Jean-Christophe Drugmand

Insect cells as factories for biomanufacturing

Insect cells (IC) and particularly lepidopteran cells are an attractive alternative to mammalian cells for biomanufacturing. Insect cell culture, coupled with the lytic expression capacity of baculovirus expression vector systems (BEVS), constitutes a powerful platform, IC-BEVS, for the abundant and versatile formation of heterologous gene products, including proteins, vaccines and vectors for gene therapy. Such products can be manufactured on a large scale thanks to the development of efficient and scaleable production processes involving the integration of a cell growth stage and a stage of cell infection with the recombinant baculovirus vector. Insect cells can produce multimeric proteins functionally equivalent to the natural ones and engineered vectors can be used for efficient expression. Insect cells can be cultivated easily in serum- and protein-free media. A growing number of companies are currently developing an interest in producing therapeutics using IC-BEVS, and many products are today in clinical trials and on the market for veterinary and human applications. This review summarizes current knowledge on insect cell metabolism, culture conditions and applications.

Microcarrier Culture of Lepidopteran Cell Lines: Implications for Growth and Recombinant Protein Production

Several microcarrier systems were screened with Sf‐9 and High‐Five cell lines as to their ability to support cell growth and recombinant (β‐galactosidase) protein production. Growth of both cell lines on compact microcarriers, such as Cytodex‐1 and glass beads, was minimal, as cells detached easily from the microcarrier surface and grew as single cells in the medium. Cell growth was also problematic on Cytopore‐1 and ‐2 porous microcarriers. Cells remained attached for several days inside the microcarrier pores, but no cell division and proliferation were observed. On the contrary, insect cells grew well in the interior of Fibra‐Cel disks mainly as aggregates at points of fiber intersection, reaching final (plateau) densities of about 4 × 106 (Sf‐9) and 2.7 × 106 (High‐Five) cells mL‐1 (8 × 106 and 5.5 × 106 cells per cm2 of projected disk area, respectively). Their growth was described well by the logistic equation, which takes into account possible inhibition effects. β‐Galactosidase (β‐gal) production of Sf‐9 cells on Fibra‐Cel disks (infected at 3.3 × 106 cells mL‐1) was prolonged (192 h), and specific protein production was similar to that of high‐density free cell infection. Cultispher‐S microcarriers were found to be a very efficient system for the growth of High‐Five cells, whereas no growth of Sf‐9 cells took place for the same system. Concentrations of about 9 × 106 cells mL‐1 were reached within 120 h, with cell growth in both microcarriers and aggregates, appearance of cellular bridges between microcarriers and aggregates, and eventual formation of macroaggregates incorporating several microcarriers. Specific protein productions after β‐gal baculovirus infection at increasing cell concentrations were almost constant, thus leading to elevated volumetric protein production: final β‐gal titers of 946, 1728, and 1484 U mL‐1 were obtained for infection densities of 3.4, 7.2, and 8.9 × 106 cells mL‐1, respectively.

Scale-up of hepatic progenitor cells from multitray stack to 2-D bioreactors

Promethera Biosciences (Mont-St-Guibert, BE) is developing cell therapies to treat several liver genetic metabolic diseases, such as the Crigler-Najjar syndrome. Human heterologous adult liver progenitors cells (HHALPCs) were initially cultivated in 2D standard cultivation devices. The present study is investigating the feasibility to cultivate HHALPCs in Xpansion bioreactors, with the following objectives: ➢ The process must be closed ➢ The growth rate and population-doubling level (i.e. the number of times the cells in the population has doubled) must be at least equivalent to the current process in multilayer trays ➢ The process must comply to the cGMP rules ➢ The cells must succeed the quality control (QC) test specifications at the end of cultivation, i.e. cells must remain undifferentiated and show the presence of HHAPLCs markers, while exhibiting the capacity to differentiate toward functional hepatocytes. Integrity® Xpansion™ multiplate bioreactors have been specifically designed to enable an easy transfer from existing multiple-tray-stack processes by offering the same cell growth environment on 2-D hydrophylized Polystyrene (PS) plates in a fully closed system. To make the bioreactors compact, the headspace between each plate has been reduced to a minimum (1.3 mm). Gas transfer is made through a semi-permeable silicone tubing mounted in the central column. Additionally, critical cell culture parameters such as pH and DO are controlled and the cell density is automatically monitored via a specific holographic microscope developed by Ovizio

Environmental Effects of Lactate on High-Five™ Insect Cell Metabolism

In batch suspension culture of High-Five™ insect cells without oxygen limitation, we observed that during the growth phase the presence of exogenous lactate in the medium seems to increase its accumulation by the cells and inhibits cell growth. At the end of culture, after glucose exhaustion from the medium, High-Five™ cells are able to use the produced lactate as a carbon source.

Synthetic peptide matrices as support for stem cells culture

Background Nowadays, stem cells draw great interest in regenerative medicine [1]. However, due to low occurrence in tissues, their in vitro expansion is required to gain access to therapeutic applications [2]. Furthermore, the existing culture systems often include xeno-derived components, present upscaling limitations [3,4] and do not necessary address important considerations, such as those related to biosafety, availability and homogeneity of (biological) raw material and process reproducibility. Moreover, the regulatory guidelines focus on the importance of defined environments, minimal handling and continuity between development and industrialization scales [5,6]. In this context, new scalable solutions a reactively considered in order to achieve production of stem cells at higher densities [7]. Among these, synthetic peptides are of particular interest [4] as potential alternatives to xeno-derived culture supports, able to mimic the physico-biochemical properties of natural cell matrices. In this way our work aims at assessing new xeno-free culture matrices based on short, synthetic and bioactive peptides and demonstrating the scalability, reproducibility and performance of such peptides-based matrices for human stem cells culture. This report is focused on a part of the work performed to study the stability of synthetic peptide coatings on polymer surfaces and the capability of such coatings to favour stem cells adhesion and growth.

New peptide-based and animal-free coatings for animal cell culture in bioreactors

Background Anchorage dependent cells require an appropriate extracellular matrix for their survival, migration, proliferation, phenotyping and/or differentiation [1-3]. These cells interact with extracellular matrix proteins, primarily through integrins, which induces focal adhesion contacts assembly and activation of signalling pathways that regulate diverse cellular processes [4]. Culture supports usually include biochemical components allowing such cells to adhere and to reconstitute an extracellular environment close to that found in vivo. Currently, this artificial environment is achieved by extracellular matrix constituents deposition, adsorption or grafting; among them collagens, fibronectin, laminin, artificial lamina propria... [5]. However, such animal proteins used in cell culture may induce pro-inflammatory stress, be unstable against proteolysis or loose activity after adsorption [6,7]. Synthetic microenvironments should be more suitable for clinical purposes: (i) improved control of physicochemical and mechanical properties, (ii) limited risks of immunogenicity, (iii) increased biosafety (animal free) and (iv) facilitated scale-up [1]. In this framework, research has recently focused on synthetic peptides or peptidomimetics that can mimic the extracellular matrix. Such molecules can be immobilized as recognition motifs on the surface of culture supports with a greater stability and easier surfaces characterization [5]. Self-assembling peptide hydrogels could mimic the chemical and mechanical aspects of the natural extracellular matrix [8,9] by undergoing large deformations, as in mammalian tissues. They have an inherent biocompatibility and should be able to direct cell behaviour [10]. They also can be functionalized with various biologically active ligands constituting good candidates to a new range of smart biomaterials, able to ensure adhesion of different cell types [11-13]. The range of biomimetic peptides that direct cell adhesion and are recognized by integrins is large. Recognition sequences derived from different extracellular matrix proteins include RGD [1], which are specific to different cell lines [1,5,6]. In this context, this work aims at designing animal-free, chemically defined and industrially scalable coatings for animal cell culture, as an alternative to collagen, fibronectin or Matrigel for laboratory and industrial large scale applications. These are based on self-assembling short peptides bearing adhesion bioactive sequences like RGDderived or other adhesion sequences developed to coat polystyrene or polyethylene terephthalate surfaces. Adhesion sequences should be recognized by cells, which should favour their anchorage and spreading.

Analysis and Modeling of the Metabolism of Lepidopteran Cell Lines

The use of BioNOC II microcarriers in a fixed bed bioreactor setup allows to produce high protein levels in a CHO-320 cell line expressing constitutively interferon-γ and also with insect cells used with a baculovirus transient expression vector.

Physiology of Insect Cells Cultured in a New Serum-Free Medium

The use of insect cells for the production of recombinant proteins by the baculovirus expression system is a well-established technology. The scale-up of this system necessitates large media quantities and serum substitution. We report here the design of a new serum-free medium and the physiological behaviour of insect cells in this medium under relevant bioprocess conditions.

Growth of High-Five™ Cells on Cultispher-S Microbarriers

Growth of High-Five™ cells on Cultispher-S microcarriers (1 g/l) produces extended clumps by adhesion, agglomeration and aggregation of cells on microcarriers and leads to increased protein productivity, two-fold for SEAP and three-fold for β-gal, using a baculovirus expression vector system.