Publications
2013
J. A. Martin, P. Parekh, Y. Kim, T. E. Morey, K. Sefah, N. Gravenstein, D. M. Dennis, W. Tan
PLOS ONE, March 2013, Volume 8, Issue 3, e57341
Adverse drug reactions, including severe patient bleeding, may occur following the administration of anticoagulant drugs. Bivalirudin is a synthetic anticoagulant drug sometimes employed as a substitute for heparin, a commonly used anticoagulant that can cause a condition called heparin-induced thrombocytopenia (HIT). Although bivalrudin has the advantage of not causing HIT, a major concern is lack of an antidote for this drug. In contrast, medical professionals can quickly reverse the effects of heparin using protamine. This report details the selection of an aptamer to bivalirudin that functions as an antidote in buffer. This was accomplished by immobilizing the drug on a monolithic column to partition binding sequences from nonbinding sequences using a low-pressure chromatography system and salt gradient elution. The elution profile of binding sequences was compared to that of a blank column (no drug), and fractions with a chromatographic difference were analyzed via real-time PCR (polymerase chain reaction) and used for further selection. Sequences were identified by 454 sequencing and demonstrated low micromolar dissociation constants through fluorescence anisotropy after only two rounds of selection. One aptamer, JPB5, displayed a dose-dependent reduction of the clotting time in buffer, with a 20 µM aptamer achieving a nearly complete antidote effect. This work is expected to result in a superior safety profile for bivalirudin, resulting in enhanced patient care.
2012
J. Lee, H. T. Gan, S. M. Abdul Latiff , C. Chuah, W. Y. Lee, Y.-S. Yang, B. Loo, S. K. Ng, P. Gagnon
Journal of Chromatography A, 1270 (2012) 162-170
We introduce a chromatography method for purification of large proteins and viruses that works by capturing them at a non-reactive hydrophilic surface by their mutual steric exclusion of polyethylene glycol (PEG). No direct chemical interaction between the surface and the target species is required. We refer to the technique as steric exclusion chromatography. Hydroxyl-substituted polymethacrylate monoliths provide a hydrophilic surface and support convective mass transport that is unaffected by the viscosity of the PEG. Elution is achieved by reducing PEG concentration. Selectivity correlates with molecular size, with larger species retained more strongly than smaller species. Retention increases with PEG size and concentration. Salts weaken retention in proportion to their concentration and Hofmeister ranking. Retention is enhanced near the isoelectric point of the target species. Virus binding capacity was measured at 9.9 × 1012 plaque forming units per mL of monolith. 99.8% of host cell proteins and 93% of DNA were eliminated. Mass recovery exceeded 90%. IgM capacity was greater than 60 mg/mL. 95% of host cell proteins were eliminated from IgM produced in protein-free media, and mass recovery was up to 90%. Bioactivity was fully conserved by both viruses and antibodies. Process time ranged from less than 30 min to 2 h depending on the product concentration in the feed stream.
P. Gagnon
Journal of Chromatography A, 1221 (2012) 57-70(2012) 57-70
This article reviews technology trends in antibody purification. Section 1 discusses non-chromatography methods, including precipitation, liquid–liquid extraction, and high performance tangential flow filtration. The second addresses chromatography methods. It begins with discussion of fluidized and fixed bed formats. It continues with stationary phase architecture: diffusive particles, perfusive particles, membranes and monoliths. The remainder of the section reviews recent innovations in size exclusion, anion exchange, cation exchange, hydrophobic interaction, immobilized metal affinity, mixed-mode, and bioaffinity chromatography. Section 3 addresses an emerging trend of formulating process buffers to prevent or correct anomalies in the antibodies being purified. Methods are discussed for preventing aggregate formation, dissociating antibody-contaminant complexes, restoring native antibody from aggregates, and conserving or restoring native disulfide pairing.
M. Žorž
ChemieXtra 3/2012 pp 30-33
Sartorius BIA Separations produziert und vertreibt kurze monolithischen Chromatografiesäulen, die auf der CIM-Convective Interaction Media-Technologie basieren. CIM-Säulen eignen sich vor allem für die Reinigung von grossen Biomolekülen wie etwa Viren (virale Vektoren und Impfstoffe), DNA (Plasmid-DNA) und grössere Proteine (Immunglobuline G und M, pegylierte Proteine). Sie weisen einzigartige Eigenschaften in Bezug auf operative Flussraten, Adsorptionsfähigkeit und Trennung grosser Biomoleküle auf. Die Säulen werden in Forschung, Labor, Pilot- und industriellen Produktionsstufen eingesetzt und sind extrem einfach zu handhaben.
R. Milačič, D. Ajlec, T. Zuliani, D. Žigon, J. Ščančar
Talanta 101 (2012) 203-210
In human milk zinc (Zn) is bound to proteins and low molecular mass (LMM) ligands. Numerous investigations demonstrated that Zn bioavailability in human milk is for infant much higher than in cow's milk. It was presumed that in the LMM human milk fraction highly bioavailable Zn-citrate prevails. However, literature data are controversial regarding the amount of Zn-citrate in human milk since analytical procedures reported were not quantitative. So, complex investigation was carried out to develop analytical method for quantitative determination of this biologically important molecule. Studies were performed within the pH range 5–7 by the use of synthetic solutions of Zn-citrate prepared in HEPES, MOPS and MES buffers. Zn-citrate was separated on weak anion-exchange convective interaction media (CIM) diethylaminoethyl (DEAE) monolithic chromatographic column using NH4NO3 as an eluent. Separated Zn species were determined by flame atomic absorption spectrometry (FAAS) or inductively coupled plasma mass spectrometry (ICP-MS). Quantitative separation of Zn-citrate complexes ([Zn(Cit)]- and [Zn(Cit)2]4-; column recoveries 94–102%) and good repeatability and reproducibility of results with relative standard deviation (RSD±3.0%) were obtained. In fractions under the chromatographic peaks Zn-binding ligand was identified by electrospray ionization tandem mass spectrometry (ESI-MS-MS). Limits of detection (LOD) for determination of Zn-citrate species by CIM DEAE-FAAS and CIM DEAE-ICP-MS were 0.01 μg Zn mL-1 and 0.0005 μg Zn mL-1, respectively. Both techniques were sensitive enough for quantification of Zn-citrate in human milk. Results demonstrated that about 23% of total Zn was present in the LMM milk fraction and that LMM-Zn corresponded to Zn-citrate. The developed speciation method represents a reliable analytical tool for investigation of the percentage and the amount of Zn-citrate in human milk.
2011
S. H. Lubbad, M. R. Buchmeiser
Journal of Chromatography A, 1218 (2011) 2362-2367
Ring-opening metathesis polymerization- (ROMP) derived monoliths were prepared from 5-norborn-2-enemethyl bromide (NBE-CH2Br) and tris(5-norborn-2-enemethoxy)methylsilane ((NBE-CH2O)3SiCH3) within the confines of surface-silanized borosilicate columns (100 × 3 mm I.D.), applying Grubbs’ first generation benzylidene-type catalyst [RuCl2(PCy3)2(CHPh)]. Monoliths were converted into weak anion exchangers via reaction with diethyl amine. The resulting monolithic anion exchangers demonstrated a very good potential for the anion-exchange separation of nucleic acids applying a phosphate buffer (0.05 mol/L, pH 7) and NaCl (1.0 mol/L) as a gradient former. Fast and efficient separations, indicated by sharp and highly symmetric analyte peaks, were established. Except for the 267 and 298 base pair fragments, the eleven fragments of a ds-pUC18 DNA Hae III digest were baseline separated within ∼8 min. Nineteen fragments of a ds-pBR322 Hae III digest were separated within ∼12 min. There, only the 192 and 213 base pair fragments and the 458, 504 and 540 base pair fragments coeluted. A ds-pUC18 DNA Hae III digest was used as a control analyte in evaluating the influence of organic additives on the mobile phase such as methanol and acetonitrile on nucleic acid separation. Methanol, and even better, acetonitrile improved the separation efficiency and shortened the analysis time.
S. Yamamoto, T. Okada, M. Abe, N. Yoshimoto
Journal of Chromatography A, 1218 (2011) 2460-2466
The peak spreading of DNAs of various sizes [12-mer, 20-mer, 50-mer and 95-mer poly(T)] in linear gradient elution (LGE) chromatography with a thin monolithic disk was investigated by using our method developed for determining HETP in LGE. Electrostatic interaction-based chromatography mode (ion-exchange chromatography, IEC) was used. Polymer-based monolithic disks of two different sizes (12 mm diameter, 3 mm thickness and 0.34 mL; 5.2 mm diameter, 4.95 mm thickness and 0.105 mL) having anion-exchange groups were employed. For comparison, a 15-μm porous bead IEC column (Resource Q, 6.4 mm diameter, 30 mm height and 0.97 mL) was also used. The peak width did not change with the flow velocity for the monolithic disks where as it became wider with increasing velocity. For the monolithic disks the peak width normalized with the column bed volume was well-correlated with the distribution coefficient at the peak position KR. HETP values were constant (ca. 0.003–0.005 cm) when KR > 5. Much higher HETP values which are flow-rate dependent were obtained for the porous bead chromatography. It is possible to obtain 50–100 plates for the 3 mm monolithic disk. This results in very sharp elution peaks (standard deviation/bed volume = 0.15) even for stepwise elution chromatography, where the peak width is similar to that for LGE of a very steep gradient slope.
Z. Jiang, N. W. Smith, Z. Liu
Journal of Chromatography A, 1218 (2011) 2350-2361
Hydrophilic interaction chromatography (HILIC) has experienced increasing attention in recent years. Much research has been carried out in the area of HILIC separation mechanisms, column techniques and applications. Because of their good permeability, low resistance to mass transfer and easy preparation within capillaries, hydrophilic monolithic columns represent a trend among novel HILIC column techniques. This review attempts to present an overview of the preparation and applications of HILIC monolithic columns carried out in the past decade. The separation mechanism of various hydrophilic monolithic stationary phases is also reviewed.
E. F. Maksimova, E. G. Vlakh, T. B. Tennikova
Journal of Chromatography A, 1218 (2011) 2425-2431
A series of macroporous monolithic methacrylate-based materials was synthesized by in situ free radical UV-initiated copolymerization of functional monomers, such as glycidyl methacrylate (GMA), butyl methacrylate (BuMA), 2-aminoethyl methacrylate (AEMA), 2-hydroxyethyl methacrylate (HEMA) and 2-cyanoethyl methacrylate (CEMA), with crosslinking agent, namely, ethylene glycol dimethacrylate (EDMA). The materials obtained were applied as the stationary phases in simple and robust technique – planar chromatography (PLC). The method of separation layer fabrication representing macroporous polymer monolith bound to the specially prepared glass surface was developed and optimized. The GMA–EDMA and BuMA–EDMA matrixes were successfully applied for the separation of low molecular weight compounds (the mixture of several dies), as well as poly(vinylpyrrolidone) and polystyrene homopolymers of different molecular weights using reversed-phase mechanism. The materials based on copolymers AEMA–HEMA–EDMA and CEMA–HEMA–EDMA were used for normal-phase PLC separation of 2,4-dinitrophenyl amino acids and polystyrene standards.
R. D. Arrua, C. I. Alvarez Igarzabal
J. Sep. Sci. 2011, 34, 1974–1987
In the early 1990s, three research groups simultaneously developed continuous macroporous rod-shaped polymeric systems to eliminate the problem of flow through the interparticle spaces generally presented by the chromatography columns that use particles as filler. The great advantage of those materials, forming a continuous phase rod, is to increase the mass transfer by convective transport, as the mobile phase is forced to go through all means of separation, in contrast to particulate media where the mobile phase flows through the interparticle spaces. Due to their special characteristics, the monolithic polymers are used as base-supports in different separation techniques, those chromatographic processes being the most important and, to a greater extent, those involving the separation of biomolecules as in the case of affinity chromatography. This mini-review reports the contributions of several groups to the development of macroporous monoliths and their modification by immobilization of specific ligands on the products for their application in affinity chromatography.
H. Shirataki, C. Sudoh, T. Eshima, Y. Yokoyama, K. Okuyama
Journal of Chromatography A, 1218 (2011) 2381–2388
It is widely recognized that membrane adsorbers are powerful tools for the purification of biopharmaceutical protein products and for this reason a novel hollow-fiber AEX type membrane adsorber has been developed. The membrane is characterized by grafted chains including DEA ligands affixed to the pore surfaces of the membrane. In order to estimate the membrane performance, (1) dynamic binding capacities for pure BSA and DNA over a range of solution conductivity and pH, (2) virus reduction by flow-through process, and (3) HCP and DNA removal from cell culture, are evaluated and compared with several other anion-exchange membranes. The novel hollow-fiber membrane is tolerant of high salt concentration when adsorbing BSA and DNA. When challenged with a solution containing IgG the membrane has high impurity removal further indicating this hollow-fiber based membrane adsorber is an effective tool for purification of biopharmaceutical protein products including IgG.
A. Trauner, M. H. Bennett, H. D. Williams
PLoS ONE 6(2): e16273. doi:10.1371/ journal.pone.0016273
We report the development of a rapid chromatographic method for the isolation of bacterial ribosomes from crude cell lysates in less than ten minutes. Our separation is based on the use of strong anion exchange monolithic columns. Using a simple stepwise elution program we were able to purify ribosomes whose composition is comparable to those isolated by sucrose gradient ultracentrifugation, as confirmed by quantitative proteomic analysis (iTRAQ). The speed and simplicity of this approach could accelerate the study of many different aspects of ribosomal biology.
2010
P. Gagnon
BioProcess International, Nov 2010
The enabling value of monoliths was strongly in evidence at the 4th International Monolith Symposium, held 29 May – 2 June in the Adriatic resort city of Portoroz, Slovenia. Forty-seven oral presentations and 34 posters highlighted important advances in vaccines, gene therapy, phage therapy for infectious disease, and monoclonal antibodies, as well as continuing advances in the performance of monoliths themselves. As these fields advance in parallel, it becomes increasingly apparent that monoliths offer industrial capabilities substantially beyond traditional methods.
A. Čevdek, M. Franko
Analytical and Bioanalytical Chemistry 398 (2010), 555-562
This work presents a comparison of convective interaction media (CIM) and controlled pore glass (CPG) as solid supports for immunoglobulin antibodies used in bioanalytical detection of allergens in foodstuffs. A flow-injection manifold with highly sensitive thermal lens spectrometric detection was used for this purpose. Using beta-lactoglobulin, a milk allergen, as a model analyte, CIM disc supports had a higher linear range (0.2–3.5 μg L-1), better reproducibility (intra-day RSD = 1%, inter-day RSD = 10%), lower consumption of reagents, and better immunocolumn stability (1 month, over 240 injections of substrate), while providing comparable LODs (0.1 μg L-1). Application of CIM discs as solid supports in immunocolumns for allergen detection enables fast and sensitive screening of allergens in foodstuffs with sample throughput of up to eight samples per hour.
P. Gagnon
Roadmap to Process Development, issue 3/2010, Sartorius BIA Separations
Introduction
The first two articles in this series addressed column selectivity and capacity. This article discusses how to apply results from these preliminary studies to create fully functional multi-step purification procedures. The principles described here can be applied to proteins, plasmids, or virus particles.
Process modeling represents a nexus at which the theoretical ideals of purification meet the practical limitations of the laboratory, or in less elegant terms: where the rubber meets the road. The key theoretical principle is the notion of developing an orthogonal purification process. Orthogonal means pertaining to right angles. In purification terms, it translates to combining purification methods that are highly complementary to one another. Its value resides in the presumption that different purification methods bind the product by different sites, along with a unique subset of contaminants. The more complementary the methods, the lower the overlap in contaminant subsets, and the higher the purification factor offered by the particular combination of methods.
Attachments
2009
P. Gagnon
Roadmap to Process Development, issue 2/2009, Sartorius BIA Separations
Introduction
Determination of column loading capacity is a critical component of purification process development. Its most obvious link is to process economics, since the more product that can be loaded per unit of media volume, the smaller the column and volume of buffers, and the smaller the process footprint (manufacturing space requirement). But binding capacity is also linked directly to loading conditions, and beyond that, loading is a key determinant of purification performance and reproducibility. In practice, determination of optimal loading is tedious, time consuming, and expensive, especially due to the large amounts of sample it requires. This makes it all the more important to get it right the first time.
The objectives of this article are to highlight the process considerations that pertain to loading, and to provide you with a set of practical tools to determine capacity values that are meaningful in your particular usage context.
Attachments
P. Gagnon
Roadmap to Process Development, issue 1/2009, BIA Separations
Introduction
Commercial purification process development involves harmonizing a complex hierarchy of safety, regulatory, and economic considerations with the unique physicochemical characteristics of the product and the suite of contaminants that must be removed. This can be challenging even with product classes that exhibit fairly consistent chromatographic behavior, such as IgG monoclonal antibodies. It is even more demanding with products that do not support a platform approach. In either case, process development requires detailed knowledge of how the product behaves relative to contaminants within the operating ranges of the methods that may be used in its purification. This knowledge can be obtained only by characterizing product retention experimentally, a process that begins with initial screening. Screening produces the first indications of what methods offer the most promising fractionation capabilities, under what conditions, and in what order different methods may be linked together to yield an integrated multi-step purification procedure.
Attachments
E.G. Vlakh, T.B. Tennikova
Journal of Chromatography A, 1216 (2009) 2637-2650
Monolithic columns were introduced in the early 1990s and have become increasingly popular as efficient stationary phases for most of the important chromatographic separation modes. Monoliths are functionally distinct from porous particle-based media in their reliance on convective mass transport. This makes resolution and capacity independent of flow rate. Monoliths also lack a void volume. This eliminates eddy dispersion and permits high-resolution separations with extremely short flow paths. The analytical value of these features is the subject of recent reviews. Nowadays, among other types of rigid macroporous monoliths, the polymethacrylate-based materials are the largest and most examined class of these sorbents. In this review, the applications of polymethacrylate-based monolithic columns are summarized for the separation, purification and analysis of low and high molecular mass compounds in the different HPLC formats, including micro- and large-scale HPLC modes.
J. L. Ammerman, J. H. Aldstadt III
Microchim Acta (2009) 164:185-196
We describe the development and optimization of a sensitive and selective screening method for the measurement of trace levels of microcystins in surface waters. Several sample preparation techniques were compared, including solid-phase microextraction (SPME), particle-based solid-phase extraction (SPE), and monolith-based SPE. A flow-injection (FI) based approach employing a reversed-phase monolithic SPE column was found to be optimal. Quantification was performed by directly interfacing the FI-based SPE system to an electrospray ionization-mass spectrometer (ESI-MS). To more safely simulate peptidyl toxins such as the microcystins, a model peptide (i.e., angiotensin II) was used for method optimization. Sample loading flow rate and volume, eluent composition, and elution flow rate were optimized. Sample throughput was six samples per hour, a detection limit of 1.31 ng angiotensin II was demonstrated for a linear dynamic range from 1–1,000 ng and 3.4% relative standard deviation (n = 4, 100 ng sample). Sample volumes up to 1,000 ml of surface water could be loaded onto the monolithic SPE disk without exceeding the sorbent’s capacity. Unlike conventional particle-based SPE methods, the monolithic SPE disk does not need to be replaced between samples and could be used indefinitely. The FI-based SPE-ESI-MS method was successfully applied to the determination of microcystin-LR, the most common of the microcystins, in environmental samples and was demonstrated for the direct monitoring of chlorinated drinking water, with trends tracked over a period of eight months.
2008
P. Gagnon
MSS2008
When monoclonal antibodies were first beginning to be commercialized, expression levels over 100 mg/L were considered outstanding, and cell culture was viewed as the bottleneck in manufacturing productivity. Antibody expression levels now commonly exceed 1 g/L and reports of 10 and 15 g/L have been recently announced. Downstream processing is now considered the bottleneck.
In one sense, the bottleneck is artificial. Cell culture production takes about two weeks (not counting preparation of seed stock) and purification takes about a week. In another sense, the bottleneck is real, and a genuine concern. Process time for the protein A capture step from 20,000 L of cell culture supernatant (CCS) commonly requires 72-96 hours. This represents multiple cycles. The long hold time for IgG produced in the early cycles increases the risk of degradation by proteolysis, deamidation, etc. It also increases the risk of contamination.