Biotechnology Letters 23: 359–364, 2001.
© 2001 Kluwer Academic Publishers. Printed in the Netherlands.
Cell size as a tool to predict the production of recombinant protein by the
insect-cell baculovirus expression system
Laura A. Palomares
, Juan Carlos Pedroza
Departamento de Bioingenier´ıa, Instituto de Biotecnolog´ıa, Universidad Nacional Aut´onoma de M´exico, A.P.
510-3, Cuernavaca Morelos, M´exico
Centro de Investigaci´on en Biotecnolog´ıa, Universidad Aut´onoma del Estado de Morelos, M´exico
Author for correspondence (Fax: +52 (7) 3138811/+52 (7) 3172388; E-mail: email@example.com)
Received 24 October 2000; Revisions requested 23 November 2000; Revisions received 11 December 2000; Accepted 13 December 2000
Key words: baculovirus, culture monitoring, insect cell size, metabolism, recombinant protein
The increase of Sf9 cell diameter after infection with a recombinant baculovirus encoding VP8 protein of ro-
tavirus can be used to predict culture productivity. A direct proportional correlation between the increase in cell
size and VP8 concentration was obtained when manipulating selected medium components. Only yeast extract
increased (38%) VP8 concentration, while fetal bovine serum increased (55%) the maximum cell concentration.
An inexpensive and simpliﬁed culture media can thus be designed without detriment to protein yields.
The insect cell-baculovirus expression system (IC-
BES) is the system of choice for selected applica-
tions, such as production of vaccines or proteins for
diagnostics, however, it is still far from being opti-
mized. Cell size is a simple and reliable parameter
that can easily be used in process monitoring and con-
trol schemes. Thus, cell size can be a powerful tool
for increasing process performance. It can be read-
ily determined in equipment commonly available in
cell culture facilities, but its utility has not been fully
exploited. Changes in cell size correlate with phases
of cell cycle or with cell death (Ramírez & Mutha-
rasan 1990, Meneses-Acosta et al. 2001). Ljunggren
et al. (1999) predicted the optimum time of infection
by following the diameter of uninfected insect cells.
Moreover, uninfected insect cell size also correlates
with the potential of a cell line to produce large quan-
tities of recombinant protein (Chai et al. 1996). The
infection of insect cells drastically increases cell di-
ameter (30–40%), as documented by some research
groups (Schopf et al. 1990, Jain et al. 1991, Kamen
et al. 1996, Taticek & Shuler 1997, Rosinski et al.
2000). Such an increase is proportional to the efﬁ-
ciency of virus replication (Taticek & Shuler 1997).
Jain et al. (1991) found a direct correlation with the
time of maximum modal cell diameter and the time of
maximum recombinant protein concentration. Thus,
harvest strategies have also been designed based on
the kinetics of cell size increase after infection.
Cell size is also determined by the cell metabolic
state, which in turn is a function of nutritional con-
ditions (Ramírez & Mutharasan 1990). Moreover, ad-
equate nutrient supply after viral infection maintains
culture viability and provides substrates for recom-
binant protein production. Glutamine, yeast extract
and fetal bovine serum are key nutrients for insect
cells, as they determine cellular metabolism (Öhman
et al. 1995, Mendonça et al. 1999), provide vitamins,
trace elements, lipids, and hormones (Drews et al.
1995, Ferrance et al. 1993), and even affect the kinet-
ics of virus attachment to cells (unpublished results).
Moreover, carbon sources are also important as they
modulate cell growth (Drews et al. 1995, Palomares
& Ramírez 1996). In this work we have evaluated
the utility of insect cell diameter to predict recombi-
nant protein productivity. Cultures were manipulated
by utilizing different media formulations which, in
turn, resulted in different recombinant protein con-