Protein A chromatography resin lifetime—impact of feed composition

Adsorbent lifetime during protein A chromatography is not readily predicted or understood, representing a key challenge to be addressed for biopharmaceutical manufacturers. This article focuses on the impact of feed composition on the performance of a typical agarose‐based protein A resin across a lifetime of 50 cycles. Cycling studies were performed using three different feed materials with varying levels of feed components including proteases, histones, DNA, and nonhistone proteins. Changes in the process and quality attributes were measured. The DBCs were not seen to vary between conditions although there was a reduction in particle porosity in all cases. Fluorescence spectroscopy and LC‐MS/MS were used to identify the contribution and extent of fouling to the observed capacity loss. Residual protein A ligand density and deposition of foulants (HCP, residual mAb, and DNA) varied between the three feed materials. Resins cycled in feed materials containing high concentrations of HCP and histones were seen to have greater extents of capacity loss. The mode of performance loss, capacity loss, or impact on product quality was seen to vary depending on the feed material. The results indicate that feed material composition may be correlated to the rate and mode of resin aging as a basis for improved process understanding. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:412–419, 2018     

Soft sensors in bioprocessing: a status report and recommendations

The following report with recommendations is the result of an expert panel meeting on soft sensor applications in bioprocess engineering that was organized by the Measurement, Monitoring, Modelling and Control (M3C) Working Group of the European Federation of Biotechnology - Section of Biochemical Engineering Science (ESBES). The aim of the panel was to provide an update on the present status of the subject and to identify critical needs and issues for the furthering of the successful development of soft sensor methods in bioprocess engineering research and for industrial applications, in particular with focus on biopharmaceutical applications. It concludes with a set of recommendations, which highlight current prospects for the extended use of soft sensors and those areas requiring development.     

Chemical and biological characterisation of a sensor surface for bioprocess monitoring

This paper describes the step-wise fabrication and characterisation of a multi-layer dual polarization interferometry (DPI) based biosensor utilising Protein G (ProG) as the bio-recognition layer for the detection of a fragment antibody (Fab'). The biosensor is capable of monitoring the concentration of Fab' product within the extracellular medium of a fed-batch fermentation after leakage from Escherichia coli (E.coli). The activity, stability and functionality of each sensor layer were analysed in situ using DPI, whilst the chemical identity and homogeneity of the chemical layers were assessed ex situ using X-ray photoelectron spectroscopy (XPS) and secondary ion mass spectrometry (SIMS). Two different biotin linkers were found to produce hugely differing surfaces after the capture of NeutrAvidin? (NA) and biotinylated Protein G (b-ProG). The hydrophilic (PEG)(4)-biotin linker resulted in a surface where the b-ProG layer was deposited and organised above the NA layer producing an active and stable surface, whilst the hydrophobic LC-biotin linker generated a surface where the b-ProG layer was buried within the NA layer leading to variable surfaces and poor binding of the Fab' target. The biosensor has a detection limit of 1.7 μg/ml with a dynamic range covering two orders of magnitude. The sensor can detect the onset of Fab' leakage as early as 2h following product induction, with high signal-to-noise ratios and little interference from extracellular components. Leakage of Fab' followed a biphasic profile, switching to a more rapid rate 20 h after induction, indicating accelerated product loss and the need for cultivation harvest.     

Cleft,Crevice, or the Inner Thigh: ‘Another Place’ for the Establishment of the Invasive Barnacle Austrominius modestus (Darwin, 1854)

The proliferation of anthropogenic infrastructure in the marine environment has aided the establishment and spread of invasive species. These structures can create novel habitats in areas normally characterised as void of suitable settlement sites. The habitat requirements of the invasive acorn barnacle Austrominius modestus (Darwin, 1854) were assessed using a novel sampling site at Crosby Beach, Liverpool. Austrominius modestus has spread rapidly around the UK since its initial introduction, becoming locally dominant in many estuarine areas including the Antony Gormley art installation, ‘Another Place’, at Crosby Beach. The installation consists of 100 replicate solid cast-iron life-size human figures, located at a range of heights on the shore. We recorded the distribution and abundance of A. modestus present on all of the statues at various positions during the summer of 2006. The positions varied in location, exposure, direction, and rugosity. Although parameters such as rugosity and exposure did influence patterns of recruitment, they were less important than interactions between shore height and direction, and specific location on the beach. The addition of a suitable substrate to a sheltered and estuarine region of Liverpool Bay has facilitated the establishment of A. modestus. Understanding the habitat requirements of invasive species is important if we are to make predictions about their spread and the likelihood of invasion success. Austrominius modestus has already become locally dominant in some regions of the UK and, with projections of favourable warming conditions and the global expansion of artificial structures, the continued spread of this species can be expected. The implications of this on the balance between native and invasive species dominance should be considered.     

Design and characterization of a microfluidic packed bed system for protein breakthrough and dynamic binding capacity determination

A 1.5 μL ion exchange chromatography column to accommodate resins used for biopharmaceutical processing has been designed to produce breakthrough curves and to quantify dynamic and maximum protein binding capacities. Channels within a glass chip were fabricated using photolithography and isotropic etching. The design includes a 1 cm long microfluidic column in which compressible, polydispersed porous agarose beads (70 μm mean diameter) were packed using a keystone method where particles aggregate in a narrow channel. The depth of the column is such that two bead layers exist. The fabrication technique used forms Cartesian geometries as opposed to circular cross sections found in standard columns. The voidage was therefore higher than standard values when measured by 3D confocal microscopy. In conjunction with microscopic techniques, the column allows visualization of events within the bed such as adsorption profiles that would otherwise be difficult to observe. In this work, the binding of fluorescently labeled protein during isocratic loading was used to generate breakthrough from the microcolumn. Useful breakthrough curves were achieved using mobile phase velocities from 60 to 270 cm h^?1. Calculated dynamic binding capacities were compared well with previously published data on conventional scale columns. The microfluidic chromatography column described here thus allows study of process scale chromatography behavior at scales 20,000 times smaller than in current practice. The work described in this article is representative of the proof of principle of a potentially powerful tool for the generation of microfluidic process bed data for the biopharmaceutical industry. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

A rational approach to improving titer in Escherichia coli-based cell-free protein synthesis reactions

Cell-free protein synthesis (CFPS) is an established method for rapid recombinant protein production. Advantages like short synthesis times and an open reaction environment make CFPS a desirable platform for new and difficult-to-express products. Most recently, interest has grown in using the technology to make larger amounts of material. This has been driven through a variety of reasons from making site specific antibody drug conjugates, to emergency response, to the safe manufacture of toxic biological products. We therefore need robust methods to determine the appropriate reaction conditions for product expression in CFPS. Here we propose a process development strategy for Escherichia coli lysate-based CFPS reactions that can be completed in as little as 48 hr. We observed the most dramatic increases in titer were due to the E. coli strain for the cell extract. Therefore, we recommend identifying a high-producing cell extract for the product of interest as a first step. Next, we manipulated the plasmid concentration, amount of extract, temperature, concentrated reaction mix pH levels, and length of reaction. The influence of these process parameters on titer was evaluated through multivariate data analysis. The process parameters with the highest impact on titer were subsequently included in a design of experiments to determine the conditions that increased titer the most in the design space. This proposed process development strategy resulted in superfolder green fluorescent protein titers of 0.686 g/L, a 38% improvement on the standard operating conditions, and hepatitis B core antigen titers of 0.386 g/L, a 190% improvement.     

Monitoring and Control of Bioproducts from Conception to Production in Real‐time Using an Optical Biosensor

The ability to observe biological interactions in real‐time using optical biosensor technology provides the scientist/engineer with a valuable analytical tool to analyze biological molecules. Production of biological products is a growing area, but the course of discovery through to production is lengthy and complex, especially for therapeutic products. However, the economics of developing new products are clear, time to market for a new product is the primary consideration. Limited patent lifetimes, the need to get a return on the investment in research and gain competitive advantage by launching products before competitors all contribute to the necessity to get products to market. The ability of biosensor technology to give rapid, direct information on key biological parameters, (such as product concentration), makes it a suitable analytical tool to help accelerate the development of biological products. It is difficult to conceive how direct measurements of this speed and selectivity can be possible with other analytical methods. This paper aims to describe how this potential can be brought to use from biological product conception and discovery all the way through to process operation and quality control.     

A framework for the prediction of scale-up when using compressible chromatographic packings

Experimental data are given for the solid pressure distributions in chromatography columns of various column aspect ratios packed with four types of agarose-based resin. The loss of column wall support at large scales can result in unexpectedly high pressures caused by the compression of the matrix via drag forces exerted by fluid flow through the bed. The need for an accurate model to predict flow conditions at increasing scale is essential for the scaling-up of chromatographic processes and for avoiding bed compression during operation. Several studies have generated correlations that allow for the prediction of column pressure drops, but they either are mathematically complex, which impairs their practical use, or require a large number of experiments to be performed before they can be used. In this study an empirical correlation was developed based on a previously proposed model, which links the critical velocity of operation of a chromatographic system (microcrit), to the gravity-settled bed height (L0), the column diameter (D), the feed viscosity (micro), and the compressibility of the chromatographic media used (micro 10%). The methodology developed in this study is straightforward to use and significantly reduces the burden of preceding laboratory-scale experimentation. The approach can be used to predict the critical velocity of any chromatographic system and will be useful in the development of chromatographic operations and for column sizing.