Vaccine Production Process Research Articles

Cultivos Celulares como Alternativa para el Aislamiento y la Producción de Biológicos Contra el Virus de Influenza

El virus de influenza ha sido reconocido como un importante patógeno en poblaciones humanas y animales, ya que es el principal causante de enfermedades respiratorias. Muchas vacunas y aislamientos de virus de influenza humana y animal son realizadas actualmente en huevos embrionados, siendo este el método usado tradicionalmente por décadas. Sin embargo, se han encontrado inconvenientes en la elaboración de vacunas ya que el proceso de fabricación es de capacidad limitada (se requiere aproximadamente un huevo para generar una dosis vacunal) y alta demanda tiempo, disminuyendo su habilidad para generar biológicos rápidamente en el caso de una pandemia. El empleo de líneas celulares continuas para la producción de vacunas virales nace como alternativa que ofrece diversas ventajas: (i) oportunidad de emplear células completamente caracterizadas y estandarizadas, (ii) producción y planeación permanente de vacunas y (iii) los biológicos pueden ser producidos de forma más rápida.El objetivo de esta revisión es analizar las diferentes alternativas empleadas en el cultivo y/o aislamiento de virus de influenza, enfatizando en el uso de cultivos celulares como sustrato para el aislamiento y la producción de biológicos destinados a la salud humana y animal.

Tetanus toxoid purification: Chromatographic procedures as an alternative to ammonium-sulphate precipitation

Given an existing demand to establish a process of tetanus vaccine production in a way that allows its complete validation and standardization, this paper focuses on tetanus toxoid purification step. More precisely, we were looking at a possibility to replace the widely used ammonium-sulphate precipitation by a chromatographic method. Based on the tetanus toxin's biochemical characteristics, we have decided to examine the possibility of tetanus toxoid purification by hydrophobic chromatography, and by chromatographic techniques based on interaction with immobilized metal ions, i.e. chelating chromatography and immobilized metal affinity chromatography. We used samples obtained from differently fragmented crude tetanus toxins by formaldehyde treatment (assigned as TTd-A and TTd-B) as starting material for tetanus toxoid purification. Obtained results imply that purification of tetanus toxoid by hydrophobic chromatography represents a good alternative to ammonium-sulphate precipitation. Tetanus toxoid preparations obtained by hydrophobic chromatography were similar to those obtained by ammonium-sulphate precipitation in respect to yield, purity and immunogenicity. In addition, their immunogenicity was similar to standard tetanus toxoid preparation (NIBSC, Potters Bar, UK). Furthermore, the characteristics of crude tetanus toxin preparations had the lowest impact on the final purification product when hydrophobic chromatography was the applied method of tetanus toxoid purification. On the other hand, purifications of tetanus toxoid by chelating chromatography or immobilized metal affinity chromatography generally resulted in a very low yield due to not satisfactory tetanus toxoid binding to the column, and immunogenicity of the obtained tetanus toxoid-containing preparations was poor.

Bioprocess optimization for cell culture based influenza vaccine production

Uncertainties and shortcomings associated with the current influenza vaccine production processes demand attention and exploration of new vaccine manufacture technologies. Based on a newly developed mammalian cell culture-based production process we investigated selected process parameters and describe three factors that are shown to impact productivity, process robustness and development time. They are time of infection, harvest time and virus input, or multiplicity of infection (MOI). By defining the time of infection as 4–5 days post cell seeding and harvest time as 2–3 days post-infection and comparing their effect on virus production, MOI is subsequently identified as the most impactful process parameter for live attenuated influenza vaccine (LAIV) manufacture. Infection at very low MOI (between 10−4 and 10−6FFU/cell) resulted in high titer virus production (up to 30-fold productivity improvement) compared to higher MOI infections (10−3 to 10−2FFU/cell). Application of these findings has allowed us to develop a platform process that can reduce the development time to approximately three weeks for an influenza vaccine manufacture process for new strains.

Development and strategies of cell-culture technology for influenza vaccine

Influenza is a pandemic contagious disease and causes human deaths and huge economic destruction of poultry in the world. In order to control and prevent influenza, mainly type A, influenza vaccine for human and poultry were available since the 1940s and 1920s, respectively. In the development of vaccine production, influenza viruses were cultured originally from chicken embryos to anchorage-dependent cell lines, such as MDCK and Vero. The anchorage-independent lines have also been used to produce influenza virus, such as PER.C6 and engineering modified MDCK and Vero. During the process of influenza vaccine production, the common problem faced by all producers is how to improve the titer of influenza virus. This paper focuses on the developments of cell culture for influenza virus vaccine production, limitations of cell culture, and relative strategies for improvement virus yields in cell-culture systems.

Differential interactions of plasmid DNA, RNA and genomic DNA with amino acid‐based affinity matrices

The development of a strategy to plasmid DNA (pDNA) purification has become necessary for the development of gene therapy and DNA vaccine production processes in recent years, since this nucleic acid and most of contaminants, such as RNA, genomic DNA and endotoxins, are negatively charged. An ideal separation methodology may be achieved with the use of affinity interactions between immobilized amino acids and nucleic acids. In this study, the binding behaviour of nucleic acids under the influence of different environmental conditions, such as the composition and ionic strength of elution buffer, and the temperature, is compared with various amino acids immobilized on chromatography resins. Supercoiled (sc) plasmid isoform was isolated with all matrices used, but in some cases preferential interactions with other nucleic acids were found. Particularly, lysine chromatography showed to be an ideal technology mainly on RNA purification using low salt concentration. On the other hand, arginine ligands have shown a greater ability to retain the sc isoform comparatively to the other nucleic acids retention, becoming this support more adequate to sc pDNA purification. The temperature variation, competitive elution and oligonucleotides affinity studies also allowed to recognize the dominant interactions inherent to biorecognition of pDNA molecule and the affinity matrices.

Evaluation of a modified vaccinia virus Ankara (MVA)-based candidate pandemic influenza A/H1N1 vaccine in the ferret model

The zoonotic transmissions of highly pathogenic avian influenza viruses of the H5N1 subtype that have occurred since 1997 have sparked the development of novel influenza vaccines. The advent of reverse genetics technology, cell-culture production techniques and novel adjuvants has improved the vaccine strain preparation, production process and immunogenicity of the vaccines, respectively, and has accelerated the availability of pandemic influenza vaccines. However, there is still room for improvement, and alternative vaccine preparations can be explored, such as viral vectors. Modified vaccinia virus Ankara (MVA), originally developed as a safe smallpox vaccine, can be exploited as a viral vector and has many favourable properties. Recently, we have demonstrated that an MVA-based vaccine could protect mice and macaques against infection with highly pathogenic influenza viruses of the H5N1 subtype. In the present study, recombinant MVA expressing the haemagglutinin (HA) gene of pandemic influenza A/H1N1 virus was evaluated in the ferret model. A single immunization induced modest antibody responses and afforded only modest protection against the development of severe disease upon infection with a 2009(H1N1) strain. In contrast, two immunizations induced robust antibody responses and protected ferrets from developing severe disease, confirming that MVA is an attractive influenza vaccine production platform.

Properly folded bacterially expressed H1N1 hemagglutinin globular head and ectodomain vaccines protect ferrets against H1N1 pandemic influenza virus.

BackgroundIn the face of impending influenza pandemic, a rapid vaccine production and mass vaccination is the most effective approach to prevent the large scale mortality and morbidity that was associated with the 1918 “Spanish Flu”. The traditional process of influenza vaccine production in eggs is time consuming and may not meet the demands of rapid global vaccination required to curtail influenza pandemic.Methodology/Principal FindingsRecombinant technology can be used to express the hemagglutinin (HA) of the emerging new influenza strain in a variety of systems including mammalian, insect, and bacterial cells. In this study, two forms of HA proteins derived from the currently circulating novel H1N1 A/California/07/2009 virus, HA1 (1–330) and HA (1–480), were expressed and purified from E. coli under controlled redox refolding conditions that favoured proper protein folding. However, only the recombinant HA1 (1–330) protein formed oligomers, including functional trimers that bound receptor and caused agglutination of human red blood cells. These proteins were used to vaccinate ferrets prior to challenge with the A/California/07/2009 virus. Both proteins induced neutralizing antibodies, and reduced viral loads in nasal washes. However, the HA1 (1–330) protein that had higher content of multimeric forms provided better protection from fever and weight loss at a lower vaccine dose compared with HA (1–480). Protein yield for the HA1 (1–330) ranged around 40 mg/Liter, while the HA (1–480) yield was 0.4–0.8 mg/Liter.Conclusions/SignificanceThis is the first study that describes production in bacterial system of properly folded functional globular HA1 domain trimers, lacking the HA2 transmembrane protein, that elicit potent neutralizing antibody responses following vaccination and protect ferrets from in vivo challenge. The combination of bacterial expression system with established quality control methods could provide a mechanism for rapid large scale production of influenza vaccines in the face of influenza pandemic threat.

Experimental RNA Therapy Shows Promise Against Ebola Virus in Monkey Studies

tionally, fragments of simian retrovirus DNA with an inactivating mutation were found in RotaTeq and also in the Vero cells (a cell line from the kidney cells from an African green monkey) from which the vaccines are derived. Delwart explained that the use of cells from other species in the production of vaccine can lead to leakage of cellular DNA and the introduction of noninfectious proviral DNA in vaccines. A possible culprit for introducing PCV1 into Rotarix may have been porcine pancreas-derived trypsin. He said that while the PCV1 contamination they found is undesirable, in some ways it is a best-case scenario because the virus has been well studied and appears to be noninfectious to humans. The discovery of unexpected viral DNA in the vaccine should not necessarily be cause for alarm, Delwart added. “We are constantly bombarded with viruses, particularly in the digestive tract [from meat, plants, and insects],” Delwart said. “We are finding them because the technology has changed.” In the future, use of these technologies early in the vaccine production process should allow them to be removed quickly, he added. Until the makers of the rotavirus vaccines are able to develop new versions of their vaccines, the FDA is requiring that the labels of Rotarix and RotaTeq be updated to disclose the presence of the PCV viruses. And Stapleton recommends that physicians inform parents that the viral DNA poses a theoretical risk, though extensive investigation has turned up no evidence of harm. Some parents may choose to decline the vaccine on this basis, he noted. “You have to be transparent and tell parents about the finding but [also inform them] that the vaccines are still recommended,” Stapleton said.

Avian influenza pandemic preparedness: developing prepandemic and pandemic vaccines against a moving target

The unprecedented global spread of highly pathogenic avian H5N1 influenza viruses within the past ten years and their extreme lethality to poultry and humans has underscored their potential to cause an influenza pandemic. Combating the threat of an impending H5N1 influenza pandemic will require a combination of pharmaceutical and nonpharmaceutical intervention strategies. The emergence of the H1N1 pandemic in 2009 emphasised the unpredictable nature of a pandemic influenza. Undoubtedly, vaccines offer the most viable means to combat a pandemic threat. Current egg-based influenza vaccine manufacturing strategies are unlikely to be able to cater to the huge, rapid global demand because of the anticipated scarcity of embryonated eggs in an avian influenza pandemic and other factors associated with the vaccine production process. Therefore, alternative, egg-independent vaccine manufacturing strategies should be evaluated to supplement the traditional egg-derived influenza vaccine manufacturing. Furthermore, evaluation of dose-sparing strategies that offer protection with a reduced antigen dose will be critical for pandemic influenza preparedness. Development of new antiviral therapeutics and other, nonpharmaceutical intervention strategies will further supplement pandemic preparedness. This review highlights the current status of egg-dependent and egg-independent strategies against an avian influenza pandemic.

Structural and functional specificity of Dichelobacter nodosus Pili: Synthesis of pilin protein D. nodosus in various recombinant bacterial strains

Sheep hoof rot is a serious infectious disease specific to this type of animal; its pathogenic agent is anaerobic gram-negative bacillus Dichelobacter nodosus. Very strict requirements for cultivation conditions and slow rate of accumulation of bacterial mass make the vaccine and diagnistcs production laborious and expensive. Fimbriae (pili) are one of the main factors of D. nodosus virulence, since they have both antigenic and immunogenic properties that allow them to be added to vaccines to intensify immunogenicity. After it was determined that the fimA gene, which encodes the fimbrial subunit, attempts were made to obtain recombinant pili. The production of genetically engineered fimbriae serves to simplify and lower the cost of the process of vaccine production, as well as to improve immune-protective qualities of the obtained preparations. The first system of bacterial expression was selected for E. coli, however, the assembly of D. nodosus pili on its surface failed due to significant differences in the biogenesis and fimbria structure of these microorganisms. The morphogenesis of D. nodosus pili succeeded in Pseudomonas aeruginosa, which is characterized by the most similar structure of pilin subunits. Sheep’s immunity after injection of recombinant fimbria, native fimbria, and whole cells of D. nodosus was almost analogous in intensity. The article presents a review of data from publications on the production of recombinant cultures of P. aeruginosa with D. nodosus pili and expression of pilin genes in various bacterial systems.

Sulfated membrane adsorbers for economic pseudo‐affinity capture of influenza virus particles

Strategies to control outbreaks of influenza, a contagious respiratory tract disease, are focused mainly on prophylactic vaccinations in conjunction with antiviral medications. Currently, several mammalian cell culture-based influenza vaccine production processes are being established, such as the technologies introduced by Novartis Behring (Optaflu) or Baxter International Inc. (Celvapan). Downstream processing of influenza virus vaccines from cell culture supernatant can be performed by adsorbing virions onto sulfated column chromatography beads, such as Cellufine sulfate. This study focused on the development of a sulfated cellulose membrane (SCM) chromatography unit operation to capture cell culture-derived influenza viruses. The advantages of the novel method were demonstrated for the Madin Darby canine kidney (MDCK) cell-derived influenza virus A/Puerto Rico/8/34 (H1N1). Furthermore, the SCM-adsorbers were compared directly to column-based Cellufine sulfate and commercially available cation-exchange membrane adsorbers. Sulfated cellulose membrane adsorbers showed high viral product recoveries. In addition, the SCM-capture step resulted in a higher reduction of dsDNA compared to the tested cation-exchange membrane adsorbers. The productivity of the SCM-based unit operation could be significantly improved by a 30-fold increase in volumetric flow rate during adsorption compared to the bead-based capture method. The higher flow rate even further reduced the level of contaminating dsDNA by about twofold. The reproducibility and general applicability of the developed unit operation were demonstrated for two further MDCK cell-derived influenza virus strains: A/Wisconsin/67/2005 (H3N2) and B/Malaysia/2506/2004. Overall, SCM-adsorbers represent a powerful and economically favorable alternative for influenza virus capture over conventional methods using Cellufine sulfate.

Substitution of Glutamine by Pyruvate To Reduce Ammonia Formation and Growth Inhibition of Mammalian Cells

In mammalian cell culture technology glutamine is required for biomass synthesis and as a major energy source together with glucose. Different pathways for glutamine metabolism are possible, resulting in different energy output and ammonia release. The accumulation of ammonia in the medium can limit cell growth and product formation. Therefore, numerous ideas to reduce ammonia concentration in cultivation broths have been developed. Here we present new aspects on the energy metabolism of mammalian cells. The replacement of glutamine (2 mM) by pyruvate (10 mM) supported cell growth without adaptation for at least 19 passages without reduction in growth rate of different adherent commercial cell lines (MDCK, BHK21, CHO-K1) in serum-containing and serum-free media. The changes in metabolism of MDCK cells due to pyruvate uptake instead of glutamine were investigated in detail (on the amino acid level) for an influenza vaccine production process in large-scale microcarrier culture. In addition, metabolite profiles from variations of this new medium formulation (1-10 mM pyruvate) were compared for MDCK cell growth in roller bottles. Even at very low levels of pyruvate (1 mM) MDCK cells grew to confluency without glutamine and accumulation of ammonia. Also glucose uptake was reduced, which resulted in lower lactate production. However, pyruvate and glutamine were both metabolized when present together. Amino acid profiles from the cell growth phase for pyruvate medium showed a reduced uptake of serine, cysteine, and methionine, an increased uptake of leucine and isoleucine and a higher release of glycine compared to glutamine medium. After virus infection completely different profiles were found for essential and nonessential amino acids.

Metabolic flux model for an anchorage‐dependent MDCK cell line: Characteristic growth phases and minimum substrate consumption flux distribution

Up to now cell-culture based vaccine production processes only reach low productivities. The reasons are: (i) slow cell growth and (ii) low cell concentrations. To address these shortcomings, a quantitative analysis of the process conditions, especially the cell growth and the metabolic capabilities of the host cell line is required. For this purpose a MDCK cell based influenza vaccine production process was investigated. With a segregated growth model four distinct cell growth phases are distinguished in the batch process. In the first phase the cells attach to the surface of the microcarriers and show low metabolic activity. The second phase is characterized by exponential cell growth. In the third phase, preceded by a change in oxygen consumption, contact inhibition leads to a decrease in cell growth. Finally, the last phase before infection shows no further increase in cell numbers. To gain insight into the metabolic activity during these phases, a detailed metabolic model of MDCK cell was developed based on genome information and experimental analysis. The MDCK model was also used to calculate a theoretical flux distribution representing an optimized cell that only consumes a minimum of carbon sources. Comparing this minimum substrate consumption flux distribution to the fluxes estimated from experiments unveiled high overflow metabolism under the applied process conditions.

Lessons Learned: Role of Influenza Vaccine Production, Distribution, Supply, and Demand—What It Means for the Provider

The Advisory Committee on Immunization Practices of the Centers for Disease Control and Prevention (CDC) has been increasing the size of the population for whom influenza vaccine is recommended to reduce the substantial and persistent annual health burden of influenza. Realization of current and future public health influenza immunization goals requires assuring vaccine supply will be adequate to meet demand. This has posed distinct challenges for the many stakeholders in the influenza vaccine program—government agencies, federal, state, and local policymakers, vaccine manufacturers and distributors, and the medical community—each of whom must make critical decisions in a constantly shifting environment. Factors such as the yearly changes in influenza virus strains, the complicated vaccine production and distribution process, revisions in vaccination recommendations, and changing demographics can all affect the delicate balance between supply and demand. While vaccine shortages and delays have been well-publicized concerns in the recent past, there has been a marked increase in supply in the past several years, with substantial growth in supply expected in the future. The primary issue today is to strengthen the demand for the influenza vaccine, which would in turn help ensure the continued availability of the vaccine to reduce disease burden. A number of strategies are discussed, including increased efforts to publicize and fully implement current CDC recommendations and to offer influenza vaccine beyond the typical vaccination season of October and November, because in the great majority of years, vaccination into January and beyond will still provide health benefits.

Stochastic population balance modeling of influenza virus replication in vaccine production processes. II. Detailed description of the replication mechanism

In a recent paper a segregated stochastic model was proposed for influenza virus replication in vaccine production processes [Sidorenko, Y., Schulze-Horsel, J., Voigt, A., Reichl, U., Kienle, A., 2008. Stochastic population balance modeling of influenza virus replication in vaccine production processes. Chemical Engineering Science 63, 157–169]. The model used a simple segregated, unstructured approach for the description of the virus replication. In particular, effects arising from limited internal cellular resources and detailed cell physiology were not taken into account. The degree of infection—corresponding to the number of virus equivalents per cell—was used as the only internal coordinate. The model was successful in describing the integral dynamics of the process, however, revealed some discrepancies with respect to the “internal dynamics” of the virus replication. Therefore, in a second step, a much more detailed description of the virus replication process is considered in this paper. Again, cell physiology is described in terms of global growth and death events. Limitations of the intracellular resources are not taken into account. It is shown that this type of model does not contribute significantly to an improvement of the prediction of the internal dynamics. Hence, it is concluded that limited intracellular resources or a detailed description of the cell physiology is required for a more realistic modeling of virus replication dynamics.

Monitoring influenza virus content in vaccine production: Precise assays for the quantitation of hemagglutination and neuraminidase activity

Robust and precise quantitation of influenza virus is a premise for the efficient development of vaccine production processes. In this article, revised assays for the determination of hemagglutination (HA) and neuraminidase (NA) activity are presented. Bias of traditional discontinuous HA assays and operator dependency was overcome by introduction of a regression procedure. At little effort, a continuous assay result is obtained with repeatability as good as +29%/−22% in the best case (95% confidence intervals reported). Similarly, neuraminidase activity determined in microtiter plates resulted in repeatability better than ±20%. NA activity decreased almost linearly for pH ranging from 5.8 to 7.8 and was enhanced by the addition of Ca 2+. Non-linearity of the assay (due to unspecific adsorption) was overcome by addition of BSA. Using 4-methylumbelliferyl- α- d- N-acetylneuraminic acid as substrate, Michaelis–Menten constants of 30 and 460 μM were determined for strains A/PR/8/34 (H1N1) and A/Equi 2/NM/1/93 (H3N8), respectively. The error introduced by approximation of Michaelis–Menten kinetics (zero and first order) was minimized by limiting substrate consumption to about 10%. Linearity of both assays was verified in dilution experiments. Applicability was demonstrated in three cases: virus propagation in mammalian cell culture, ultrafiltration and precipitation of nucleic acids.

Stochastic population balance modeling of influenza virus replication in vaccine production processes

A distributed population balance model of influenza A virus replication in adherent Madin-Darby canine kidney cells has been developed to reproduce and interpret flow cytometry data for virus propagation in microcarrier culture. The population of cells is differentiated into uninfected, infected and degraded cells. As an internal coordinate the number of intracellular viral components is considered. The main focus of the model is to link the time course of intracellular virus protein accumulation monitored by flow cytometry with the total yield of virus particles measured by the hemagglutination assay. The model allows simulating the extracellular virus dynamics for multiplicities of infection in the range 0.025–3.0. Shape of predicted histograms is in general agreement with distributions obtained by flow cytometry. Differences in time course at about 12–14 and 20 h post infection indicate that additional assumptions on intracellular virus dynamics are required to fully explain experimental data. Furthermore, prerequisites for virus replication, like receptor binding sites, the number of endosomes or the demand for free amino acids and nucleotides for virus synthesis can be estimated and compared with cellular resources available. Simulation results suggest that intracellular pools of free amino acids as well as early cell death due to influenza virus-induced apoptosis can limit virus yields. It is expected that based on a better understanding of the infectivity status of cells and the spreading of viruses in population of cells in bioreactors strategies on design and optimization of vaccine production processes can be developed.

Establishment of a mink enteritis vaccine production process in stirred-tank reactor and Wave ® Bioreactor microcarrier culture in 1–10 L scale

A scale-up and process optimization scheme for the growth of adherent embryonic feline lung fibroblasts (E-FL) on microcarriers and the propagation of a mink enteritis virus (MEV) strain for the production of an inactivated vaccine is shown. Stirred-tank cultivations are compared with results obtained from Wave ® Bioreactors. Transfer from a roller bottle-based production process into large-scale microcarrier culture with starting concentrations of 2 g/L Cytodex™ 1 microcarriers and 2.0 × 10 5 cells/mL was successful. A maximum cell yield of 1.2 × 10 6 cells/mL was obtained in stirred-tank microcarrier batch culture while cell numbers in the Wave ® Bioreactor could not be determined accurately due to the fast sedimentation of microcarriers under non-rocking conditions required for sampling. Detailed off-line analysis was carried out to understand the behaviour of the virus–host cell system in both cultivation systems. Metabolic profiles for glucose, lactate, glutamine, and ammonium showed slight differences for both systems. E-FL cell growth was on the same level in stirred-tank and Wave ® Bioreactor with a higher volumetric cell yield compared to roller bottles. Propagation of MEV, which can only replicate efficiently in mitotic cells, was characterized in the Wave ® Bioreactor using a multiple harvest strategy. Maximum virus titres of 10 6.6 to 10 6.8 TCID 50/mL were obtained, which corresponds to an increase in virus yield by a factor of about 10 compared to cultivations in roller bottles. As a consequence, a single Wave ® Bioreactor cultivation of appropriate scale can replace hundreds of roller bottles. Thus, the Wave ® Bioreactor proved to be a suitable system for large-scale production of an inactivated MEV vaccine.

Differential interactions of plasmid DNA, RNA and endotoxin with immobilised and free metal ions

Separation of negatively charged molecules, such as plasmid DNA (pDNA), RNA and endotoxin forms a bottleneck for the development of pDNA vaccine production process. The use of affinity interactions of transition metal ions with these molecules may provide an ideal separation methodology. In this study, the binding behaviour of pDNA, RNA and endotoxin to transition metal ions, either in immobilised or free form, was investigated. Transition metal ions: Cu 2+, Ni 2+, Zn 2+, Co 2+ and Fe 3+, typically employed in the immobilised metal affinity chromatography (IMAC), showed very different binding behaviour depending on the type of metal ions and their existing state, i.e. immobilised or free. In the alkaline cell lysate, pDNA showed no binding to any of the IMAC chemistries tested whereas RNA interacted significantly with Cu 2+-iminodiacetic acid (IDA) and Ni 2+-IDA but showed no substantial binding to the rest of the IMAC chemistries. pDNA and RNA, however, interacted to varying degrees with free metal ions in the solution. The greatest selectivity in terms of pDNA and RNA separation was achieved with Zn 2+ which enabled almost full precipitation of RNA while keeping pDNA soluble. For both immobilised and free metal ions, ionic strength of solution affected the metal ion-nucleic acid interaction significantly. Endotoxin, being more flexible, was able to interact better with the immobilised metal ions than the nucleic acids and showed binding to all the IMAC chemistries. The specific interactions of immobilised and/or free metal ions with pDNA, RNA and endotoxin showed a good potential, by selectively removing RNA and endotoxin at high efficiency, to develop a simplified pDNA purification process with improved process economics.

High stability of yellow fever 17D-204 vaccine: A 12-year restrospective analysis of large-scale production

We have retrospectively analyzed 12 bulk lots of yellow fever vaccine Stamaril™, produced between 1990 and 2002 and prepared from the same seed lot that has been in continuous use since 1990. All vaccine batches displayed identical genome sequence. Only four nucleotide substitutions were observed, compared to previously published sequence, with no incidence at amino-acid level. Fine analysis of viral plaque size distribution was used as an additional marker for genetic stability and demonstrated a remarkable homogeneity of the viral population. The total virus load, measured by qRT-PCR, was also homogeneous pointing out reproducibility of the vaccine production process. Mice inoculated intracerebrally with the different bulks exhibited a similar average survival time, and ratio between in vitro potency and mouse LD 50 titers remained constant from batch-to-batch. Taken together, these data demonstrate the genetic stability of the strain at mass production level over a period of 12 years and reinforce the generally admitted idea of the safety of YF17D-based vaccines.