Rabies is a viral zoonosis caused by negative-stranded RNA viruses of the Lyssavirus genus. It can affect all mammals including humans. Dogs are the main source of human rabies deaths, contributing up to 99% of all rabies transmissions to humans. Vaccination against rabies is still the sole efficient way to fight against the disease.
Cell culture vaccines are recommended by World Health Organization (WHO) for pre and post exposure prophylaxis; among them Vero cell rabies vaccines which are used worldwide. In this work we studied the purification of inactivated rabies virus produced in Vero cells grown in animal component free conditions, using different methods. Cells were grown in VP-SFM medium in stirred bioreactor, then infected at an MOI of 0.05 with the LP2061 rabies virus strain. Collected harvests were purified by zonal centrifugation, and by chromatography supports, namely the Capto Core 700 and the monolithic CIM-QA column. Generated data were compared in terms of residual DNA level, host cell proteins (HCP) level and the overall recovery yield.
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.
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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.
Laura M. Fischer, Michael W. Wolff, Udo Reichl, Vaccine 2017 July 17
The continuously increasing demand for potent and safe vaccines and the intensifying economic pressure on health care systems underlines the need for further optimization of vaccine manufacturing. Here, we focus on downstream processing of human influenza vaccines, investigating the purification of serum free cell culture-derived influenza virus (A/PR/8/34 H1N1) using continuous chromatography. Therefore, quaternary amine anion exchange monoliths (CIM QA) were characterized for their capacity to capture virus particles from animal cells cultivated in different media and their ability to separate virions from contaminating host cell proteins and DNA. The continuous chromatography was implemented as simulated moving bed chromatography (SMB) in a three zone open loop configuration with a detached high salt zone for regeneration.
SMBs exploiting 10% and 50% of the monoliths’ dynamic binding capacity, respectively, allowed the depletion of >98% of the DNA and >52% of the total protein. Based on the hemagglutination assay (HA assay), the virus yield was higher at 10% capacity use (89% vs. 45%). Both SMB separations resulted in a ratio of total protein to hemagglutinin antigen (based on single radial diffusion assay, SRID assay) below the required levels for manufacturing of human vaccines (less than 100 mg of protein per virus strain per dose). The level of contaminating DNA was five-times lower for the 10% loading, but still exceeded the required limit for human vaccines. A subsequent Benzonase treatment step, however, reduced the DNA contamination below 10 ng per dose. Coupled to continuous cultivations for virus propagation, the establishment of integrated processes for fully continuous production of vaccines seems to be feasible.
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.
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.
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
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.
Alicia T Lucero, Sergio A Mercado, Anamaría C Sánchez,Carolina A Contador, Barbara A Andrews and Juan A Asenjo, Journal of chemical technology and biotechnology, (2017)
BACKGROUND: Gene therapy is a potent alternative for long-lasting inhibition of alcohol consumption. This study compares the purification of a recombinant adenoviral vector serotype 5 (rAdV5) for use in gene therapy against alcoholism using two anion-exchange methods.
RESULTS: Two anion-exchange chromatography methods using fast protein liquid chromatography were compared using a packed-bed column (Q-Sepharose™ XL) and two monolithic columns (CIM™ QA-1 and CIM™ DEAE-1). An improved and reproducible separation of recombinant adenovirus type 5 from cell lysate contaminants was achieved using the two strong anion-exchange columns in a two-step gradient chromatography. Higher adenovirus yields were achieved using the CIM QA-1 tube monolithic column at sample volumes of both 1 and 10 mL compared with the Q-Sepharose XL column. At higher flow rates, the CIM QA-1 tube monolithic column achieved better separation of the target fraction. Process recovery was improved from 28% using the Q-Sepharose XL column to 34% with the CIM QA-1 tube monolithic column quantified as vector genome. Analysis by SDS-PAGE demonstrated a purity of 70% for purified adenovirus using the CIM QA-1 tube monolithic column.
CONCLUSION: This study indicated that the use of a CIM QA-1 tube monolithic column is a better alternative than Q-Sepharose XL, and CIM DEAE-1 tube monolithic columns for the primary purification process of rAdV5 carrying the human aldehyde dehydrogenase-2 antisense gene. This purification strategy has been used as a basis to scale-up a GLP process for the production of material at the National Research Council of Canada to be used in preclinical trials of this gene therapy against alcoholism
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.
David Vincent, Petra Kramberger, Rosana Hudej, Aleš Štrancar, Yaohe Wang,Yuhong Zhou, Ajoy Velayudhan
The purification of large viruses remains an important field of research and development. The development of efficient purification trains is limited by limited analytical methods, as well as by the complexity of large viruses, as well as the high variability in starting material from cell culture. Vaccinia virus holds great potential as an oncolytic and immunotherapeutic vaccine against a broad spectrum of cancers. In this work, monolith-based capture and polishing chromatographic steps for vaccinia virus Lister strain has been developed. Virus produced in CV-1 cells was harvested and passed through a 0.8μm pre-filter before loading onto CIEX, AIEX and HIC CIM monoliths. Without the need for nuclease treatment, up to 99% of the total DNA loaded can be removed from the vaccinia feed stream by the CIM OH monolith, which also reduces the total protein concentration in the product pool to LLOQ levels, and achieves infectious virus recoveries of 90%. Binding capacities of greater than 1x109 pfu of vaccinia per mL of matrix were obtained on both CIM SO3 and CIM OH monoliths. Multiple orthogonal analytical methods have been used to develop process knowledge and understanding.
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.
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.
P. Stepperta, D. Burgstallera, M. Klausbergera, E. Bergerb, P.P. Aguilara, T.A. Schneiderb, P. Krambergerc, A. Toverd, K. Nöbauere, E. Razzazi-Fazelie, A. Jungbauer, Journal of Chromatography A, 1455 (2016)
Enveloped virus-like particles (VLPs) are increasingly used as vaccines and immunotherapeutics. Frequently, very time consuming density gradient centrifugation techniques are used for purification ofVLPs. However, the progress towards optimized large-scale VLP production increased the demand for fast, cost efficient and scaleable purification processes. We developed a chromatographic procedure for purification of HIV-1 gag VLPs produced in CHOcells. The clarified and filtered cell culture supernatant was directly processed on an anion-exchange monolith. The majority of host cell impurities passed throughthe column, whereas the VLPs were eluted by a linear or step salt gradient; the major fraction of DNA waseluted prior to VLPs and particles in the range of 100–200nm in diameter could be separated into two fractions. The earlier eluted fraction was enriched with extracellular particles associated to exosomes or microvesicles, whereas the late eluting fractions contained the majority of most pure HIV-1 gag VLPs. DNA content in the exosome-containing fraction could not be reduced by Benzonase treatment which indicated that the DNA was encapsulated. Many exosome markers were identified by proteomic analysisin this fraction. We present a laboratory method that could serve as a basis for rapid downstream processing of enveloped VLPs. Up to 2000 doses, each containing 1×109 particles, could be processed witha 1mL monolith within 47 min. The method compared to density gradient centrifugation has a 220-fold improvement in productivity.
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.
I. T. Akmacic,B. Nemec,U. Vidic, S. Malcic, K. Miklic, U. Cernigoj, J. Vidic, N. L. Krajnc, A. Strancar, G. Lauc, T. L. Rovis, M. P. Bakovic. Croat. Chem. Acta 2016, 89(2)
Changes in protein glycosylation are related to different diseases and have a potential as diagnostic and prognostic disease biomarkers. Transferrin (Tf) glycosylation changes are common marker for congenital disorders of glycosylation. However, biological interindividual variability of Tf N-glycosylation and genes involved in glycosylation regulation are not known. Therefore, high-throughput Tf isolation method and large scale glycosylation studies are needed in order to address these questions. Due to their unique chromatographic properties, the use of chromatographic monoliths enables very fast analysis cycle, thus significantly increasing sample preparation throughput. Here, we are describing characterization of novel immunoaffinity-based monolithic columns in a 96-well plate format for specific highthroughput purification of human Tf from blood plasma. We optimized the isolation and glycan preparation procedure for subsequent ultra performance liquid chromatography (UPLC) analysis of Tf N-glycosylation and managed to increase the sensitivity for approximately three times compared to initial experimental conditions, with very good reproducibility.
J. Transfiguracion, A. P. Manceur, E. Petiot, C. M. Thompson, A. A. Kamen
The influenza virus continuously undergoes antigenic evolution requiring manufacturing, validation and release of new seasonal vaccine lots to match new circulating strains. Although current production processes are well established for manufacturing seasonal inactivated influenza vaccines, significant limitations have been underlined in the case of pandemic outbreaks. The World Health Organization called for a global pandemic influenza vaccine action plan including the development of new technologies. A rapid and reliable method for the quantification of influenza total particles is crucially needed to support the development, improvement and validation of novel influenza vaccine manufacturing platforms. This work presents the development of an ion exchange-high performance liquid chromatography method for the quantification of influenza virus particles. The method was developed using sucrose cushion purified influenza viruses A and B produced in HEK 293 suspension cell cultures. The virus was eluted in 1.5 M NaCl salt with 20 mM Tris–HCl and 0.01% Zwittergent at pH 8.0. It was detected by native fluorescence and the total analysis time was 13.5 min. A linear response range was established between 1 × 109 and 1 × 1011 virus particle per ml (VP/ml) with a correlation coefficient greater than 0.99. The limit of detection was between 2.07 × 108 and 4.35 × 109 whereas the limit of quantification was between 6.90 × 108 and 1.45 × 1010 VP/ml, respectively. The coefficient of variation of the intra- and inter-day precision of the method was less than 5% and 10%. HPLC data compared well with results obtained by electron microscopy, HA assay and with a virus counter, and was used to monitor virus concentrations in the supernatant obtained directly from the cell culture production vessels. The HPLC influenza virus analytical method can potentially be suitable as an in-process monitoring tool to accelerate the development of processes for the manufacturing of influenza vaccines.
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.