Eduardo Doménech Martínez

Large-scale production of recombinant hepatitis B surface antigen from Pichia pastoris

The ability of the Pichia pastoris-based technology for large-scale production of recombinant hepatitis B virus surface antigen (HBsAg) and both reproducibly purify HBsAg and remove most of the relevant contaminants was ascertained by evaluating ten industrial production batches, five in 1993 and five in 1998. At an early stage, the clarification of mechanically disrupted yeast cells by acid precipitation renders HBsAg with a purity as low as 3.8 +/- 0.6%. However, by adsorption/desorption from diatomaceous earth matrix, the purity of HBsAg rapidly increases to 18.8 +/- 5%, which is suitable for chromatographic processing. This step also eliminates non-particulated forms of HBsAg, significantly lowers the amount of carbohydrates and lipids, and concentrates the HBsAg 4.8-fold. Finally, a sequential purification procedure that includes large-scale immunoaffinity, ion-exchange, and size-exclusion chromatographies further purifies the preparation, resulting in a product (HBsAg at a concentration of 1.3 +/- 0.2 g l-1) with a purity of 95% or more. Furthermore, each of the other contaminants measured reaches the following low levels per 20 micrograms HBsAg: host deoxyribonucleic acid (< 10 pg), carbohydrates (1.2 +/- 0.02 micrograms), lipids (14 +/- 0.28 micrograms), immunopurification-released immunoglobulin G (less than 100 ppm), and endotoxins (106.7 +/- 19.3 pg). These values are below those specified for recombinant DNA hepatitis B vaccines according to World Health Organization (WHO) guidelines.

Extrauterine life duration and ontogenic EEG parameters in preterm newborns with and without major ultrasound brain lesions

OBJECTIVE To determine whether extrauterine life duration is associated with different ontogenic electroencephalographic (EEG) patterns in premature infants compared with neonates born at later gestational ages (GAs). METHODS We included 97 preterm infants of 24-36 weeks GA. We performed neurosonography and EEG recordings at week 1 and 2 of postnatal life, then every two weeks until term conceptional age (CA). We analyzed background EEG and sleep states and compared them at matched CA. RESULTS In the group of infants without major ultrasound lesion (MUL), those <29 weeks GA showed increased sawtooth at 32-36 weeks CA and delta brushes from 34 to 40 weeks CA compared to infants > or =29 weeks GA. Infants with MUL compared to those without MUL showed significantly longer interburst intervals, shorter minimum burst duration at 2 and 4 weeks postnatal life, and higher incidence of sawtooth and delta brushes from 34 weeks CA to term. In addition, those of <29 weeks GA had higher rates of indeterminate sleep until 32 weeks CA. The type of MUL most frequently associated with dysmature EEG patterns was persistent ventricular dilatation. CONCLUSIONS Premature infants without MUL showed prolonged dysmature EEG patterns as from early CAs (> or =29 weeks). This finding was more evident in those with MUL. SIGNIFICANCE Extrauterine EEG development in prematures may differ from that of babies born at a later GA.

Positive temporal sharp waves in preterm infants with and without brain ultrasound lesions

OBJECTIVE Clinical significance of neonatal positive temporal sharp waves (PTS) is controversial. The aim of this work is to study (1) PTS incidence in preterm infants with or without major ultrasound lesion (MUL) per gestational age (GA), and (2) the relationship between PTS in both sleep states and other electroencephalographic (EEG) findings with poor prognoses. METHODS 97 preterm infants of <27-36 weeks GA, and 12 full-term healthy infants were presented. Prospective study included (1) neurodevelopmental assessment at 40-42 weeks conceptional age (CA), (2) serial neurosonography, and (3) EEG recording at postnatal week 1, 2, 4 and at 40-42 weeks CA. RESULTS In 50 neonates without MUL, peak PTS was at 31-32 weeks GA. In 47 neonates with MUL, PTS increased significantly from week 2 after birth, descending at the 4th. Neonates of <33 weeks GA with MUL showed significantly increased PTS at term. A significant relationship was found between PTS and other EEG abnormalities with poor neurologic prognoses. PTS incidence varied with sleep states, being predominant in indeterminate sleep in neonates with MUL. CONCLUSIONS PTS increased significantly in infants with MUL, mainly at week 2 of postnatal life, persisting high until term CA, and correlated with other abnormal EEG findings. SIGNIFICANCE PTS are highly sensitive to MUL.