Publications
2015
J. Transfiguracion, A. P. Manceur, E. Petiot, C. M. Thompson, A. A. Kamen
Vaccine (2014)
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.
J-P Pirnay et al.
Pharm Res, Springer, 14 Jan 2015
The worldwide antibiotic crisis has led to a renewed interest in phage therapy. Since time immemorial phages control bacterial populations on Earth. Potent lytic phages against bacterial pathogens can be isolated from the environment or selected from a collection in a matter of days. In addition, phages have the capacity to rapidly overcome bacterial resistances, which will inevitably emerge.
To maximally exploit these advantage phages have over conventional drugs such as antibiotics, it is important that sustainable phage products are not submitted to the conventional long medicinal product development and licensing pathway. There is a need for an adapted framework, including realistic production and quality and safety requirements, that allows a timely supplying of phage therapy products for 'personalized therapy' or for public health or medical emergencies.
This paper enumerates all phage therapy product related quality and safety risks known to the authors, as well as the tests that can be performed to minimize these risks, only to the extent needed to protect the patients and to allow and advance responsible phage therapy and research.
J. Ruscic, I. Gutiérrez-Aguirre, M. Tusek Znidaric, S. Kolundzija, A. Slana, M. Barut, M. Ravnikar, M. Krajacic
Journal of Chromatography A, 1388 (2015) 69–78
The emergence of next-generation "deep" sequencing has enabled the study of virus populations with much higher resolutions. This new tool increases the possibility of observing mixed infections caused by combinations of plant viruses, which are likely to occur more frequently than previously thought. The bio-logical impact of co-infecting viruses on their host has yet to be determined and fully understood, and the first step towards reaching this goal is the separation and purification of individual species. Ion-exchange monolith chromatography has been used successfully for the purification and concentration of different viruses, and number of them have been separated from plant homogenate or bacterial and eukaryoticlysate. Thus, the question remained as to whether different virus species present in a single sample could be separated. In this study, anion-exchange chromatography using monolithic supports was optimized for fast and efficient partial purification of three model plant viruses: Turnip yellow mosaic virus, Tomato bushy stunt virus, and Tobacco mosaic virus. The virus species, as well as two virus strains, were separated from each other in a single chromatographic experiment from an artificially mixed sample. Based on A260/280 ratios, we were able to attribute specific peaks to a certain viral morphology/structure (icosa-hedral or rod-shaped). This first separation of individual viruses from an artificially prepared laboratory mixture should encourage new applications of monolithic chromatographic supports in the separation of plant, bacterial, or animal viruses from all kinds of mixed samples.
M. Zaveckas, S. Snipaitis, H. Pesliakas, J. Nainys, A. Gedvilaite
Journal of Chromatography B, 991 (2015) 21–28
Diseases associated with porcine circovirus type 2 (PCV2) infection are having a severe economic impacton swine-producing countries. The PCV2 capsid (Cap) protein expressed in eukaryotic systems self-assemble into virus-like particles (VLPs) which can serve as antigens for diagnostics or/and as vaccinecandidates. In this work, conventional adsorbents as well as a monolithic support with large pore sizeswere examined for the chromatographic purification of PCV2 Cap VLPs from clarified yeast lysate. QSepharose XL was used for the initial separation of VLPs from residual host nucleic acids and some hostcell proteins. For the further purification of PCV2 Cap VLPs, SP Sepharose XL, Heparin Sepharose CL-6Band CIMmultus SO3 monolith were tested. VLPs were not retained on SP Sepharose XL. The purity of VLPsafter chromatography on Heparin Sepharose CL-6B was only 4–7% and the recovery of VLPs was 5–7%.Using ion-exchange chromatography on the CIMmultus SO3 monolith, PCV2 Cap VLPs with the purityof about 40% were obtained. The recovery of VLPs after chromatography on the CIMmultus SO3 mono-lith was 15–18%. The self-assembly of purified PCV2 Cap protein into VLPs was confirmed by electronmicroscopy. Two-step chromatographic purification procedure of PCV2 Cap VLPs from yeast lysate wasdeveloped using Q Sepharose XL and cation-exchange CIMmultus SO3 monolith.
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.
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.
M. M. Segura, M. Puig, J. Piedra, S. Miravet
Adenovirus: Methods and protocols, Methods in Molecular Biology, vol. 1089
Adenovirus vectors are efficient gene delivery tools. A major caveat with vectors derived from common human adenovirus serotypes is that most adults are likely to have been exposed to the wild-type virus and exhibit active immunity against the vectors. This preexisting immunity limits their clinical success. Strategies to circumvent this problem include the use of nonhuman adenovirus vectors. Vectors derived from canine adenovirus type 2 (CAV-2) are among the best-studied representatives. CAV-2 vectors are particularly attractive for the treatment of neurodegenerative disorders. In addition, CAV-2 vectors have shown great promise as oncolytic agents in virotherapy approaches and as vectors for recombinant vaccines. The rising interest in CAV-2 vectors calls for the development of scalable GMP compliant production and purification strategies. A detailed protocol describing a complete scalable downstream processing strategy for CAV-2 vectors is reported here. Clarification of CAV-2 particles is achieved by microfiltration. CAV-2 particles are subsequently concentrated and partially purified by ultrafiltration–diafiltration. A Benzonase® digestion step is carried out between ultrafiltration and diafiltration operations to eliminate contaminating nucleic acids. Chromatography purification is accomplished in two consecutive steps. CAV-2 particles are first captured and concentrated on a propyl hydrophobic interaction chromatography column followed by a polishing step using DEAE anion exchange monoliths. Using this protocol, high-quality CAV-2 vector preparations containing low levels of contamination with empty viral capsids and other inactive vector forms are typically obtained. The complete process yield was estimated to be 38–45 %.
F. W. Krainer, R. Pletzenauer, L. Rossetti, C. Herwig, A. Glieder, O. Spadiut
Protein Expression and Purification 95 (2014) 104–112
The plant enzyme horseradish peroxidase (HRP) is used in several important industrial and medical applications, of which especially biosensors and diagnostic kits describe an emerging field. Although there is an increasing demand for high amounts of pure enzyme preparations, HRP is still isolated from the plant as a mixture of different isoenzymes with different biochemical properties. Based on a recent next generation sequencing approach of the horseradish transcriptome, we produced 19 individual HRP isoenzymes recombinantly in the yeast Pichia pastoris. After optimizing a previously reported 2-step purification strategy for the recombinant isoenzyme HRP C1A by substituting an unfavorable size exclusion chromatography step with an anion exchange step using a monolithic column, we purified the 19 HRP isoenzymes with varying success. Subsequent basic biochemical characterization revealed differences in catalytic activity, substrate specificity and thermal stability of the purified HRP preparations. The preparations of the isoenzymes HRP A2A and HRP A2B were found to be highly interesting candidates for future applications in diagnostic kits with increased sensitivity.
M.-C. Claudepierrea, et al.
Journal of Virology, February 2014
To identify novel stimulators of the innate immune system, we constructed a panel of eight HEK293-cells lines, double-positive for human Toll-like receptors (TLR) and a NF-κB-inducible reporter gene. Screening a large variety of compounds and cellular extracts detected a TLR3 activating compound in a microsomal yeast extract. Fractionation of this extract identified a RNA molecule of 4.6 kb, named Nucleic Acid Band 2 (NAB2) that was sufficient to confer the activation of TLR3. Digests with single- and double-strand-specific RNases showed the double-strand nature of this RNA, and its sequence was found to be identical to the genome of the dsRNA L-BC virus of Saccharomyces cerevisiae. A large scale production and purification process of this RNA was established based on chemical cell lysis and dsRNA-specific chromatography. NAB2 complexed with the cationic lipid Lipofectin, but neither NAB2 nor Lipofectin alone, induced the secretion of IL-12(p70), IFNα, IP-10, Mip-1β and IL-6 in human monocyte-derived dendritic cells. While NAB2 activated TLR3, Lipofectin-stabilized NAB2 signaled also via the cytoplasmic sensor for RNA recognition MDA-5. Significant increase of RMA-MUC1 tumor rejection and survival was observed in C57BL/6 mice after prophylactic vaccination with MUC1-encoding MVA and NAB2+Lipofectin. This combination of immunotherapeutics strongly increased the percentage of infiltrating Natural Killer (NK) cells and plasmacytoid dendritic cells (pDCs) at the injection sites, cell types which can modulate innate and adaptive immune responses.
M. Banjac, E. Roethl, F. Gelhart, P. Kramberger, B. Lah Jarc, M. Jarc, A. Štrancar, T. Muster, M. Peterka
Vaccine 2014
We explored the possibilities for purification of various ΔNS1 live, replication deficient influenza viruses on ion exchange methacrylate monoliths. Influenza A ΔNS1-H1N1, ΔNS1-H3N2, ΔNS1-H5N1 and ΔNS1-influenza B viruses were propagated in Vero cells and concentrated by tangential flow filtration. All four virus strains adsorbed well to CIM QA and CIM DEAE anion exchangers, with CIM QA producing higher recoveries than CIM DEAE. ΔNS1-influenza A viruses adsorbed well also to CIM SO3 cation exchanger at the same pH, while ΔNS1-influenza B virus adsorption to CIM SO3 was not complete. Dynamic binding capacity (DBC) for CIM QA, DEAE and SO3 methacrylate monoliths for influenza A ΔNS1-H1N1 virus were 1.9E+10 TCID50/ml, 1.0E+10 TCID50/ml and 8.9E+08 TCID50/ml, respectively. Purification of ΔNS1 viruses on CIM QA was scaled up and reproducibility was confirmed. Yields of infectious virus on CIM QA were between 70.8±32.3% and 87±30.8%. Total protein removal varied from 93.3±0.4% to 98.6±0.2% and host cell DNA removal efficiency was ranging from 76.4% to 99.9% and strongly depended on pretreatment with deoxyribonuclease.
F. W. Krainer, R. Pletzenauer, L. Rossetti, C. Herwig, A. Glieder, O. Spadiut
Protein Expression and Purification 95 (2014) 104–112
The plant enzyme horseradish peroxidase (HRP) is used in several important industrial and medical applications, of which especially biosensors and diagnostic kits describe an emerging field. Although there is an increasing demand for high amounts of pure enzyme preparations, HRP is still isolated from the plant as a mixture of different isoenzymes with different biochemical properties. Based on a recent next generation sequencing approach of the horseradish transcriptome, we produced 19 individual HRP isoenzymes recombinantly in the yeast Pichia pastoris. After optimizing a previously reported 2-step purification strategy for the recombinant isoenzyme HRP C1A by substituting an unfavorable size exclusion chromatography step with an anion exchange step using a monolithic column, we purified the 19 HRP isoenzymes with varying success. Subsequent basic biochemical characterization revealed differences in catalytic activity, substrate specificity and thermal stability of the purified HRP preparations. The preparations of the isoenzymes HRP A2A and HRP A2B were found to be highly interesting candidates for future applications in diagnostic kits with increased sensitivity.
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.
M. Bartolini, I. W. Wainer, C. Bertucci, V. Andrisano
Journal of Pharmaceutical and Biomedical Analysis 73 (2013) 77-81
Adenosine nucleotides are involved as substrates or co-factors in several biochemical reactions, catalyzed by enzymes, which modulate energy production, signal transduction and cell proliferation. We here report the development and optimization of an ion exchange liquid chromatography (LC) method for the determination of ATP, ADP and AMP. This method is specifically aimed at the determination of the ATP-ase activity of human heat shock protein 90 (Hsp90), a molecular chaperone that has emerged as target enzyme in cancer therapy. Separation of the three nucleotides was achieved in a 15-min run by using a disk shaped monolithic ethylene diamine stationary phase of small dimensions (2 mm × 6 mm i.d.), under a three-solvent gradient elution mode and UV detection at 256 nm. The described direct LC method resulted highly specific as a consequence of the baseline separation of the three adenosine nucleotides and could be applied to the determination of the enzymatic activity of ADP/ATP generating or consuming enzymes (such as kinases). Furthermore, comparison of the LOD and LOQ values of the LC method with those obtained with the malachite green assay, which is one of the most used indirect screening methodologies for ATP-ase activity, showed that the LC method has a similar range of application without presenting the drawbacks related to contamination by inorganic phosphate ions and glycerol, which are present in Hsp90 commercial samples.
F. Ibrahim, C. Andre, R. Aljhni, T. Gharbi, Y. C. Guillaume
Journal of Molecular Catalysis B: Enzymatic 94 (2013) 136-140
Acetylcholinesterase (AChE) is a serine protease that hydrolyzes the neurotransmitter acetylcholine. Here, the effects of hydroxyl radical (OH•) and nitric oxide (NO) on AChE activity were studied using a biochromatographic process. The enzyme was immobilized on an ethylenediamine (EDA) monolithic convective interaction media (CIM) disk. The AChE enzymatic mechanism was demonstrated from the chromatographic peak shape. A decrease in AChE activity was observed for each concentration of NO, while OH• dot radical formation led to an increase in the rate of enzymatic catalysis. Michaelis–Menten and Lineweaver–Burk plots were obtained in the presence or absence of the free radicals and their effects on Km and Vmax were evaluated. Our results indicated classical deactivation/activation kinetics without significant influence on the rate of substrate binding. The variation in transition state energies (ΔΔGES) induced by the free radicals indicated that a conformational change was occurring in the active site, while changes in the binding site were negligible. These results clearly demonstrate the direct role of OH• dot and NO on AChE activity and confirm the role they may play in Alzheimer's disease.
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.
P. Fernandes, C. Peixoto, VM Santiago, EJ Kremer, AS Coroadinha and PM Alves
Gene Therapy (2012), 1–8
Canine adenovirus type 2 (CAV-2) vectors overcome many of the clinical immunogenic concerns related to vectors derived from human adenoviruses (AdVs). In addition, CAV-2 vectors preferentially transduce neurons with an efficient traffic via axons to afferent regions when injected into the brain. To meet the need for preclinical and possibly clinical uses, scalable and robust production processes are required. CAV-2 vectors are currently produced in E1-transcomplementing dog kidney (DK) cells, which might raise obstacles in regulatory approval for clinical grade material production. In this study, a GMP-compliant bioprocess was developed. An MDCK-E1 cell line, developed by our group, was grown in scalable stirred tank bioreactors, using serum-free medium, and used to produce CAV-2 vectors that were afterwards purified using column chromatographic steps. Vectors producedin MDCK-E1 cells were identical to those produced in DK cells as assessed by SDS-PAGE and dynamic light scatering measurements (diameter and Zeta potential). Productivities of ~109 infectious particles (IP) ml-1 and 2x103 IP per cell were possible. A downstream process using technologies transferable to process scales was developed, yielding 63% global recovery. The total particles to IP ratio in the purified product (<20:1) was within the limits specified by the regulatory authorities for AdV vectors. These results constitute a step toward a scalable process for CAV-2 vector production compliant with clinical material specifications.