Publications
2019
This discussion introduces new analytical approaches that enable in-line chromatographic detection of exosomes. One approach can discriminate extracellular vesicles from nonvesicle contaminants, and one potentially can discriminate exosomes from other vesicles. Examples illustrate how they enable development of more effective and better documented purification methods. The special qualifications of monolithic chromatography media for exosome purification are discussed. New process tools designed to accommodate some of the special challenges of exosome purification are introduced.
Feel free to download the eBook by clicking on the link to attachment below.
Attachments
2018
The purpose of this book is to provide you with a guide to developing monoclonal antibody purification procedures taht meet the requirements of both research and commercial applications. It is based on successful purifications developed for over 250 monoclonal-based products, addressing a wide range of diagnostic and therapeutic applications. it is supported by nearly 1000 citations from the scientific literature and enriched by the insights of skilled practitioners from throught the industry. It incorporates over 100 figures and tables to illustrate key concepts.
Attachments
Miladys Limonta, Lourdes Zumalacarregui, Urska Vidic, Nika Lendero Krajnc
The main component of the Center for Genetic Engineering and Biotechnology (CIGB) candidate vaccine against Hepatitis C virus (HCV) is the pIDKE2 plasmid. The current designed downstream process for the production of pIDKE2 fulfils all regulatory requirements and renders the required quantities of pharamceutical-grade plasmid DNA (pDNA)with 95% purity. The advantages of this procedure include high plasmid purity and the elimination of undesirable additives. such as toxic organic extractants and animal-derived enzymes. However, yields and consequently the productivity of the process are low. Previous work demonstrated that the most critical step of the process is the reverse phase chromatography, where conventional porous particle resins are used. Therefore, to increase the process productivity alternative technologies such as membranes and chromatographic monoliths were tested as alternative options for this critical step. Here, a comparison between the behaviours of CIM® C4-HLD and Sartobind phenyl matrices was performed.
Attachments
2017
Marina Naldi, Urh Černigoj, Ales Štrancar, Manuela Bartolini
Reducing experimental variability, limiting contamination and increasing automation are essential goals in the development of reliable analytical platforms for mass spectrometry (MS)-based proteomics. In this work novel trypsin-based monolithic immobilized enzyme reactors (tryp-IMERs), obtained by covalent immobilization on convective interaction media (CIMac™) analytical columns (5 mm×5.2 mm I.D.), were developed. Notwithstanding the small dimensions, column format allowed the insertion in common high performance liquid chromatography (HPLC) systems, thus avoiding the use of expensive micro- or nano-platforms. Monolith pore diameter and surface chemistry were optimized to achieve high digestion efficiency even with high molecular weight proteins and to avoid protein/peptide adsorption, peak broadening and sample loss. A full characterization of the tryp-IMERs was undertaken to select the best protocol for preparation and type of trypsin. Optimization of the operational and storage conditions was carried out by an off-line approach. On-line studies were performed by setting a multidimensional analytical platform, which included the tryp-IMER, a trapping column, an analytical C4 column and a high resolution hybrid mass spectrometer (ESI-Q-TOF). In the optimized conditions rapid protein digestion (90 ± 9 s), high protein coverage (≥60%) and high score values were achieved for five selected sample proteins (cytochrome c, myoglobin and albumins from different sources) differing in molecular size, isoelectric point and accessibility to cleavage sites as well as for a protein mixture of 200 ng. The best performing tryp-IMERs showed high sensitivity down to the pmole level. The platform also resulted suitable for the analysis of high-molecular weight proteins such as a pool of human immunoglobulins G (hIgG) and for the high molecular weight fraction of human plasma proteins, which were digested in less than two minutes to an extent similar to that achieved by overnight incubation in a classical in solution protocol. Finally, underestimated key procedural issues were also highlighted during the study. Such aspects are of general interest both for tryp-IMER users and tryp-IMER developers.
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.
2016
M. Naldi, M. Baldassarre, M. Domenicali, F. A. Giannone, M. Bossic, J. Montomoli,T. D. Sandahl, E. Glavind, H. Vilstrup, P. Caraceni, C. Bertucci
Journal of Pharmaceutical and Biomedical Analysis, Volume 122 (2016) 141-147
Human serum albumin (HSA) is the most abundant plasma protein, endowed with several biological properties unrelated to its oncotic power, such as antioxidant and free-radicals scavenging activities, binding and transport of many endogenous and exogenous substances, and regulation of endothelial function and inflammatory response. These non-oncotic activities are closely connected to the peculiarly dynamic structure of the albumin molecule. HSA undergoes spontaneous structural modifications, mainly by reaction with oxidants and saccharides; however, patients with cirrhosis show extensive post-transcriptional changes at several molecular sites of HSA, the degree of which parallels the severity of the disease. The present work reports the development and application of an innovative LC–MS analytical method for a rapid and reproducible determination of the relative abundance of HSA isoforms in plasma samples from alcoholic hepatitis (AH) patients. A condition of severe oxidative stress, similar to that observed in AH patients, is associated with profound changes in circulating HSA microheterogeneity. More interestingly, the high resolution provided by the analytical platform allowed the monitoring of novel oxidative products of HSA never reported before.
Karla Mayolo-Deloisa, Jose Gonzalez-Valdez, and Marco Rito-Palomares
Biotechnol. Prog., 2016, Vol. 00, No. 00
Protein hydrophobicity can be modified after a PEGylation process. However, hydrophobic interaction chromatography (HIC) has been used to separate PEGylation reaction products less frequently than other techniques. In this context, chromatographic monoliths represent a good alternative to continue exploring the separation of PEGylated proteins with HIC. In this work, the separation of PEGylated proteins using C4 A monolith as well as Toyopearl Butyl 650C and Butyl Sepharose was analyzed. Three proteins were used as models: RNase A, b-lactoglobulin, and lysozyme. All proteins were PEGylated in the Nterminal amino groups with 20 kDa methoxy poly(ethylene glycol) propionaldehyde. The concentration of ammonium sulfate (1 M) used was the same for all stationary phases. The results obtained demonstrated that the C4 A monolith could better resolve all protein PEGylation reaction mixtures, since the peaks of mono- and di-PEGylated proteins can be clearly distinguished in the chromatographic profiles. On the contrary, while using Butyl Sepharose media only the PEGylation reaction mixtures of RNase A could be partially separated at 35 and 45 CVs. PEGylated proteins of b-lactoglobulin and lysozyme could not be resolved when Toyopearl Butyl 650C and Butyl Sepharose were used. It is then clear that monoliths are an excellent choice to explore the purification process of PEGylated proteins exploiting the advantages of HIC.
Tarasova, I. A., Lobas, A. A., Černigoj, U., Solovyeva, E. M., Mahlberg, B., Ivanov, M. V., Panić-Janković, T., Nagy, Z., Pridatchenko, M. L., Pungor, A., Nemec, B., Vidič, U., Gašperšič, J., Krajnc, N. L., Vidič, J., Gorshkov, M. V. and Mitulović, G. ELECTROPHORESIS. Accepted Author Manuscript. doi:10.1002/elps.201500489.
Affinity depletion of abundant proteins such as human serum albumin (HSA) is an important stage in routine sample preparation prior to tandem mass spectrometry (MS/MS) analysis of biological samples with high range of concentrations. Due to the charge competition effects in electrospray ion source that results in discrimination of the low-abundance species, as well as limited dynamic range of MS/MS, restricted typically by three orders of magnitude, the identification of low-abundance proteins becomes a challenge unless the sample is depleted from high concentration compounds. This dictates a need for developing efficient separation technologies allowing fast and automated protein depletion. In this study we performed evaluation of a novel immunoaffinity-based CIMac depletion column with specificity to HSA (CIMac-αHSA). Because of the convective flow-through channels, the polymethacrylate CIMac monoliths afford flow rate-independent binding capacity and resolution that results in relatively short analysis time compared with traditional chromatographic supports. Seppro IgY14 depletion kit was used as a benchmark to control the results of depletion. Bottom-up proteomic approach followed by label-free quantitation using normalized spectral indexes were employed for protein quantification in G1/G2 and Cleavage/Blastocyst IVF culture media widely utilized in clinics for embryo growth in vitro. The results revealed approximately equal HSA level of 100% ± 25% in albumin-enriched fractions relative to the non-depleted samples for both CIMac-αHSA column and Seppro kit. In the albumin-free fractions concentrated 5.5-fold by volume, serum albumin was identified at the levels of 5 to 30% and 20 to 30% for the CIMac-αHSA and Seppro IgY14 spin columns, respectively.
U. Cernigoj, U. Vidic, B. Nemec, J. Gaspersic, J. Vidic,N. L. Krajnc, A. Strancar, A. Podgornik. Journal of Chromatography A, 1464 (2016) 72–78
We investigated effect of immobilization procedure and monolith structure on chromatographic performance of methacrylate monoliths bearing affinity ligands. Monoliths of different pore size and variousaffinity ligands were prepared and characterized using physical and chromatographic methods. When testing protein A monoliths with different protein A ligand densities, a significant non linear effect ofligand density on dynamic binding capacity (DBC) for IgG was obtained and accurately described by Langmuir isotherm curve enabling estimation of protein A utilization as a function of ligand density. Maximal IgG binding capacity was found to be at least 12 mg/mL exceeding theoretical monolayer adsorption value of 7.8 mg/mL assuming hexagonal packing and IgG hydrodynamic diameter of 11 nm. Observed discrepancy was explained by shrinkage of IgG during adsorption on protein A experimentally determined through calculated adsorbed IgG layer thickness of 5.4 nm from pressure drop data. For monoliths with different pore size maximal immobilized densities of protein A as well as IgG dynamic capacitylinearly correlates with monolith surface area indicating constant ligand utilization. Finally, IgGs toward different plasma proteins were immobilized via the hydrazide coupling chemistry to provide oriented immobilization. DBC was found to be flow independent and was increasing with the size of bound protein. Despite DBC was lower than IgG capacity to immobilized protein A, ligand utilization was higher.
2015
A.M. Almeida, J.A. Queiroz, F. Sousa, A. Sousa
Journal of Chromatography B, 978–979 (2015) 145–150
The progress of DNA vaccines is dependent on the development of suitable chromatographic procedures to successfully purify genetic vectors, such as plasmid DNA. Human Papillomavirus is associated with the development of tumours due to the oncogenic power of E6 and E7 proteins, produced by this virus. The supercoiled HPV-16 E6/E7 plasmid-based vaccine was recently purified with the arginine monolith, with 100% of purity, but only 39% of recovery was achieved. Therefore, the present study describes the application of experimental design tools, a newly explored methodology in preparative chromatography, in order to improve the supercoiled plasmid DNA recovery with the arginine monolith, maintaining the high purity degree. In addition, the importance and influence of pH in the pDNA retention to the arginine ligand was also demonstrated. The Composite Central Face design was validated and the recovery of the target molecule was successfully improved from 39% to 83.5%, with an outstanding increase of more than double, while maintaining 100% of purity.
Zunyang Ke, Yu Wang and Zhongming Li
Anion-exchange chromatography is a key capture step in downstream processing plasmid DNA (pDNA). Separation of pDNA using traditional particle-based anion-exchange supports is usually slow and has a low capacity for pDNA due to steric exclusion effects. Due to convective mass transfer properties, and large flow-through channels for binding large biomolecules, monoliths have been shown to provide a fast and efficient alternative for pDNA purification. This study describes the use of monoliths for purification of a therapeutic pDNA vaccine against multidrug resistant tuberculosis (MDR TB).
Attachments
L. Hernandez, D. Stewart, L. Zumalacarregui, D. Amaro
Chinese Journal of Chromatography A, 1000-8713 (2015) 642-646
Affinity and ion exchange conventional chromatography have been used to capture erythropoietin (EPO) from mammalian cell culture supernatant. Currently, chromatographic adsorbent perfusion is available, however a limited number of applications have been found in the literature. In this work, three anion exchange chromatographic supports (gel, membrane and monolithic) were evaluated in the capture step of the recombinant erythropoietin purification process. The influences of load and flow rate on each support performance were analyzed. Also the purity of the EPO molecules was determined. A productivity analysis, as a decision tool for larger scale implementation, was done. As a conclusion, the evaluated supports are technically suitable to capture EPO with adequate recovery and good purity. However, the monolithic column admits high operating velocity, showing the highest adsorption capacity and productivity.
Urh Černigoj, Urška Martinuč, Sara Cardoso, Rok Sekirnik, Nika Lendero Krajnc, Aleš Štrancar
Sample displacement chromatography (SDC) is a chromatographic technique that utilises different rela-tive binding affinities of components in a sample mixture and has been widely studied in the context ofpeptide and protein purification. Here, we report a use of SDC to separate plasmid DNA (pDNA) isoformsunder overloading conditions, where supercoiled (sc) isoform acts as a displacer of open circular (oc) orlinear isoform. Since displacement is more efficient when mass transfer between stationary and mobilechromatographic phases is not limited by diffusion, we investigated convective interaction media (CIM)monoliths as stationary phases for pDNA isoform separation. CIM monoliths with different hydrophobic-ities and thus different binding affinities for pDNA (CIM C4 HLD, CIM-histamine and CIM-pyridine) weretested under hydrophobic interaction chromatography (HIC) conditions. SD efficiency for pDNA isoformseparation was shown to be dependent on column selectivity for individual isoform, column efficiencyand on ammonium sulfate (AS) concentration in loading buffer (binding strength). SD and negative modeelution often operate in parallel, therefore negative mode elution additionally influences the efficiencyof the overall purification process. Optimisation of chromatographic conditions achieved 98% sc pDNAhomogeneity and a dynamic binding capacity of over 1 mg/mL at a relatively low concentration of AS.SDC was successfully implemented for the enrichment of sc pDNA for plasmid vectors of different sizes,and for separation of linear and and sc isoforms, independently of oc:sc isoform ratio, and flow-rate used.This study therefore identifies SDC as a promising new approach to large-scale pDNA purification, whichis compatible with continuous, multicolumn chromatography systems, and could therefore be used toincrease productivity of pDNA production in the future.
Attachments
2014
P. Leblebici, M. E. Leblebici, F. Ferreira-da-Silva, A. E.Rodrigues, L. S. Pais
Journal of Chromatography B, 962 (2014) 89-93
Monolithic columns have attracted significant attention for the purification of large biomolecules. In the present study, a step gradient elution method was evaluated for the separation of human immunoglobulinG (hIgG) into its subclasses on CIM (convective interaction media) r-protein A (recombinant protein A)monolithic column. hIgG was loaded onto the column and bound protein was eluted with a pH gra-dient. The subclass content of the eluted fractions was analyzed by enzyme-linked immunosorbentassay (ELISA). Results showed that separation of IgG3 from the other three subclasses can be success-fully achieved with high selectivity (100%) and throughput on monolithic media. It was also revealedthat enriched fractions of IgG1 and IgG2 could be obtained from purified hIgG in a 28 min long chro-matographic run. Three fractions with high IgG1 content (89.1%, 94.3% and 88.8%) were recovered. Furthermore, IgG2 was enriched to 64% successfully. A rapid step gradient elution scheme without any additives in buffers was proven to obtain enriched preparations of the two important subclasses with high throughput. The separation time can be reduced even more by increasing the flow rate without anyloss in selectivity, which will be beneficial in industrial scale applications.
M. M. St. Amand, B. A. Ogunnaike, A.S. Robinson
Published online in Wiley Online Library, 2013
One major challenge currently facing the biopharmaceutical industry is to understand how MAb microheterogeneity affects therapeutic efficacy, potency, immunogenicity, and clearance. MAb micro-heterogeneity can result from post-translational modifications such as sialylation, galactosylation, C-terminal lysine cleavage, glycine amidation, and tryptophan oxidation, each of which can generate MAb charge variants; such heterogeneity can affect pharmacokinetics (PK) considerably. Implementation of appropriate on-line quality control strategies may help to regulate bioprocesses, thus enabling more homogenous material with desired posttranslational modifications and PK behavior. However, one major restriction to implementation of quality control strategies is the availability of techniques for obtaining on-line or at line measurements of these attributes. In this work, we describe the development of an at-line assay to separate MAb charge variants in near real-time, which could ultimately be used to implement on-line quality control strategies for MAb production. The assay consists of a 2DHPLC method with sequential in-line Protein A and WCX-10 HPLC column steps. To perform the 2D-HPLC assay at-line, the two columns steps were integrated into a single method using
a novel system configuration that allowed parallel flow over column 1 or column 2 or sequential flow from column 1 to column 2. A bioreactor system was also developed such that media samples could be removed automatically from bioreactor vessels during production and delivered
to the 2D-HPLC for analysis. With this at-line HPLC assay, we have demonstrated that MAb microheterogeneity occurs throughout the cell cycle whether the host cell line is grown under different or the same nominal culture conditions.
2013
A. Martinčič, M. Cemazar, G. Sersa, V. Kovač, R. Milačič, J. Ščančar
Talanta 116 (2013)141-148
Conjoint liquid chromatography (CLC) on monolithic convective interaction media (CIM) disks coupled on-line to UV and inductively coupled plasma mass spectrometry (ICP-MS) detectors was used for the first time in speciation analysis of Pt in human serum spiked with Pt-based chemotherapeutics. CIM Protein G and CIM DEAE disks were assembled together in a single housing forming a CLC monolithic column. Such a set-up allows rapid two-dimensional separation by affinity and ion-exchange (IE) modes to be carried out in a single chromatographic run. By applying isocratic elution with Tris–HCl–NaHCO3 buffer (pH 7.4) in the first minute, followed by gradient elution with 1 mol L-1 NH4Cl (pH 7.4) in the next 9 min, immunoglobulins (IgG) were retained by the Protein G disk enabling subsequent separation of unbound Pt from Pt bound to transferrin (Tf) and albumin (HSA) on the CIM DEAE disk. Further elution with acetic acid (AcOH) in the next 3 min allowed separation of Pt associated with IgG. Separated Pt species were quantified by post-column isotope dilution—ICP-MS. Pt recovery on the CLC column was close to 100%. In comparison to commonly applied procedures that involve separation of protein peaks by size-exclusion chromatography (SEC) followed by IE separation of metal-based chemotherapeutic fractions bound to serum proteins, the CLC method developed is much faster and simpler. Its sensitivity (LOQs adequate for quantification of all separated Pt species, lower than 2.4 ng Pt mL-1), good selectivity and method repeatability (RSD±3%) enabled investigation of the kinetics of interaction of Pt-based chemotherapeutics with serum proteins and the distribution of Pt species in spiked human serum. Pt species present in spiked serum were bound preferentially to HSA. The proportion of Pt associated with IgG and Tf was lower than 13%. Cisplatin and especially oxaliplatin react rapidly with serum proteins, while carboplatin much less. The method developed may be reliably applied in preclinical and clinical studies of the kinetics of the interaction and distribution of different metallodrugs with proteins in blood serum.
H. G. Schwelberger, J. Feurle, F. Ahrens
Journal of Neural Transmission 120 (2013) 983-986
Diamine oxidase (DAO) was purified to homogeneity from human seminal plasma by consecutive chromatographic fractionation on heparin-sepharose, phenyl-sepharose, CIM-QA, and Superdex 200. Human seminal plasma DAO behaves electrophoretically similar to DAO proteins from other human tissues and has very similar enzymatic properties with histamine and aliphatic diamines being the preferred substrates as well as significant conversion of polyamines. The cellular source and functional importance of DAO in human semen remain to be determined.
O. Zitka, S. Skalickova, S. Krizkova, M. Vlkova, V. Adam, R. Kizek
Chromatographia (2013) 76:611-619
In this study, we optimized method for the isolation and detection of lactoferrin from human saliva using 3 mm short monolithic disc. We optimized the conditions for separation as flow rate 4 mL min-1 and ionic strength of effluent as 2 M·NaCl. We estimated limit of detection of whole method, which was hyphenated to the Bradford’s assay, down to 100 ng mL-1. The purity of the isolated fractions was verified by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and recovery of isolation was found to be 51 % using minimally processed sample of saliva. Further, we tested the optimized method on group of healthy volunteers (n = 7). We were able to distinguish between the healthy subjects and subject suffering from celiac disease, which reported at least 2.5× higher level of lactoferrin in comparison to healthy ones. The results were correlated with standard enzyme-linked immunosorbent assay (ELISA) kit with obtained correlation coefficient R2 = 0.8446. Analysis of lactoferrin in saliva by monolithic disc and subsequent offline photometric detection is faster and cheaper method compared to ELISA commercial kit. The total analysis of one sample takes.
D. A. Ribeiro, D. F. Passos, H. C. Ferraz, L. R. Castilho
Journal of Chromatography B, 938 (2013) 111-118
Both recombinant and plasma-derived factor IX concentrates are used in replacement therapies for the treatment of haemophilia B. In the present work, the capture step for a recombinant FIX (rFIX) purification process was investigated. Different strong anion-exchange chromatography media (the resins Q Sepharose® FF and Fractogel® TMAE, the monolith CIM® QA and the membrane adsorber Sartobind® Q) were tested for their rFIX binding capacity under dynamic conditions. In these experiments, crude supernatant from CHO cells was used, thus in the presence of supernatant contaminants and mimicking process conditions. The highest dynamic binding capacity was obtained for the monolith, which was then further investigated. To study pseudoaffinity elution of functional rFIX with Ca2+ ions, a design of experiments to evaluate the effects of pH, NaCl and CaCl2 on yield and purification factor was carried out. The effect of pH was not statistically significant, and a combination of no NaCl and 45 mM CaCl2 yielded a good purification factor combined with a high yield of active rFIX. Under these conditions, activity yield of rFIX was higher than the mass yield, confirming selective elution of functional, γ-carboxylated rFIX. Scaling-up of this process 8 fold resulted in very similar process performance. Monitoring of the undesired activated FIX (FIXa) revealed that the FIXa/FIX ratio (1.94%) was higher in the eluate than in the loaded sample, but was still within an acceptable range. HCP and DNA clearances were high (1256 and 7182 fold, respectively), indicating that the proposed process is adequate for the intended rFIX capture step.
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.