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July 24, 2008

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Kimberley Buytaert-Hoefen, Ph.D.

An Alternative to Transferrin for Cell Culture in Animal-Free Conditions: Recombinant Human Lactoferrin

The use of transferrin in cell culture is a method for providing iron to the cells. The delivery of iron using transferrin has been well documented for cell culture systems and in biomanufacturing of therapeutic proteins, such as monoclonal antibodies. Transferrin is derived from both animal and human serum sources for use in these processes. Recently, a move is being made towards animal and human-free cell culture systems. Iron chelators have been used although many chelators do not control the redox cycling of iron that contributes to oxidative stress. The employment of a human recombinant alternative to transferrin would prove optimal for cell culture as well as for biomanufacturing.

Recombinant human lactoferrin is a 80 kD glycoprotein belonging to the transferrin family of proteins. Lactoferrin has an amino-acid homology and similar iron binding properties to human transferrin. It is present in human milk and epithelial cell surfaces and in blood plasma and tears. Lactoferrin is a multi-functional protein, as such it is a cell growth factor, a source of iron, delays apoptosis and displays anti-microbial properties. Lactoferrin has been reported to have synergistic effects with both EGF (Kohno et al., Acta. Paedietr., 1993) and bFGF on cell growth (Shinoda et al., Adv. Exp. Med. Biol., 1994).

Lacromin™

Lacromin™ (InVitria) is a cell culture grade recombinant human lactoferrin that is produced from an animal-free plant based expression system, ExpressTec (Nandi et al., Transgen. Res., 2005). The ExpressTec system achieves protein expression using the self-pollinating crops of rice and grain. The advantages of ExpressTec include high and stable expression of recombinant proteins, tissue specific expression in grain endosperm, rapid scalability to metric-ton quantities, prevention of gene flow with self pollinating crops, low capital investment and production costs, and efficient processing and recovery (Huang, BioProcess, 2004). Since Lacromin™ is derived from plants, it is free of any viral or prion contaminant risks.

Lacromin™ has been shown to increase cell growth in many cell lines, including chinese hamster ovary (CHO) cells, human embryonic kidney (HEK) cells and mouse hybridoma cells when compared with transferrin. In hybridoma cells, Lacromin™ increased IgG productivity by 50% compared to transferrin (InVitria Lacromin™ product brochure, www.InVitria.com).

The use of Lacromin™ as a replacement for serum derived transferrin would provide higher cell density and reduction of cell doubling time under animal-free conditions (Haung et al., In Vitro Cell. Dev. Biol.-Animal., 2008). The employment of a human recombinant alternative to transferrin would prove optimal for cell culture as well as for biomanufacturing.

Forbes.com, Press Release: http://www.forbes.com/prnewswire/feeds/prnewswire/2008/10/10/prnewswire200810101418PR_NEWS_USPR_____AQF037.html.

Kimberley Buytaert-Hoefen, Ph.D.

An Alternative to Knockout Serum Replacement for Culturing Human Embryonic Stem Cells in Animal-Free Conditions: Recombinant Human Serum Albumin

Human embryonic stem cells (hES) due to their ability to differentiate into all cell types offer great promise for clinical cell based therapies. The majority of hES cell lines available to date have been either directly or indirectly exposed to animal material. This exposure can increase the risk of graft vs. host disease and the transfer of animal pathogens. The elimination of all animal material for both the derivation and long term culture of hES cells is essential for future hES cell based therapies to be produced under good manufacturing practice (GMP). GMP quality assurance used in the pharmaceutical industry, is required by both the European Medicines Agency and the Food and Drug Administration, offers optimal defined quality and safety for cellular based therapies.

Serum-Free Media

Knockout serum replacement (Ko-SR, Invitrogen) is frequently used in hES cell culture. Although, Ko-SR contains the animal derived product bovine serum albumin and hence is not animal-free (Price et al., Internat. Patent Applic. WO98/30679, 1998).

Albumin has been defined as the essential component of serum for stimulating hES cell self-renewal (Garcia-Gonzalo and Belmonte, Plos One, 2008, Xu et al., Stem Cells, 2005). A new product, recombinant human serum albumin, is available under the commercial name Cellastim (InVitria). The use of Cellastim in the culturing of hES cells would allow for animal-free culture conditions.

Cellastim

Cellastim (InVitria) is a cell culture grade recombinant human serum albumin that is produced from an animal-free plant based expression system, ExpressTec (Nandi et al., Transgen. Res., 2005). The ExpressTec system achieves protein expression using the self-pollinating crops of rice and grain. The advantages of ExpressTec include high and stable expression of recombinant proteins, tissue specific expression in grain endosperm, rapid scalability to metric-ton quantities, prevention of gene flow with self pollinating crops, low capital investment and production costs, and efficient processing and recovery (Huang, BioProcess, 2004).

Cellastim has similar biophysical characteristics and identical amino acid sequence as plasma derived human serum albumin. Cellastim is animal-free and immunoglobulin free. Studies have shown that Cellastim enhances cell growth and productivity better than or equivalent to plasma derived human serum albumin (InVitria Cellastim product brochure, www.InVitria.com). Recently, Ying et al., Nature Letters, 2008, reported using Cellastim as a substitute for bovine serum albumin in combination with transferrin and insulin for culturing mouse ES cells. This combination displayed both bulk and clonal propagation of the mouse ES cells.

The use of Cellastim as a replacement for serum derived albumin may prove beneficial for cellular expansion and maintenance of undifferentiated hES cells and would provide for optimal animal-free cell culture conditions that could be used for processing hES cell lines under GMP conditions.

Kimberley Buytaert-Hoefen, Ph.D.

Replacing Animal Serum in Cell Culture and Bioprocessing

Fetal bovine serum (FBS) is a common supplement for in vitro cell culture and bioprocessing. The composition of FBS is unknown and varies between batches, as well may contain animal viruses or prions. Because of these safety risks the elimination of all animal material is essential and offers optimal defined quality and safety for cellular based therapies and bioprocessing (Asher, Dev. in Biol. Standard., 1999).

Albumin is used in cell culture and in bio-manufacturing of therapeutic monoclonal antibodies and recombinant proteins. It is an important component of many serum-free cell culture systems such as those that utilize hybridoma or Chinese Hamster Ovary (CHO) cells. A new product, recombinant human serum albumin, is available under the commercial name Cellastim™ (InVitria). The use of Cellastim™ in the culturing of cell lines and in bioprocessing would allow for animal-free culture conditions.

Recombinant Human Serum Albumin: Cellastim™

Cellastim™ (InVitria) is a cell culture grade recombinant human serum albumin that is produced from an animal-free plant based expression system, ExpressTec (Nandi et al., Transgen. Res., 2005). The ExpressTec system achieves protein expression using the self-pollinating crops of rice and grain. The advantages of ExpressTec include high and stable expression of recombinant proteins, tissue specific expression in grain endosperm, rapid scalability to metric-ton quantities, prevention of gene flow with self pollinating crops, low capital investment and production costs, and efficient processing and recovery (Huang, BioProcess, 2004).

Cellastim™ has similar biophysical characteristics and identical amino acid sequence as plasma derived human serum albumin. Cellastim™ is animal-free and immunoglobulin free. Studies have shown that Cellastim™ enhances cell growth in hybridoma cells and antibody productivity in CHO cells better than or equivalent to plasma derived human serum albumin (InVitria Cellastim™ product brochure, www.InVitria.com).

Adapting Cells to Serum-Free Environment

Conversion of cells to a serum-free media is optimal for cell culture under animal-free conditions. This process takes 2-6 weeks to ensure the high cellular survival. Two methods have been sited for this process, sequential adaptation and adaptation with condition medium (www.invitrogen.com). The first method, sequential adaptation, calls for serum-free media supplementation at 25% for the first passage, 50% for the second passage, 75% for the third passage and 100% for the forth passage. The second process involves the adaptation with condition media. For the first passage 100% of serum supplemented media is used. For the second passage 50% of the media from passage 1 and 50% serum-free supplement is used, this process is repeated for passages 3 and 4. For the fifth passage the media is supplemented with 100% serum-free media. If cell doubling time and viability decreases prior to 100% serum-free conversion, cells should be passaged at the previous concentration for 2-3 passages to allow for cellular adjustment to the serum-free conditions. The use of a higher density cellular cultures is recommended for adaptation to serum-free conditions.

Conclusions

The conversion from serum supplemented media to serum-free media for cell culture and bioprocessing is essential for the elimination of animal components in the culturing media. The recombinant human serum albumin, Cellastim™, has been shown to enhance cell growth and productivity in cell lines under animal-free conditions and offers optimal defined quality and safety for cellular based therapies and bioprocessing (InVitria Cellastim™ product brochure, www.InVitria.com).

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