A sensitive method for the detection of residual cell DNA in a recombinant hepatitis B vaccine prepared from Chinese hamster ovary cells

Yoneyama, Tetsuo; Qi, Zi-bai; Miyamura, Tatsuo

doi:10.1016/s0092-1157(89)80008-3

Cited by 4 publications

(1 citation statement)

References 11 publications

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“…Standards and the extracted samples were blotted onto a nylon membrane (Nytran 0.45, Schleicher and Schuell) and fixed using UV radiation. Radiolabeled CR2-DNA probe (Prime It II kit, Stratagene) was incubated with the membrane (Yoneyama et al, 1989). Following exposure to Kodak Biomax film for 6 h the film was developed (X-OGRAPH Compact X2) and scanned using a densitometer (Molecular Dynamics).…”

Section: Complementing Cell Cr2-dna Assaymentioning

confidence: 99%

The affinity adsorptive recovery of an infectious Herpes Simplex Virus vaccine

O'Keeffe

Johnston

Slater

1999

Biotechnol. Bioeng.

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The chromatographic purification of a recombinant Herpes Simplex Virus (type 2) from salt‐ and heparin‐released harvests of infected complementing Vero (CR2) cells is addressed. Functionalized matrices and process operating conditions are identified that provide adequate virus titres in eluates that are significantly reduced in CR2 cell protein and DNA and possess a low level of HSV‐2 protein. Virus from diluted salt‐released harvests (0.14 M NaCl) was not appreciably adsorbed onto either heparin‐Sepharose or Cellufine‐heparin matrices but was virtually completely adsorbed onto Cellufine‐sulfate and heparin‐HP matrices. Virus was recovered by either a linear salt gradient elution (0.14–2 M NaCl) or by a single‐step elution with 1.5 M NaCl in phosphate buffer. Recoveries of infectious virus with step elution were 21% and 89%, respectively, for these matrices. Virus from undiluted salt‐released harvest (0.8 M NaCl) was substantially adsorbed onto Cellufine‐sulfate gel (44% adsorption) and completely adsorbed onto heparin‐HP matrices. This virus was recovered with high yield by either gradient or step elution with phosphate‐buffered saline. Finally, heparin‐harvested virus was fed directly to these matrices and quantitatively adsorbed. The virus could be completely recovered from the heparin‐HP matrix with 1.5 M NaCl buffer to provide a purified preparation containing only 0.05 pg protein/pfu and 1.2 × 10−4 pg DNA/pfu. © 1999 John Wiley & Sons, Inc. Biotechnol Bioeng 62: 537–545, 1999.

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Section: Complementing Cell Cr2-dna Assaymentioning

confidence: 99%