All cultures were followed by analysis of collagen II, C-P4H enzymatic activity and product-related mRNAs. The amount of type II collagen chains (139 kD) derived from correctly assembled protease-resistant triple-helical collagen II was analyzed by reducing SDS-PAGE after HCl extraction with pepsin-treatment according to Myllyharju et al. [8] . Stable and correctly assembled collagens are resistant against pepsin. Collagen II showed a clear band ( Figure 4 ) which was significantly stronger in the cultivations with the quasi-continuous feeding profile, compared to the standard pulse feeding profile. The results were reproducible (see Table 1 ) despite experiment to experiment variations. Generally, more correctly assembled triple-helical collagen was produced in the experiments with quasi-continuous feeding of methanol.