Catalase HPII from aerobically grown Escherichia coli normally contains heme d but cultures grown with poor or no aeration produce HPII containing a mixture of heme d and protoheme IX. The protoheme component of HPII from anaerobically grown cells is converted into heme d during treatment of the purified enzyme with hydrogen peroxide. It is concluded that heme d found in catalase HPII is formed by the cis-hydroxylation of protoheme in a reaction catalyzed by catalase HPII using hydrogen peroxide as a substrate. The distal His128 residue of HPII is absolutely required for the protoheme to heme d conversion. Two mutant enzymes, Ala128 and Asn128, are catalytically inactive and contain only protoheme, which is unaffected by hydrogen peroxide treatment. The Asn201 residue is not an absolute requirement for heme conversion. The mutant enzyme Ala201 contains predominantly heme d and is partially active. However, insertion of a histidyl residue to give the His201 enzyme interferes with the heme conversion reaction. This mutant form is isolated as a protoheme enzyme with limited activity, and a reversible conversion to a heme d-like species occurs in vitro in the presence of continuously generated hydrogen peroxide.

No abstract

In mammalian cell culture producing therapeutic proteins, one of the important challenges is the use of several complex raw materials whose compositional variability is relatively high and their influences on cell culture is poorly understood. Under these circumstances, application of spectroscopic techniques combined with chemometrics can provide fast, simple, and non-destructive ways to evaluate raw material quality, leading to more consistent cell culture performance. In this study, a comprehensive data fusion strategy of combining multiple spectroscopic techniques is investigated for the prediction of raw material quality in mammalian cell culture. To achieve this purpose, four different spectroscopic techniques of near-infrared, Raman, 2D fluorescence, and X-ray fluorescence spectra were employed for comprehensive characterization of soy hydrolysates which are commonly used as supplements in culture media. First, the different spectra were compared separately in terms of their prediction capability. Then, ensemble partial least squares (EPLS) was further employed by combining all of these spectral datasets in order to produce a more accurate estimation of raw material properties, and compared with other data fusion techniques. The results showed that data fusion models based on EPLS always exhibit best prediction accuracy among all the models including individual spectroscopic methods, demonstrating the synergetic effects of data fusion in characterizing the raw material quality.

Understanding variability in raw materials and their impacts on product quality is of critical importance in the biopharmaceutical manufacturing processes. For this purpose, several spectroscopic techniques have been studied for raw material characterization, providing fast and nondestructive ways to measure quality of raw materials. However, investigations of correlation between spectra of raw materials and cell culture performance have been scarce due to their complexity and uncertainty. In this study, near-infrared spectra and bioassays of multiple soy hydrolysate lots manufactured by different vendors were analyzed using chemometrics approaches in order to address variability of raw materials as well as correlation between raw material properties and corresponding cell culture performance. Principal component analysis revealed that near-infrared spectra of different soy lots contain enough physicochemical information about soy hydrolysates to allow identification of lot-to-lot variability as well as vendor-to-vendor differences. The identified compositional variability was further analyzed in order to estimate cell growth and protein production of two mammalian cell lines under the condition of varying soy dosages using partial least square regression combined with optimal variable selection. The performance of the resulting models demonstrates the potential of near-infrared spectroscopy as a robust lot selection tool for raw materials while providing a biological link between chemical composition of raw materials and cell culture performance.

Although glutamine is a major carbon source for mammalian cells in culture, its chemical decomposition or cellular metabolism leads to an undesirable excess of ammonia. This limits the shelf-life of glutamine-supplemented media and may reduce the cell yield under certain conditions. We have attempted to develop a less ammoniagenic medium for the growth of BHK-21 cells by a mole-to-mole substitution of glutamine by glutamate. This results in a medium that is thermally stable but unable to support an equivalent growth yield. However, supplementation of the glutamate-based medium with asparagine (3 mM) and a minimal level of glutamine (0.5 mM) restored the original growth capacity of the cultures. Substitution of the low level of glutamine with the glutamine dipeptides, ala-gln (1 mM), or gly-gln (3 mM) resulted in an equivalent cell yield and in a thermally stable medium. The ammonia accumulation in cultures with glutamate-based medium was reduced significantly (>60%). Factors mediating growth and adaptation in medium substituted with glutamate were also investigated. The maximum growth capacity of the BHK-21 cells in glutamate-based medium (without glutamine) was achieved after a period of adaptation of 5 culture passages from growth in glutamine-based cultures. Adaptation was not influenced by increases in glutamate uptake which was constitutively high in BHK cells. Adaptation was associated with changes in the activities of enzymes involved in glutamate or glutamine metabolism. The activities of glutamine synthetase (GS) and alanine aminotransferase (ALT) increased significantly and the activity of phosphate-activated glutaminase (PAG) decreased significantly. The activity of glutamate dehydrogenase (GDH) showed no significant change after adaptation to glutamate. These changes resulted in an altered metabolic profile which included a reduced ammonia production but an increased alanine production. Alanine production is suspected of being an alternative route for removal of excess nitrogen.