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Griffiths I.M.,Mathematical Institute | Kumar A.,Pall Life science | Stewart P.S.,University of Glasgow
Journal of Membrane Science | Year: 2016

Asymmetric multilayered filters, comprising a series of membranes with varying pore sizes stacked on top of one another, allow filtration to be tailored in a variety of novel ways. We develop a network model that systematically captures the complex filtration behaviour in such multilayer filters. The model allows us to understand the response of the system when challenged with a particular feed composition, characterized through the particle size and adhesivity to the membrane. We show how the model enables comprehensive and time-efficient sweeps in parameter space to be conducted that determine the optimal multilayered filter configuration for a given filtration challenge, classified by the number of membrane layers, the change in pore size between each layer (filter taper angle), and the level of trans-pore interconnectivity between each layer. The model allows us to isolate and analyse the effect of each of the specific filter characteristics and identify the practical merits and disadvantages. In particular, we predict that the optimal arrangement for maximizing throughput through the filter is to have pore radius gradually decreasing with depth, and a slight level of pore interconnectivity, with the precise set-up a function of the particle size, adhesivity and number of filter layers. The results of the analysis are used to draw conclusions on the design of membrane filters for optimal filter performance. © 2016.


Griffiths I.M.,Mathematical Institute | Kumar A.,Pall Life science | Stewart P.S.,University of Glasgow
Journal of Colloid and Interface Science | Year: 2014

Membrane fouling during particle filtration occurs through a variety of mechanisms, including internal pore clogging by contaminants, coverage of pore entrances, and deposition on the membrane surface. Each of these fouling mechanisms results in a decline in the observed flow rate over time, and the decrease in filtration efficiency can be characterized by a unique signature formed by plotting the volumetric flux, Q^, as a function of the total volume of fluid processed, V^. When membrane fouling takes place via any one of these mechanisms independently the Q^V^ signature is always convex downwards for filtration under a constant transmembrane pressure. However, in many such filtration scenarios, the fouling mechanisms are inherently coupled and the resulting signature is more difficult to interpret. For instance, blocking of a pore entrance will be exacerbated by the internal clogging of a pore, while the deposition of a layer of contaminants is more likely once the pores have been covered by particulates. As a result, the experimentally observed Q^V^ signature can vary dramatically from the canonical convex-downwards graph, revealing features that are not captured by existing continuum models. In a range of industrially relevant cases we observe a concave-downwards Q^V^ signature, indicative of a fouling rate that becomes more severe with time. We derive a network model for membrane fouling that accounts for the inter-relation between fouling mechanisms and demonstrate the impact on the Q^V^ signature. Our formulation recovers the behaviour of existing models when the mechanisms are treated independently, but also elucidates the concave-downward Q^V^ signature for multiple interactive fouling mechanisms. The resulting model enables post-experiment analysis to identify the dominant fouling modality at each stage, and is able to provide insight into selecting appropriate operating regimes. © 2014 Elsevier Inc.


Angelo J.M.,University of Delaware | Cvetkovic A.,Pall Life science | Gantier R.,Pall Life science | Lenhoff A.M.,University of Delaware
Journal of Chromatography A | Year: 2013

The structural characteristics of the HyperCel family of cellulosic ion-exchange materials (Pall Corporation) were assessed using methods to gauge the pore dimensions and the effect of ionic strength on intraparticle architecture. Inverse size exclusion chromatography (ISEC) was applied to the S and STAR AX HyperCel derivatives. The theoretical analysis yielded an average pore radius for each material of about 5. nm, with a particularly narrow pore-size distribution. Electron microscopy techniques were used to visualize the particle structure and relate it to macroscopic experimental data. Microscopy of Q and STAR AX HyperCel anion exchangers presented some qualitative differences in pore structure that can be attributed to the derivatization using conventional quaternary ammonium and salt-tolerant ligands, respectively. Finally, the effect of ionic strength was studied through the use of salt breakthrough experiments to determine to what extent Donnan exclusion plays a role in restricting the accessible pore volume for small ions. It was determined that Donnan effects were prevalent at total ionic strengths (TIS) less than 150. mM, suggesting the presence of a ligand-containing partitioning volume within the pore space. © 2013 Elsevier B.V.


Toueille M.,Pall Life science | Uzel A.,Pall Life science | Depoisier J.-F.,NovImmune | Gantier R.,Pall Life science
Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences | Year: 2011

Current platforms for purification of monoclonal antibodies, mostly relying on Protein A as a first capture step, are robust and efficient but significantly increase downstream purification costs, mainly due to Protein A resins. To decrease manufacturing costs, industry is increasingly considering the use of purification schemes without affinity Protein A resins. Mixed-mode chromatography can be used as a powerful alternative to standard purification platforms as it offers new selectivity and separation mechanisms exploiting a combination of both ionic and hydrophobic characteristics of antibodies and contaminating proteins. By using a design of experiments (DoE) approach and high throughput screening in 96-well plates, we developed four different two-steps MAb purification processes, based on the use of mixed-mode sorbents. Finally, three of the tested processes resulted in final purified Mab fractions containing less than 100. ppm of residual CHO proteins (CHOP), with overall process yields above 70%. These data show that mixed-mode chromatography sorbents, used at capture or intermediate purification steps, really expand the options of MAb purification process development with or without Protein A affinity chromatography. © 2011 Elsevier B.V.


Langdon B.B.,University of Colorado at Boulder | Mirhossaini R.B.,University of Colorado at Boulder | Mabry J.N.,University of Colorado at Boulder | Sriram I.,University of Colorado at Boulder | And 5 more authors.
ACS Applied Materials and Interfaces | Year: 2015

Although polymeric membranes are widely used in the purification of protein pharmaceuticals, interactions between biomolecules and membrane surfaces can lead to reduced membrane performance and damage to the product. In this study, single-molecule fluorescence microscopy provided direct observation of bovine serum albumin (BSA) and human monoclonal antibody (IgG) dynamics at the interface between aqueous buffer and polymeric membrane materials including regenerated cellulose and unmodified poly(ether sulfone) (PES) blended with either polyvinylpyrrolidone (PVP), polyvinyl acetate-co-polyvinylpyrrolidone (PVAc-PVP), or polyethylene glycol methacrylate (PEGM) before casting. These polymer surfaces were compared with model surfaces composed of hydrophilic bare fused silica and hydrophobic trimethylsilane-coated fused silica. At extremely dilute protein concentrations (10-3-10-7 mg/mL), protein surface exchange was highly dynamic with protein monomers desorbing from the surface within ∼1 s after adsorption. Protein oligomers (e.g., nonspecific dimers, trimers, or larger aggregates), although less common, remained on the surface for 5 times longer than monomers. Using newly developed super-resolution methods, we could localize adsorption sites with ∼50 nm resolution and quantify the spatial heterogeneity of the various surfaces. On a small anomalous subset of the adsorption sites, proteins adsorbed preferentially and tended to reside for significantly longer times (i.e., on "strong" sites). Proteins resided for shorter times overall on surfaces that were more homogeneous and exhibited fewer strong sites (e.g., PVAc-PVP/PES). We propose that strong surface sites may nucleate protein aggregation, initiated preferentially by protein oligomers, and accelerate ultrafiltration membrane fouling. At high protein concentrations (0.3-1.0 mg/mL), fewer strong adsorption sites were observed, and surface residence times were reduced. This suggests that at high concentrations, adsorbed proteins block strong sites from further protein adsorption. Importantly, this demonstrates that strong binding sites can be modified by changing solution conditions. Membrane surfaces are intrinsically heterogeneous; by employing single-molecule techniques, we have provided a new framework for understanding protein interactions with such surfaces. © 2015 American Chemical Society.


Pezzini J.,University of Bordeaux 1 | Joucla G.,University of Bordeaux 1 | Gantier R.,Pall Life science | Toueille M.,Pall Life science | And 5 more authors.
Journal of Chromatography A | Year: 2011

We evaluated mixed mode chromatography for the capture of recombinant antibodies from CHO cell culture supernatants. We studied PPA HyperCel, HEA HyperCel, MEP HyperCel and Capto adhere resins, which all contain hydrophobic and cationic groups. A microplate approach combined with DoE modeling allowed the exploration of the complex behaviors of these mixed mode resins. Optimal conditions for antibody purification and host cell proteins (HCPs) elimination were determined and then directly up-scaled to laboratory columns. Then we used mass spectrometry to identify the major HCPs potentially coeluted with the antibody. Differences between the four resins in terms of amount, complexity and identity of the HCPs present in the elution fractions were investigated. © 2011 Elsevier B.V.


Pueschel L.,Pall Life science
Journal of visualized experiments : JoVE | Year: 2011

We describe the complete process of AcroPrep Advance Filter Plates for 96 plasmid preparations, starting from prokaryotic culture and ending with high purity DNA. Based on multi-well filtration for bacterial lysate clearance and DNA purification, this method creates a streamlined process for plasmid preparation. Filter plates containing silica-based media can easily be processed by vacuum filtration or centrifuge to yield appreciable quantities of plasmid DNA. Quantitative analyses determine the purified plasmid DNA is consistently of high quality with average OD(260/280;) ratios of 1.97. Overall, plasmid yields offer more pure DNA for downstream applications, such as sequencing and cloning. This streamlined method of using AcroPrep Advance Filter Plates allows for manual, semi-automated or fully-automated processing.


Lajmi A.R.,Pall Life science | Nochumson S.,Pall Life science | Berges A.,Wyatt Technology Corporation
Biotechnology Progress | Year: 2010

The impact of typical anion-exchange flowthrough conditions on the IgG mass loading of an anion-exchange membrane scale-down unit (Mustang® Q coin) was investigated. High performance size-exclusion chromatography and multiangle laser light scattering results suggested the presence of a small fraction of IgG aggregates with average radius >100 nm under anion-exchange flowthrough conditions. The small filtration area presented by the 0.35 mL membrane volume Mustang® Q coin limited the membrane throughput due to fouling from the aggregates at higher antibody loading. Data in this report indicated that a 0.2 μm hybrid polyethersulfone and polyvinylidene fluoride membrane in-line prefilter with a minimum filtration area of 20 sq cm alleviated the Mustang® Q coin fouling. The combined cake filtration and intermediate blocking model was proposed as the most likely membrane pore blocking mechanism. Increasing the filtration area in the in-line prefilter resulted in higher IgG mass throughput. Thus, using an appropriately sized in-line prefilter could provide more robust antibody throughput performance on scale-down membrane anion-exchange units. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010 © 2010 American Institute of Chemical Engineers (AIChE).


Rawlings B.,Pall Life science
VTT Symposium (Valtion Teknillinen Tutkimuskeskus) | Year: 2010

The presentation will provide an overview of the current technologies and approaches most widely implemented within industry in the field of single-use systems. Furthermore, we will show real examples of specific applications including the benefits of implementation from the end-users perspective. A short review of other key areas will also be discussed such as regulatory position, challenges for implementation and key considerations prior to implementation. Finally we will present an outlook for the future trends and requirements for single-use systems.


Champagne J.,Pall Life science | Balluet G.,Pall Life science | Gantier R.,Pall Life science | Toueille M.,Pall Life science
Protein Expression and Purification | Year: 2013

The present study describes the use of the new HyperCel STAR AX "salt tolerant" anion exchange sorbent for the capture from Chinese Hamster Ovary (CHO) cell culture supernatant (CCS) of an acidic model protein (α-amylase). HyperCel STAR AX sorbent and other conventional anion exchangers were evaluated to purify biologically-active enzyme. Salt tolerance of the sorbent allowed reaching 5-fold higher dynamic binding capacity than conventional anion exchange during capture of the enzyme from neat (undiluted) CCS. After optimization of operating conditions, HyperCel STAR AX turned out to be the only sorbent allowing efficient protein capture directly from both neat and diluted CCS with consistent and satisfying purity, yield and productivity. Therefore implementation of the salt tolerant sorbent in industrial purification processes should allow avoiding time and cost consuming steps such as dilution or UF/DF that exclusively aim at establishing suitable conditions for ion exchange step without bringing any added value to the purification process performance. Altogether this study highlights the flexibility and cost-reduction potential brought in process design by the HyperCel STAR AX salt tolerant sorbent.© 2013 Elsevier Inc. All rights reserved.

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