Efficient Lipid Delivery to Hybridoma Culture by Use of Cyclodextrin in a Novel Granulated Dry‐Form Medium Technology

Abstract

Lipids are critical nutrients for high density eukaryotic cell cultivation, but inclusion of lipid components into dry-form media has been technically challenging. Lipid supplements are usually supplied for separate addition after powder reconstitution and filtration, which increases manipulation and risk in a biopharmaceutical manufacturing facility. Advanced Granulation Technology (AGT) is a novel dry-form media format designed for large-scale biopharmaceutical manufacture. All components of a complex formulation are homogeneously incorporated into a single granulated medium that simply requires water addition to yield a complete reconstituted 1X medium. We investigated whether cyclodextrin technology could be combined with the AGT process to deliver usable lipid in a dry medium format. The test lipids were cholesterol and several fatty acids supplied either as an aseptic supplement to liquid media or as part of a complete AGT formulation. The test system utilized a cholesterol auxotroph, NS0, and a protein-free basal formulation (CD Hybridoma Medium), with or without supplemental lipid. Post-filtration recovery of cholesterol from AGT-processed materials compared favorably with media supplemented with liquid lipid concentrates. Cell growth and viability and expression of recombinant protein were equivalent in all test media.

Synthetic Low-Density Lipoprotein, a Novel Biomimetic Lipid Supplement for Serum-Free Tissue Culture

“…However, lipids are intrinsically hydrophobic, possessing low water solubility, but are required by cells at higher concentrations (13) than hormones. Lipid supplementation in serum‐free media is therefore problematical and currently relies on methods to enhance lipid solubility either as free fatty acids, surfactant mixed micelles, cyclodextrin (14), or protein‐based complexes and liposomes (13). These systems, however, do not present the lipids to cells in a natural form (i.e., lipoproteins) and therefore have a limited utility with potential toxicity.…”

Compaction of chemically defined cell culture media increases its dissolution rate through an increase of solvent accessible surface area

“…Granulation of cell culture media by fluid bed granulation did not influence the expression of recombinant protein or virus titer when tested with multiple cell lines [15]. Fluid bed granulation of cyclodextrin with lipids was found to be an efficient delivery method for cholesterol and other fatty acids to cell lines sensitive to these ingredients [16]. Further research is underway to evaluate roller compaction of individual ingredients including lipids, to improve their availability to cultured cells.…”

“…These results thus indicate that compaction of CHO210 and Feed210 does not impact its performance in cell culture, further suggesting no change to media composition. Studies have been carried out to investigate the influence of processing of CCM on cell culture [13,15,16]. Granulation of cell culture media by fluid bed granulation did not influence the expression of recombinant protein or virus titer when tested with multiple cell lines [15].…”

Design of Culture Media

“…Although molecules such as fatty acids and lipids may be beneficial, they often have poor solubility in aqueous solutions. These components are usually complexed with agents such as cyclodextrin or with emulsifying agents [32].…”