Publications by authors named "Evan Shave"

12 Publications

  • Page 1 of 1

Intensified Downstream Processing of Monoclonal Antibodies Using Membrane Technology.

Biotechnol J 2021 Mar 19;16(3):e2000309. Epub 2020 Oct 19.

Australian Research Council Training Centre for Biopharmaceutical Innovation, Australian Institute for Bioengineering and Nanotechnology, The University of Queensland, Corner College and Cooper Roads, Brisbane, Queensland, 4072, Australia.

The need to intensify downstream processing of monoclonal antibodies to complement the advances in upstream productivity has led to increased attention toward implementing membrane technologies. With the industry moving toward continuous operations and single use processes, membrane technologies show promise in fulfilling the industry needs due to their operational flexibility and ease of implementation. Recently, the applicability of membrane-based unit operations in integrating the downstream process has been explored. In this article, the major developments in the application of membrane-based technologies in the bioprocessing of monoclonal antibodies are reviewed. The recent progress toward developing intensified end-to-end bioprocesses and the critical role membrane technology will play in achieving this goal are focused upon.
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http://dx.doi.org/10.1002/biot.202000309DOI Listing
March 2021

Progression of continuous downstream processing of monoclonal antibodies: Current trends and challenges.

Biotechnol Bioeng 2018 12 18;115(12):2893-2907. Epub 2018 Oct 18.

Australian Research Council Training Centre for Biopharmaceutical Innovation, Australian Institute for Bioengineering and Nanotechnology, The University of Queensland, Brisbane, Queensland, Australia.

Rapid advances in intensifying upstream processes for biologics production have left downstream processing as a bottleneck in the manufacturing scheme. Biomanufacturers are pursuing continuous downstream process development to increase efficiency and flexibility, reduce footprint and cost of goods, and improve product consistency and quality. Even after successful laboratory trials, the implementation of a continuous process at manufacturing scale is not easy to achieve. This paper reviews specific challenges in converting each downstream unit operation to a continuous mode. Key elements of developing practical strategies for overcoming these challenges are detailed. These include equipment valve complexity, favorable column aspect ratio, protein-A resin selection, quantitative assessment of chromatogram peak size and shape, holistic process characterization approach, and a customized process economic evaluation. Overall, this study provides a comprehensive review of current trends and the path forward for implementing continuous downstream processing at the manufacturing scale.
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http://dx.doi.org/10.1002/bit.26812DOI Listing
December 2018

The Biflow: an instrument for transfer-loop mediated, continuous, preparative-scale isoelectric trapping separations.

Electrophoresis 2007 Jul;28(13):2291-9

Chemistry Department, Texas A&M University, College Station, TX 77842-3012, USA.

The Biflow, a new isoelectric trapping instrument was designed to obtain a narrow DeltapI fraction from a complex feed in one step. The Biflow contains two identical separation units, each unit houses: an anode and cathode compartment, an anodic and cathodic membrane, an anodic and cathodic separation compartment, and a separation membrane. The separation units are connected to independent power supplies. The anodic membranes in Units 1 and 2 typically buffer at the same pH value and so do the cathodic membranes. The separation membranes in Units 1 and 2 buffer at different pH values, these determine the pI range (DeltapI) of the product. The cathodic separation compartments in Units 1 and 2 contain the feed and harvest streams. The two anodic separation compartments, connected through an electrically insulating air gap, form the transfer loop through which the transfer stream is recirculated between Units 1 and 2. Ampholytic components in the feed, with pI values lower than the pH of the buffering membrane in Unit 1, pass into the transfer stream and are shuttled into Unit 2. In Unit 2, components in the transfer stream which have pI values higher than the pH of the buffering membrane in Unit 2, pass into the harvest stream. This double transfer of the target component, oppositely directed, guarantees the complete exclusion of products outside the desired DeltapI range from the harvest stream. The utility of the Biflow unit was demonstrated by isolating carnosine from a mixture of UV-absorbing ampholytes and ovalbumin isoforms as well as 4.4
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http://dx.doi.org/10.1002/elps.200600770DOI Listing
July 2007

Use of a preparative-scale, recirculating isoelectric trapping device for the isolation and enrichment of acidic proteins in bovine serum.

J Chromatogr A 2007 Jul 4;1155(2):237-41. Epub 2007 Feb 4.

Department of Chemistry, Texas A&M University, College Station, TX 77842-3012, USA.

A recirculating, preparative-scale isoelectric trapping device, developed for the binary isoelectric trapping separation of proteins has been used to desalt, isolate and enrich the pI<4 protein fraction from a 150 mL sample of bovine serum. Subsequent re-separation of the 2
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http://dx.doi.org/10.1016/j.chroma.2007.01.125DOI Listing
July 2007

pH transients during salt removal in isoelectric trapping separations: a curse revisited.

Electrophoresis 2007 Feb;28(4):587-94

Chemistry Department, Texas A&M University, College Station, TX 77842-3012, USA.

The pH transients that occur during isoelectric trapping separations as a result of the removal of nonampholytic ionic components have been re-examined. Salts containing strong electrolyte anions and cations, both with equal and dissimilar mobilities, have been studied using anodic and cathodic buffering membranes whose pH values were both equidistant and nonequidistant from pH 7. The direction and magnitude of the pH transient (acidic or basic) was found to depend on both the mobilities of the anion and cation (mu(anion)/mu(cation)) and the pH difference between pH 7 and the pH of the buffering membranes (|pH(memb) (anodic) - 7|/|7 - pH(memb) (cathodic)|). When |pH(memb) (anodic) - 7|/|7 - pH(memb) (cathodic)| = 1, mu(anion)/mu(cation)<1 leads to an acidic pH transient, mu(anion)/mu(cation) = 1 eliminates the pH transient and mu(anion)/mu(cation)>1 leads to a basic pH transient. When mu(anion)/mu(cation) = 1, |pH(memb) (anodic) - 7|/|7 - pH(memb) (cathodic)|<1 leads to a basic pH transient, |pH(memb) (anodic) - 7|/|7 - pH(memb) (cathodic)| = 1 eliminates the pH transient and |pH(memb) (anodic) - 7|/|7 - pH(memb) (cathodic)|>1 leads to an acidic pH transient. By selecting appropriate anodic and cathodic buffering membranes to adjust the |pH(memb) (anodic) - 7|/|7 - pH(memb) (cathodic)| value, pH transients caused by dissimilar anion and cation mobilities can be avoided.
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http://dx.doi.org/10.1002/elps.200600738DOI Listing
February 2007

Synthesis of UV-absorbing and fluorescent carrier ampholyte mixtures and their application for the determination of the operational pH values of buffering membranes used in isoelectric trapping separations.

J Chromatogr A 2006 Oct 19;1130(2):232-7. Epub 2006 Apr 19.

Department of Chemistry, Texas A&M University, College Station, 77842-3012, USA.

Success in isoelectric trapping separations critically depends on the knowledge of the accurate operational pH value of the buffering membranes used. Currently, due to a lack of easy, rapid, accurate methods that can be used for the post-synthesis determination of the operational pH value of a buffering membrane, only nominal pH values calculated from the amounts of the reagents used in the synthesis of the membranes and their acid-base dissociation constants are available. To rectify this problem, UV-absorbing and fluorescent carrier ampholyte mixtures were prepared by alkylating pentaethylenehexamine with a chromophore and a fluorophore, followed by Michael addition of acrylic acid and itaconic acid to the resulting oligoamine. Carrier ampholyte mixtures, with evenly distributed absorbance values across the 3
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http://dx.doi.org/10.1016/j.chroma.2006.03.054DOI Listing
October 2006

High-buffering capacity, hydrolytically stable, low-pI isoelectric membranes for isoelectric trapping separations.

Electrophoresis 2004 Oct;25(20):3323-30

Department of Chemistry, Texas A&M University, College Station, TX, USA.

Hydrolytically stable, low-pI isoelectric membranes have been synthesized from low-pI ampholytic components, poly(vinyl alcohol), and a bifunctional cross-linker, glycerol-1,3-diglycidyl ether. The low-pI ampholytic components used contain one amino group and at least two weakly acidic functional groups. The acidic functional groups are selected such that the pI value of the ampholytic component is determined by the pK(a) values of the acidic functional groups. When the concentration of the ampholytic component incorporated into the membrane is higher than a required minimum value, the pI of the membrane becomes independent of variations in the actual incorporation rate of the ampholytic compound. The new, low-pI isoelectric membranes have been successfully used as anodic membranes in isoelectric trapping separations with pH < 1.5 anolytes and replaced the hydrolytically less stable polyacrylamide-based isoelectric membranes. The new low-pI isoelectric membranes have excellent mechanical stability, low electric resistance, good buffering capacity, and long life time, even when used with as much as 50 W power and current densities as high as 33 mA/cm(2) during the isoelectric trapping separations.
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http://dx.doi.org/10.1002/elps.200406045DOI Listing
October 2004

Alkali-stable high-pI isoelectric membranes for isoelectric trapping separations.

Electrophoresis 2004 Jul;25(14):2128-38

Department of Chemistry, Texas A&M University, College Station, TX 778452-3012, USA.

Alkali-stable, high-pI isoelectric membranes have been synthesized from quaternary ammonium derivatives of cyclodextrins and poly(vinyl alcohol), and bifunctional cross-linkers, such as glycerol-1,3-diglycidyl ether. The new, high-pI isoelectric membranes were successfully applied as cathodic membranes in isoelectric trapping separations in place of the hydrolytically more labile, polyacrylamide-based cathodic isoelectric membranes, and permitted the use of catholytes as alkaline as 1 M NaOH. The new high-pI isoelectric membranes have shown excellent mechanical stability, low electric resistance and long life times, even when subjected to electrophoresis with current densities as high as 80 mA/cm2.
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http://dx.doi.org/10.1002/elps.200305944DOI Listing
July 2004

Preparative-scale isoelectric trapping separations in methanol-water mixtures.

J Chromatogr A 2004 May;1036(1):3-6

Chemistry Department, Texas A&M University, MS 3255, College Station, TX 77842-3012, USA.

The typically low aqueous solubilities of small, hydrophobic organic ampholytic molecules limit the production rates that can be achieved in their isoelectric trapping (IET) separations and call for the use of hydro-organic mixtures as solvents. The compatibility of methanol-water mixtures and poly(ethylene terephthalate) substrate-supported isoelectric polyacrylamide hydrogels, developed for binary IET separations in a Gradiflow BF200IET unit, was investigated. The isoelectric polyacrylamide-based hydrogels retained their functional and mechanical integrities when the methanol concentration in the hydro-organic solvent mixture was kept at or below 25% (v/v). The utility of the hydro-organic media was demonstrated in the purification of a hydrophobic ampholytic compound, technical grade 4-hydroxy-3-(morpholinomethyl) benzoic acid. Production rates as high as 7 mg/h were achieved using small, 15 cm2 active surface area isoelectric membranes.
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http://dx.doi.org/10.1016/j.chroma.2003.11.008DOI Listing
May 2004

Preparative-scale, recirculating, pH-biased binary isoelectric trapping separations.

Electrophoresis 2004 Jan;25(2):381-7

Chemistry Department, Texas A&M University, College Station, TX 77842, USA.

In order to improve the production rates and lower the specific electrophoretic energy consumption values in preparative-scale, recirculating, binary isoelectric trapping separations, we propose to add an auxiliary isoelectric agent to the solution in the anodic separation compartment and another to the solution in the cathodic separation compartment to implement pH-biased isoelectric trapping. The auxiliary isoelectric agents are selected such that they are trapped in the respective anodic and cathodic separation compartments and also, have isoelectric point (pI) values that are different from the pI values of the analytes of interest. By proper selection of the auxiliary isoelectric agents and their concentrations, the analytes of interest can be kept in nonisoelectric, charged state during the entire course of the preparative-scale, recirculating, binary isoelectric trapping separation. This results in higher electrophoretic mobilities and solubilities for the analytes than in their isoelectric or near-isoelectric states, and leads to faster binary isoelectric trapping separations.
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http://dx.doi.org/10.1002/elps.200305752DOI Listing
January 2004

Preparative-scale isoelectric trapping enantiomer separations.

J Chromatogr A 2003 Mar;989(1):73-8

Department of Chemistry, MS 3255, Texas A&M University, PO Box 30012, College Station, TX 77842-3012, USA.

The new Gradiflow BF200 IET unit, developed for isoelectric trapping protein separations has been modified and used to carry out preparative-scale enantiomer separations. Hydroxypropyl beta-cyclodextrin was used as the chiral resolving agent to induce an isoelectric point difference between the enantiomers. Three isoelectric membranes with isoelectric points below, in between and above the isoelectric points of the complexed enantiomers were used to trap the separated enantiomers in the anodic and cathodic separation compartments of the Gradiflow BF200 IET apparatus, respectively. The production rates were about 15 times higher than those previously obtained with another isoelectric trapping device and about 30% higher than those obtained in a continuous free-flow electrophoretic device operated in the isoelectric focusing mode. The remarkable separation speed observed in the modified Gradiflow BF200 IET unit is attributed to the favorable interplay of the short electrophoretic transfer distance, the high electric field strength and the large effective surface areas of the isoelectric membranes.
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http://dx.doi.org/10.1016/s0021-9673(02)01607-2DOI Listing
March 2003

Preparative-scale isoelectric trapping separations using a modified Gradiflow unit.

J Chromatogr A 2002 Dec;979(1-2):155-61

Gradipore, French's Forest, NSW 2086, Australia.

The Gradiflow BF200 preparative electrophoretic unit (Gradipore), which has been developed for size-based and charge-sign-based protein separations and in which the hydraulic flow path of the recirculating sample stream in the separation cartridge is orthogonal to the electric field, has been modified to carry out binary protein separations using the principles of isoelectric trapping. The disposable separation cartridge contained three isoelectric membranes which, along with the cartridge holder, formed the anode and cathode compartments and the anodic and cathodic separation compartments. The utility of the modified instrument was demonstrated by effecting a binary separation of chicken egg white across an isoelectric point 5.5 isoelectric membrane. The desalting and subsequent binary separation steps proved to be remarkably rapid, due to the favorable combination of short electrophoretic path, high electric field strength and large effective isoelectric membrane surface area.
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http://dx.doi.org/10.1016/s0021-9673(02)01601-1DOI Listing
December 2002