Quantification of rotavirus-like particles by gel permeation chromatography

Mena, Jimmy A.; Ramı́rez, Octavio T.; Palomares, Laura A.

doi:10.1016/j.jchromb.2005.07.034

Cited by 26 publications

(31 citation statements)

References 31 publications

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“…Throughout the development of this work and others from our groups (Mena et al, 2005;Peixoto et al, 2007) we never observed by electron microscopy a significant TLP or DLP aggregation during short period of time (<1 month). Ludert et al (2002), who studied in vitro TLP (1.5 mg mL…”

Section: Discussionmentioning

confidence: 47%

Impact of physicochemical parameters on in vitro assembly and disassembly kinetics of recombinant triple‐layered rotavirus‐like particles

Mellado¹,

Mena²,

Lopes³

et al. 2009

Biotech & Bioengineering

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Virus-like particles constitute potentially relevant vaccine candidates. Nevertheless, their behavior in vitro and assembly process needs to be understood in order to improve their yield and quality. In this study we aimed at addressing these issues and for that purpose triple- and double-layered rotavirus-like particles (TLP 2/6/7 and DLP 2/6, respectively) size and zeta potential were measured using dynamic light scattering at different physicochemical conditions, namely pH, ionic strength, and temperature. Both TLP and DLP were stable within a pH range of 3-7 and at 5-25 degrees C. Aggregation occurred at 35-45 degrees C and their disassembly became evident at 65 degrees C. The isoelectric points of TLP and DLP were 3.0 and 3.8, respectively. In vitro kinetics of TLP disassembly was monitored. Ionic strength, temperature, and the chelating agent employed determined disassembly kinetics. Glycerol (10%) stabilized TLP by preventing its disassembly. Disassembled TLP was able to reassemble by dialysis at high calcium conditions. VP7 monomers were added to DLP in the presence of calcium to follow in vitro TLP assembly kinetics; its assembly rate being mostly affected by pH. Finally, DLP and TLP were found to coexist under certain conditions as determined from all reaction products analyzed by capillary electrophoresis. Overall, these results contribute to the design of new strategies for the improvement of TLP yield and quality by reducing the VP7 detachment from TLP.

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Section: Discussionmentioning

confidence: 47%

Impact of physicochemical parameters on in vitro assembly and disassembly kinetics of recombinant triple‐layered rotavirus‐like particles

Mellado¹,

Mena²,

Lopes³

et al. 2009

Biotech & Bioengineering

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“…They are produced by the recombinant expression of the structural proteins that, in the absence of non-structural proteins and genetic material, assemble into structures identical to the native virus [1,2]. Viruslike particles (VLP) have a wide range of applications, including E-mail address: mrito@itesm.mx (M. Rito-Palomares).…”

Section: Introductionmentioning

confidence: 99%

“…The structure of dlRLP makes their production and purification a unique challenge [2,6,7]. However, in order for dlRLP to be a viable alternative for immunization, economical production of complete particles of good quality at large scale needs to be achieved.…”

Section: Introductionmentioning

confidence: 99%

Rotavirus-like particles primary recovery from insect cells in aqueous two-phase systems☆

Benavides

Mena

Cisneros-Ruiz

et al. 2006

Journal of Chromatography B

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“…To obtain pure complete VLP several purification steps and a combination of various methods have to be employed. Ultracentrifugation methods such as CsCl or sucrose density gradients and various microfiltrations are commonly used, often followed by one or more chromatographic steps [5,[16][17][18][19][20]. Such a purification procedure does not, however, provide large batches of sufficiently homogenous and pure material [5].…”

Section: Introductionmentioning

confidence: 99%

Purification of recombinant adenovirus type 3 dodecahedric virus-like particles for biomedical applications using short monolithic columns

Urbas

Jarc

Barut

et al. 2011

Journal of Chromatography A

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Adenovirus type 3 dodecahedric virus-like particles (Ad3 VLP) are an interesting delivery vector. They penetrate animal cells in culture very efficiently and up to 300,000 Ad3 VLP can be observed in one cell. The purification of such particles usually consists of several steps. In these work we describe the method development and optimization for the purification of Ad3 VLP using the Convective Interaction Media analytical columns (CIMac). Results obtained with the CIMac were compared to the already established two-step purification protocol for Ad3 VLP based on sucrose density gradient ultracentifugation and the Q-Sepharose ion-exchange column. Pure, concentrated and bioactive VLP were obtained and characterized by several analytical methods. The recovery of the Ad3 VLP was more than 50% and the purified fraction was almost completely depleted of DNA; less than 1% of DNA was present. The purification protocol was shortened from five days to one day and remarkably high penetration efficacy of the CIMac-purified vector was retained. Additionally, CIMac QA analytical column has proven to be applicable for the final and in-process control of various Ad3 VLP samples.

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Quantification of rotavirus-like particles by gel permeation chromatography

Cited by 26 publications

References 31 publications

Impact of physicochemical parameters on in vitro assembly and disassembly kinetics of recombinant triple‐layered rotavirus‐like particles

Impact of physicochemical parameters on in vitro assembly and disassembly kinetics of recombinant triple‐layered rotavirus‐like particles

Rotavirus-like particles primary recovery from insect cells in aqueous two-phase systems☆

Purification of recombinant adenovirus type 3 dodecahedric virus-like particles for biomedical applications using short monolithic columns

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