffusion of individual protein particles in three dimensions. This is not the case for membrane proteins, as the membrane itself forms a continuum that joins the proteins within the membrane with one another.

Studies related to antibody fragment (Fab) production in Escherichia coli W3110 fed-batch fermentation processes using multiparameter flow cytometry.

Microbiology is important to industry therefore rapid and statistically representative measurements of cell physiological state, proliferation, and viability are essential if informed decisions about fermentation bioprocess optimization or control are to be made, because process performance will depend largely on the number of metabolically active viable cells. Samples of recombinant Escherichia coli W3110, containing the gene for the D1.3 anti-lysozyme Fab fragment under the control of the lac-based expression system, were taken at various stages from fed-batch fermentation processes and stained with a mixture of bis-(1,3-dibutylbarbituric acid) trimethine oxonol and propidium iodide (PI/BOX).

Lumped parameter model for prediction of initial breakthrough profiles for the chromatographic capture of antibodies from a complex feedstock.

A simple mathematical model to predict initial breakthrough profiles from preparative chromatographic separations of biological macromolecules has been developed. A lumped parameter approach was applied, employing Langmuirian adsorption kinetics to describe the rate of mass transfer (MT) from the bulk liquid in the column to the bound state. Equilibrium and kinetic adsorption data were determined for six different packed bed chromatographic adsorbents: two derivatised with rProtein A; and four functionalised with synthetic low molecular weight ligands.

Protein purification using magnetic adsorbent particles.

The application of functionalised magnetic adsorbent particles in combination with magnetic separation techniques has received considerable attention in recent years. The magnetically responsive nature of such adsorbent particles permits their selective manipulation and separation in the presence of other suspended solids. Thus, it becomes possible to magnetically separate selected target species directly out of crude biological process liquors (e.

Fractionation, solid-phase immobilization and chemical degradation of long pectin oligogalacturonides. Initial steps towards sequencing of oligosaccharides.

This work presents the optimized separation of pectin oligomers, their analysis by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS), their subsequent immobilization to supports, and our initial steps towards solid-support assisted sequencing. The ambient pressure strong anion-exchange resin Source 15Q combined with ammonium formate buffer (AF) was used for the separation of unsaturated and saturated pectic oligogalacturonides (OGAs) derived from enzymatic digestion of pectin. Routinely, multi-milligram quantities of defined sizes OGAs with DPs from 5 to 19 were produced in excellent purity (>95%).

A novel system for continuous protein refolding and on-line capture by expanded bed adsorption.

A novel two-step protein refolding strategy has been developed, where continuous renaturation-bydilution is followed by direct capture on an expanded bed adsorption (EBA) column. The performance of the overall process was tested on a N-terminally tagged version of human beta2-microglobulin (HAT-hbeta2m) both at analytical, small, and preparative scale. In a single scalable operation, extracted and denatured inclusion body proteins from Escherichia coli were continuously diluted into refolding buffer, using a short pipe reactor, allowing for a defined retention and refolding time, and then fed directly to an EBA column, where the protein was captured, washed, and finally eluted as soluble folded protein.

Demonstration of a strategy for product purification by high-gradient magnetic fishing: recovery of superoxide dismutase from unconditioned whey.

A systematic approach for the design of a bioproduct recovery process employing magnetic supports and the technique of high-gradient magnetic fishing (HGMF) is described. The approach is illustrated for the separation of superoxide dismutase (SOD), an antioxidant protein present in low concentrations (ca. 0.

Use of laminar flow patterning for miniaturised biochemical assays.

Laminar flow in microfluidic chambers was used to construct low (one dimensional) density arrays suitable for miniaturized biochemical assays. By varying the ratio of flows of two guiding streams flanking a sample stream, precise focusing and positioning of the latter was achieved, and reactive species carried in the sample stream were deposited on functionalized chip surfaces as discrete 50 microm wide lanes. Using different model systems we have confirmed the method's suitability for qualitative screening and quantification tasks in receptor-ligand assays, recording biotin-streptavidin interactions, DNA-hybridization and DNA-triplex formation.

Fractionation of whey proteins with high-capacity superparamagnetic ion-exchangers.

In this study we describe the design, preparation and testing of superparamagnetic anion-exchangers, and their use together with cation-exchangers in the fractionation of bovine whey proteins as a model study for high-gradient magnetic fishing. Adsorbents prepared by attachment of trimethyl amine to particles activated in sequential reactions with allyl bromide and N-bromosuccinimide yielded a maximum bovine serum albumin binding capacity of 156 mg g(-1) combined with a dissociation constant of 0.60 microM, whereas ion-exchangers created by linking polyethylene imine through superficial aldehydes bound up to 337 mg g(-1) with a dissociation constant of 0.