RESUMO
The quality and stability of recombinant adenovirus preparations for gene therapy trials are currently analyzed by anion-exchange HPLC. It was shown that the retention time of the adenovirus peak increased over the course of time upon storage in current liquid formulations. The number of isoaspartate residues at the outer surface of the particles also increased in parallel in these preparations. A recombinant adenovirus AV3760 was constructed with a modified hexon protein in which all four accessible Asn residues engaged in Asn-Gly pairs were changed into Leu (Asn244, Asn255, Asn437) or Ala (Asn276). The retention time of AV3760, as measured by HPLC, was unchanged after 2 months at +20 degrees C as opposed to the control adenovirus. This demonstrates that the shift in retention time is caused by an increase in carboxyl groups on the outer surface of the virion due to deamidation of the above Asn residues. The modifications introduced in the hexon protein reduced the rate of Asn deamidation, without adverse effects on the infectivity of the virions. By reducing microheterogeneity in recombinant adenovirus, this work represents a significant advance in the development of a stable pharmaceutical vector for gene therapy.
Assuntos
Adenoviridae/química , Adenoviridae/genética , Proteínas do Capsídeo , Capsídeo/genética , DNA Recombinante/genética , Mutagênese Sítio-Dirigida/genética , Adenoviridae/isolamento & purificação , Adenoviridae/fisiologia , Asparagina/química , Asparagina/genética , Capsídeo/química , Cromatografia Líquida de Alta Pressão , Cromatografia por Troca Iônica , Terapia Genética/métodos , Vetores Genéticos/química , Vetores Genéticos/genética , Ácido Isoaspártico/metabolismo , Cinética , Manejo de EspécimesRESUMO
For cancer gene therapy, a recombinant adenovirus serotype 5 named RPR/INGN201 has been constructed by susbtitution of the E1 region with human tumor suppressor gene p53. The protein components of RPR/INGN201 virions were separated by reversed-phase HPLC and were individually identified by electrospray time-of-flight mass spectrometry and N-terminal sequencing, both on intact proteins and on their proteolytic fragments after trypsin digestion. Twenty-five peptide components of the proteome (including fiber) with greater than 0.25-0.5% contribution to the protein content of the virus were identified and characterized. Fiber was confirmed to be partially glycosylated (both the non-glycosylated and the monoglycosylated states were identified), and two proteins were isolated and identified as phosphorylation derivatives, namely protein V (non-phosphorylated and monophosphorylated) and protein IIIa (mono- and diphosphorylated). This new analytical tool proved to be very useful not only for refining our current knowledge of the polypeptide repertoire of purified infectious virions but also for monitoring and very rapidly identifying structural modifications resulting from changes in the manufacturing process. It was also used successfully for the characterization of various adenoviral constructs.
Assuntos
Adenoviridae/genética , Capsídeo/química , Terapia Genética , Neoplasias/terapia , Peptídeos/análise , Capsídeo/isolamento & purificação , Cromatografia Líquida de Alta Pressão , Cromatografia por Troca Iônica , Eletroforese em Gel de Poliacrilamida , Humanos , Espectrometria de Massas por Ionização por ElectrosprayRESUMO
We have developed an anion-exchange high-performance liquid chromatography (HPLC) method using Q Sepharose XL (Amersham Pharmacia Biotech) as adsorbent to analyze samples containing adenovirus. This method has several major advantages over the HPLC method previously described for quantitating particles, namely (1) a >10-fold improvement in the detection limit of adenovirus in crude preparations; (2) absence of interferences originating from nucleic acids and proteins which usually contaminate crude samples; (3) unprecedented sharpness and symmetry of adenovirus peak, rendering the identification of the viral peak unambiguous, even in extremely crude and dilute preparations; and (4) no enzymatic treatment required even for crude samples. This assay was used to quantitate particles in samples taken at the transfection and amplification stages of production of various recombinant adenovirus, and in cultures of wild-type adenovirus of different serotypes. A modification of this analytical method was also developed for the purification of infectious adenovirus particles, including fiber-modified and third-generation recombinant viruses, giving highly purified preparations from low-titer crude lysates with an excellent overall recovery (50-74%).
