Tsutomu Arakawa, Pete Gagnon

Journal of Pharmaceutical Sciences 107 (2018) 2297-2305

The concept of cosolvent exclusion was developed by a group of Timasheff's laboratory in 1970-1990 and is currently used widely to explain the effects of a variety of cosolvents on the stability and solubility of macromolecules. Not surprisingly, these concepts have had substantial influence in the fields of formulation, protein folding and unfolding, but they have perhaps more surprisingly found their way into the field of chromatography. A variety of excluded cosolvents have been used to enhance binding and resolution of proteins and other macromolecules in ion exchange, hydroxyapatite, affinity, and hydrophobic interaction chromatography. These cosolvents include salting-out salts, amino acids and polymers, and frequently polyethylene glycol (PEG). A new mode of chromatography, termed “steric exclusion chromatography,” was recently introduced. It employs hydroxylated solid phase surfaces. Steric exclusion of the PEG stabilizes the association of macromolecules with the solid phase. Elution is achieved by reducing the PEG concentration. Magnetic particles are also used in this chromatography. This review summarizes the concepts of preferential cosolvent exclusion and its applications in column chromatography.

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Antonio M. Munoz, Paul Yourik, Vaishnavi Rajagopal, Jagpreet S. Nanda, Jon R. Lorsch, Sarah E. Walker

RNA Biology, 2017, VOL. 14, NO. 2, 188–196

In vitro studies of translation provide critical mechanistic details, yet purification of large amounts of highly active eukaryotic ribosomes remains a challenge for biochemists and structural biologists. Here, we present an optimized method for preparation of highly active yeast ribosomes that could easily be adapted for purification of ribosomes from other species. The use of a nitrogen mill for cell lysis coupled with chromatographic purification of the ribosomes results in 10-fold-increased yield and less variability compared with the traditional approach, which relies on sedimentation through sucrose cushions. We demonstrate that these ribosomes are equivalent to those made using the traditional method in a host of in vitro assays, and that utilization of this new method will consistently produce high yields of active yeast ribosomes.

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Sebastijan Peljhan, Tina Jakop, Dunja Šček, Vid Skvarča, Blaž Goričar, Romina Žabar, Nina Mencin. Electrophoresis 2017 July 20

The plasma-derived IgG used either for diagnostic purpose or intravenous application (in form of IVIG) in various medical therapies is certainly gaining more and more attention on annual basis. Different manufacturing processes are used to isolate immunoglobulins from human plasma. However, a quest for alternative paths in IgG isolation not only requires development of the most efficient isolation process, but also a rapid and reliable analytics to track the purification. Fast and reliable fingerprint based method for characterization of IgG prepared from Cohn I+II+III paste is presented in this paper. The fingerprint method bases on partial separation of proteins in linear gradient on CIMacTM quaternary amine, strong anion exchange group (QA) 0.1 mL column. Partial separation of proteins does not allow simple quantitative analysis of the samples during the IgG isolation from Cohn I+II+III fraction paste, but very accurate qualitative information about the composition of the sample can be obtained in less than 5 min. From the differences in the chromatograms of various samples, the ratio between IgG and impurities in each sample can be easily assessed. The method is suitable for input material control, in-line monitoring of the downstream processing, final control of the products, as well as in stability studies and enables taking fast and accurate decisions during fractionation process.

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V.Rajamanickam, D.Wurm, C.Slouka, C.Herwig, O.Spadiut

Anal Bioanal Chem (2016)

The bacterium Escherichia coli is a well-studied recombinant host organism with a plethora of applications in biotechnology. Highly valuable biopharmaceuticals, such as antibody fragments and growth factors, are currently being produced in E. coli. However, the high metabolic burden during recombinant protein production can lead to cell death, consequent lysis, and undesired product loss. Thus, fast and precise analyzers to monitor E. coli bioprocesses and to retrieve key process information, such as the optimal time point of harvest, are needed. However, such reliable monitoring tools are still scarce to date. In this study, we cultivated an E. coli strain producing a recombinant single-chain antibody fragment in the cytoplasm. In bioreactor cultivations, we purposely triggered cell lysis by pH ramps. We developed a novel toolbox using UV chromatograms as fingerprints and chemometric techniques to monitor these lysis events and used flow cytometry (FCM) as reference method to quantify viability offline. Summarizing, we were able to show that a novel toolbox comprising HPLC chromatogram fingerprinting and data science tools allowed the identification of E. coli lysis in a fast and reliable manner. We are convinced that this toolbox will not only facilitate E. coli bioprocess monitoring but will also allow enhanced process control in the future

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P. Kramberger, U. Lidija, A. Štrancar
Human Vaccines & Immunotherapeutics, 11:4 (2015) 1010-1021

Downstream processing of nanoplexes (viruses, virus-like particles, bacteriophages) is characterized by complexity of the starting material, number of purification methods to choose from, regulations that are setting the frame for the final product and analytical methods for upstream and downstream monitoring. This review gives an overview on the nanoplex downstream challenges and chromatography based analytical methods for efficient monitoring of the nanoplex production.

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D. Buzzi, A. Štrancar

Chimica Oggi-Chemistry Today; Vol 33(1) January/February 2015

The importance of the monitoring of a process all along its steps by means of PAT has been defined by FDA in 2002. How can be defined the product quality and what are the parameters that should be checked by means of different analysis techniques, being focused in particular on the application of high pressure liquid chromatography techniques (HPLC) as high value tool for the process monitoring. From the first introduction of Process Analytical Technology to the "state of the art": how can be PAT implemented in order to ensure the final product quality.

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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.

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Roy N D‘Souza, Ana M Azevedo, M Raquel Aires-Barros, Nika Lendero Krajnc, Petra Kramberger, Maria Laura Carbajal, Mariano Grasselli, Roland Meyer & Marcelo Fernández-Lahore

Vol. 1, No. 5, Pharmaceutical Bioprocessing (2013)

Downstream processing is currently the major bottleneck for bioproduct generation. In contrast to the advances in fermentation processes, the tools used for downstream processes have struggled to keep pace in the last 20 years. Purification bottlenecks are quite serious, as these processes can account for up to 80% of the total production cost. Coupled with the emergence of new classes of bioproducts, for example, virus-like particles or plasmidic DNA, this has created a great need for superior alternatives. In this review, improved downstream technologies, including aqueous two-phase systems, expanded bed adsorption chromatography, convective flow systems, and fibre-based adsorbent systems, have been discussed. These adaptive methods are more suited to the burgeoning downstream processing needs of the future, enabling the cost-efficient production of new classes biomaterials with a high degree of purity, and thereby hold the promise to become indispensable tools in the pharmaceutical and food industries.

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E. Maksimova, E. Vlakh, E. Sinitsyna, T. Tennikova
J. Sep. Sci. 2013, 36, 3741–3749

Ultrashort monolithic columns (disks) were thoroughly studied as efficient stationary phases for precipitation–dissolution chromatography of synthetic polymers. Gradient elution mode was applied in all chromatographic runs. The mixtures of different flexible chain homopolymers, such as polystyrenes, poly(methyl methacrylates), and poly(tert-butylmethacrylates) were separated according to their molecular weights on both commercial poly(styrene-co divinylbenzene).
disks (12 id × 3 mm and 5 × 5 mm) and lab-made monolithic columns (4.6 id × 50 mm) filled with supports of different hydrophobicity. The experimental conditions were optimized to reach fast and highly efficient separation. It was observed that, similar to the separation of monoliths of other classes of (macro)molecules (proteins, DNA, oligonucleotides), the length of column did not affect the peak resolution.
A comparison of the retention properties of the poly(styrene-co-divinylbenzene) diskshaped monoliths with those based on poly(lauryl methacrylate-co-ethylene dimethacrylate), poly(butyl methacrylate-co-ethylene dimethacrylate), and poly(glycidyl methacrylate-co-ethylene dimethacrylate) supports demonstrated the obvious effect of surface chemistry on the resolution factor. Additionally, the results of the discussed chromatographic mode on the fast determination of the molecular weights of homopolymers used in this study were compared to those established by SEC on columns packed with sorbent beads of a similar nature to the monoliths.

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A. G. Lopes

FBP-461, Food and Bioproducts Processing (2014)

As the biopharmaceutical industry matures, the trend towards increased flexibility and productivity, faster time tomarket and greater profitability are driving the replacement of traditional stainless steel equipment by single-use technology (SUT). The use of SUT in the biopharmaceutical industry can significantly impact the manufacturing process efficiency by reducing capital costs, improving plant flexibility, reducing start-up times and costs, and elim-inating both non-value added process steps and the risk of cross-contamination. In addition it significantly reduces process liquid waste, labour costs and on-site quality and validation requirements. This paper reviews the current status of the technology and the impact of SUT in the biopharmaceutical industry, with the aim of identifying the challenges and limitations that still need to be addressed for further adoption of these technologies. Even tough SUT has a multitude of systems available, its components and assemblies have little standardisation as well as alack of harmonised tests and procedures among suppliers, with an array of guidelines from a variety of sourcesand no critical limits have been established. In addition, the use of SUT has new validation requirements such as leachables and extractables, suppliers’ qualification and SUT lot-to-lot variability. The lack of expertise in these areas and the new training requirements when using SUT also need to be addressed. To date the majority of the avail-able literature regarding SUT is found in trade journals where typically suppliers are the main contributors. There is still a lack of engagement of the academic community, which contributes to very limited scientific proof from independent peer-reviewed research to support performance of SUT. This is particularly the case during operation and integrity testing of SUT, during for example on-site testing, transport and disposal. Another area where no work has been undertaken concerns conceptual approaches for facility clean-room requirement and appropriate layout design using SUT. Investment in novel technologies, research, standardisation and training is paramount for further development and implementation of SUTs across all sectors of the biopharmaceutical industry.

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K. Pflegerl, A. Podgornik, E. Berger, A. Jungbauer

Biotechnology and Bioengineering 79 (2002) 733-740

Screening of peptide ligands for affinity chromatography usually involves incubation with the target protein in a batch system. In an additional step, peptides with fast binding kinetics have to be selected in respect to satisfactory performance under flow conditions on a support ensuring optimal three-dimensional presentation of the peptide. We have developed a rapid screening system based on peptide synthesis and screening on CIM® disks. The disk size was minimized to fit into microplates usually applied for solid-phase extraction. In combination with a vacuum manifold, semi-automated peptide synthesis and screening for binding to a target protein under simulated chromatography conditions are possible. Various analytical methods can be applied for parallel and automated determination of the quantity, integrity, or activity of the target protein in the flow through or bound to the affinity support. This system also allows parallel screening for suitable chromatographic conditions like running buffer, washing, and elution conditions.

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H. LeThanh, B. Lendl

Analytica Chimica Acta 422 (2000) 63–69

A fully automated method for the rapid determination of organic acids (citric-, malic- and tartaric acid) and sugars (glucose, fructose, and sucrose) in soft drinks by sequential injection Fourier transform infrared (FTIR) spectroscopy is presented. A convective interaction media (CIM) disc carrying quaternary amino moieties was added as a solid phase extraction column to the flow system. Upon injection of a sample the organic acids were completely retained on the CIM disc whereas sugars passed to the flow cell. The organic acids were subsequently eluted by injection of an alkaline (pH 8.5) 1 M sodium chloride solution and recorded in their fully deprotonated form as a second flow injection peak. In both cases, the FTIR spectra corresponding to the peak maxima were selected for data evaluation. Two partial least squares models, one for sugars and the other for organic acids, were constructed based on the analysis of standards containing all six analytes. The developed method was applied to natural samples yielding results which were in good agreement with those obtained by an external reference method (enzymatic test kits). Deviations in the results were 3.4. and 4.1% for citric and malic acid and ranged from 4.7–5.1% for the sugars. The developed method is characterized by its short analysis time, experimental simplicity and its potential applications in routine analysis and process control.

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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.

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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.

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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.

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M. Žorž

ChemieXtra 3/2012 pp 30-33

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.

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D. G. Glover, M. Barut, A. Podgornik, M. Peterka, A. Štrancar

BioProcess International, Oct 2004, 58-63

The sequencing of the human genome and the rise of proteomics have increased the numbers of potential therapeutic targets. Biotechnology companies need to increase productivity, decrease discovery and production costs, and use technologies that easily transfer across departments if they wish to remain competitive. The most important tools are those for separation (purification) of target substance(s). They should be easy to use and offer an identical performance and purification profile no matter where they are implemented — in discovery, production, or quality assurance (QA).

CIM Convective Interaction Media short monolithic columns are just such a unifying technology. Produced in shapes and sizes from microliter to liter scale, they represent an evolutionary approach to meeting biochromatographic separation requirements in research and product development. Able to withstand 1 M NaOH with no loss of capacity or resolution, these easily scalable columns have been optimized for analysis and cGMP production of complex biomolecules ranging from oligonucleotides and plasmid DNA (pDNA) to proteins and viruses.

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I. Mihelič, T. Koloini, A. Podgornik, M. Barut, A. Štrancar

Acta Chim. Slov. 2001, 48, 551-564

Monolithic stationary phases are becoming very important field of liquid chromatography. Methacrylate based CIM Convective Interaction Media® monolithic columns and are produced via radical polymerization, which results in a rigid and chemically very stable porous monolithic structure. Some characteristics of small-scale monolithic columns and an example of extremely fast separation of biomolecules are presented in the paper. However, the preparation of large and homogeneous monolithic columns represents a big problem, because the heat released during the polymerization causes distortion of the monolithic structure. A mathematical model employing the polymerization kinetics for the prediction of the temperature profiles and a comparison with the experimental results is presented with the emphasis on the conversion and the rate od the heat release profiles. Finally, the characteristics of a large-scale monolithic column are presented.

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P. Kramberger, D. Glover, A. Štrancar

American Biotechnology Laboratory, 2003, 27-28

Research in molecular and cell biology has shown that macromolecules such as pDNA and virus vectors, together called nanoparticles, have the potential to assist in the prevention and treatment of some human diseases. The most important step in their production is the downstream processing (isolation and cleaning). Precipitation, ultrafiltration, and LC techniques are the most widely used for these purposes, but only LC can purify the product so that it is recognized as safe for therapeutic use.

Apart from reduced yield, downstream processing can cause minor or even major modifications in the structure of the biomolecule. Usually these modifications do not affect the activity of the product, but may change its antigenicity. Minimizing these changes to maintain product safety is the main objective in the downstream processing of nanoparticles. For the efficient isolation of labile biomolecules, liquid chromatographic supports should provide fast and efficient separation in order to decrease biomolecule degradation; have high, preferably flow-unaffected capacity and resolution; and exhibit low backpressure. They should be stable, even if harsh conditions are applied during sanitation (e.g., 1 MNaOH), and should be easy to handle and operate.

CIM® (Convection Interaction Media) monolithic chromatographic columns (BIA Separations, Ljubljana, Slovenia) meet all of these requirements. This application note will discuss the columns and their use on human models and plant viruses and pDNA.

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P. Gagnon

Roadmap to Process Development, issue 3/2010, Bia Separations

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.

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