Showing papers by "Paul F. Greenfield published in 1997"

The indirect effects of multiplicity of infection on baculovirus expressed proteins in insect cells: secreted and non-secreted products.

Abstract: The baculovirus expression vector system was employed to produce human apolipoprotein E and β-galactosidase in order to study the effect of multiplicity of infection on secreted and non-secreted recombinant protein production. Prior knowledge of the influence of other cell culture and infection parameters, such as the cell density at time of infection and the time of harvest, allowed determination of the direct and indirect influences of multiplicity of infection on recombinant protein synthesis and degradation in insect cells. Under non-limited, controlled conditions, the direct effect of multiplicity of infection (10−1−10 pfu/cell) on specific recombinant product yields of non-secreted β-galactosidase was found to be insignificant. Instead, the observed increased in accumulated product was directly correlated to the total number of infected cells during the production period and therefore ultimately dependent on an adequate supply of nutrients. Only the timing of recombinant virus and protein production was influenced by, and dependent on the multiplicity of infection. Evidence is presented in this study that indicates the extremely limited predictability of post-infection cell growth at very low multiplicities of infection of less than 0.1 pfu/cell. Due to the inaccuracy of the current virus quantification techniques, combined with the sensitivity of post-infection cell growth at low MOI, the possibility of excessive post-infection cell growth and subsequent nutrient limitation was found to be significantly increased. Finally, as an example, the degree of product stability and cellular and viral protein contamination at low multiplicity of infection is investigated for a secreted recombinant form of human apolipoprotein E. Comparison of human apolipoprotein E production and secretion at multiplicities of infection of 10−4−10 pfu/cell revealed increased product degradation and contamination with intracellular proteins at low multiplicities of infection.

Substrate limitation in the baculovirus expression vector system.

Abstract: The inability to infect insect cell cultures at the highest achievable cell densities has imposed major limitations to both the fundamental understanding of the Baculovirus Expression Vector System (BEVS) as well as full exploitation of its potential productive capacity for recombinant (β-galAcNPV) products. The current literature does not characterize and identify the exact nature of the observed limitations, which therefore has become the major objective and contribution of the following study. Critical densities for infection of Spodoptera frugiperda (Sf9) cells with nuclear polyhedrosis virus expressing β-galactosidase (Autographa californica) grown in media both containing fetal calf serum (FCS) and free of serum were found to be at 2 × 106 and 5 × 106 cells/ml respectively. Medium exchange was found to completely reverse the effect if renewed up to 24 hours post-infection (HPI). The inevitable arrest of uninfected cell growth and decreased production of recombinant products at high cell densities of infection were both correlated to nutrient depletion. Cystine was found to be depleted in uninfected insect cell cultures at the onset of the stationary phase and in serum-free insect cell cultures infected with baculovirus above a cell density of 5 × 106 cells/ml. Neither glucose depletion nor accumulation of possible inhibitory metabolites such as alanine, ammonia, or lactate could be correlated to growth arrest or decreased recombinant product yields. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng56: 32–44, 1997.

The impact of generic competitive strategies on corporate R&D: an empirical study in Japan

Abstract: This project sought to explore the impact of generic competitive strategies on corporate research and development (R&D) in Japanese technology-based companies through an extensive survey. It results in the generation of information on how Japanese firms approach the integration of R&D and corporate strategic management. Research hypotheses are empirically tested on the basis of the feedback provided by R&D managers from eighty-five leading companies in Japan. The results suggest that both cost leadership and differentiation have considerable impact on corporate R&D. However, Japanese managers particularly place great emphasis on the overall differentiation, because the strategy enables companies to satisfy the increasing demand of customers from a broad range of market segments. We also find out that Japanese managers in different types of industries and different sizes of firms tend to perceive each generic strategic alternative similarly important. Finally, we conclude that consistency with a firm's competitive strategy is considered essential for strategic R&D management.

Major considerations in the corporate development of R&D strategies within Australian technology-based firms

Abstract: The work reported in this paper empirically examines major considerations in corporate R&D decision making in Australia. A set of hypotheses is tested through analysing the data obtained from one hundred and twelve technology-based companies with R&D activities. Australian R&D managers generally perceive that consistency with customer requirements, a firm's competitive strategy and the competitive market environment is critical for strategic R&D decision making. They also believe that R&D decisions must take into account both current technological trends and a firm's resources and capability. Australian companies, while taking advantage of government tax incentives for industrial R&D, do not regard such incentives as key to formulating corporate R&D strategies.