For Culture Media Use - Trehalose SG by Nagase Viita

Founded as a starch syrup manufacturer in 1883, Nagase Viita (Formerly Hayashibara Co., Ltd.) has grown into a company with strong research and development expertise, engaging in original and creative research. We present the latest brochure “For Culture Media Use – Trehalose SG by Nagase Viita”:

What is TREHALOSE SG？

TREHALOSE SG is a dihydrous, crystalline and non-reducing disaccharide consisting of two glucose molecules linked by an α,α-1,1 bond.
TREHALOSE SG is white crystalline powder.
TREHALOSE SG is very soluble in water and very heat stable.
TREHALOSE SG is an injectable grade of pharmaceuticals and is monographed as being low endotoxin.

Evaluation of the suppression of protein aggregation in CHO cell culture ①

Materials & Methods (1)

1. Protein solution of a bispecific antibody (bispecific
single-chained diabody with Fc, scDb-Fc, 0.8 mg/mL)
was prepared with or without addition of
trehalose (200 mM) in culture medium.
2. Circular dichroism (CD) spectroscopy of the
scDb-Fc protein contained in the supernatant was
measured.

Materials & Methods (2)

1. The scDb-Fc (0.8 mg/mL) solution prepared in the
same manner as described in M & M (1) was
heat-treated at 60℃ for 5 minutes.
2. The precipitate was removed by centrifugation.
3. The soluble fraction of scDb-Fc protein in the
supernatant was measured.

Fig.1 CD spectrum analysis

Results

▲ Addition of trehalose (200 mM) had no effects on the structure of scDb-Fc protein (Fig. 1).
▲ Protein aggregation caused by heat denaturation was greatly reduced by trehalose (Fig. 2).

Evaluation of the suppression of protein aggregation in CHO cell culture ②

Materials & Methods

The CHO Top-H cell line producing a bispecific antibody (single-chained diabody with Fc: scDb-Fc) was grown
in cell culture media containing 150 mM trehalose. The cells were then cultured in an animal cell culture
bioreactor (1 L scale, medium capacity 0.7 L) with or without addition of 150 mM trehalose.
After purifying the scDb-Fc protein from the culture supernatant by protein A affinity chromatography, the
secondary structures (monomer, dimer and large aggregates), and their cohesiveness were evaluated by
circular dichroism (CD)/fluorescence spectroscopy and gel filtration column chromatography, respectively.

Results

▲ A decrease in the ratio of large aggregates was observed by adding trehalose to the culture medium as compared with the culture medium without trehalose. On the other hand, the ratio of the monomeric scDb-Fc protein, which is an indication of no aggregation, increased while no effect on the dimer was observed (Fig. 3).

▲ The secondary structure of the dimer was similar to that of the monomer with antibody-like β-strand structure (Fig. 4).

▲ The large aggregates had both a non-native β-strand structure and a misfolded structure in which a hydrophobic region is largely exposed (Fig. 5).

Fig. 3 Gel filtration column chromatogram

Conclusion

It was possible to culture the antibody-producing CHO cell line in the presence of TREHALOSE SG.
TREHALOSE SG can suppress antibody aggregation, especially the formation of high-order aggregates, during the cell culture process.
Use of TREHALOSE SG appears to increase efficiency of functional antibody production

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Source: Nagase Viita, Masayoshi Onitsuka and Takeshi Omasa: Institute of Technology and Science, The University of Tokushima. 16th Trehalose Symposium (2012), “For Culture Media Use – Trehalose SG by Nagase Viita”

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