Chemistry: molecular biology and microbiology – Animal cell – per se ; composition thereof; process of... – Insect cell – per se
Reexamination Certificate
1994-08-24
2002-06-11
Lankford, Jr., Leon B. (Department: 1651)
Chemistry: molecular biology and microbiology
Animal cell, per se ; composition thereof; process of...
Insect cell, per se
Reexamination Certificate
active
06403375
ABSTRACT:
FIELD OF THE INVENTION
The invention pertains to the field of insect cell lines. More particularly, the invention pertains to
Trichoplusia ni
cell lines established in serum-free medium.
BACKGROUND OF THE INVENTION
Since Grace (1962) established the first insect cell line, insect cell culture has been progressing at a rapid rate. Insect cells have been used successfully to produce recombinant proteins, pharmaceutical and new biopesticides, due to the development of the baculovirus expression vector system (BEVS) (Ribeiro and Crook, 1993). This system utilizes the strong polyhedrin and p10 promoters (Miller, 1988) to produce large amounts of protein. To date, more than 200 foreign genes from viral, bacterial, invertebrate, mammalian and plant species have been expressed successfully with the BEVS. However, cell lines derived from different insect species differ in their ability to produce virus or express recombinant proteins. Furthermore, culture conditions and media type play major roles in choosing the appropriate cell line and system for a particular application.
One of the insect cell lines which has shown great promise is BTI-Tn-5B1-4, established at Boyce Thompson Institute, Ithaca, N.Y. and commercially available as High Five™ cells from Invitrogen Corp. The cell line is on deposit at the American Type Culture Collection as ATCC CRL 10859. This novel cell line is the subject of U.S. Pat. No. 5,300,435, granted Apr. 5, 1994. These cells were derived from eggs of the Cabbage Looper (
Trichoplusia ni
). They have been found to be particularly susceptible to baculoviruses, which are adaptable to genetic modifications which lead to high levels of production and secretion of proteins.
Recent data suggests that different insect cell lines differ in their capacity to produce occlusion bodies (OBs) and recombinant proteins. It has been previously reported that BTI-Tn-5B1-4 (Tn-5B1) cells produced a minimum for 20-fold more secreted alkaline phosphatase (SEAP) on a per cell basis than the IPLB-Sf-21 (Sf-21) cell line derived from
Spodoptera frugiperda.
Along with the development of baculovirus expression vectors, the development of serum-free media (SFM) for the growth of insect cells and for virus replication, has become critical to the success of large-scale production operations. The primary reasons for this are the high cost of fetal bovine serum (FBS) and the various components of FBS that make the downstream purification and recovery process difficult. At present, most of the attention is being focused on the adaptation of cell lines to serum-free media and the development of new serum-free media which will broadly support the replication of baculoviruses and the expression of recombinant proteins. One approach has been to investigate the use of commercial serum replacements in place of serum.
Stiles et al. (1992) recently established the first Coleopteran cell lines in serum-free medium; however, no information on the cell lines ability to support the replication of baculoviruses and express recombinant proteins was reported. While this was the first known invertebrate cell line to be established in serum-free medium, it was unknown whether an attempt to establish a Lepidopteran cell line in serum-free medium would be successful. Those skilled in the art have always believed that it is necessary to establish cell lines in medium containing serum and then adapt the cell lines to serum-free conditions.
SUMMARY OF THE INVENTION
New cell lines from the cabbage looper,
Trichoplusia ni
, have been established in a commercially-available, serum-free medium.
The cell lines were screened with, and found to be susceptible to, AcMNPV-1A and TnSNPV. Selected cell lines demonstrating superior growth qualities and virus susceptibility were chosen for further screening. A cell line designated BTI-Tn4B (Tn4B) and a daughter cell line designated BTI-Tn4B31, produced more occlusion bodies per cell than any other cell line tested and were approximately three-fold more productive than IPLB-Sf-21 cells. Bioassay results showed that the occlusion bodies produced in Tn4B cells were more infectious than those derived from IPLB-sF-21 cells and
T.ni
larvae. To evaluate the expression capacity of the new cell lines for recombinant protein, two recombinant AcMNPV viruses, E2-&bgr;-Gal and AcSEAP, were used and compared with IPLB-SF-21 and another
T.ni
cell line, BTI-Tn-5B1-4, available through Invitrogen under the trademark, High Five™, which is known to express recombinant proteins at high levels. Under the same conditions, &bgr;-galactosidase and alkaline phosphatase production in two cell lines—Tn4B and Tn4B31—produced more of these proteins than IPLB-Sf-21 cells, but were not significantly different from BTI-Tn-5B1-4 cells.
The establishment of these new cell lines in serum-free medium is a great step forward in the art of insect cell lines. The new cell lines make the cost of producing recombinant proteins and large scale quanities of viral pesticides much less expensive. This decrease in production expenses is an important step towards more economical and safer insect control.
More advantages and features of the new cell lines will become apparent from the drawings and following detailed description.
REFERENCES:
patent: 5348877 (1994-09-01), McKenna et al.
Boyce Thompson Institute for Plant Research Inc.
Brown & Michaels PC
Lankford , Jr. Leon B.
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