Quality assurance after process changes of the production of a therapeutic antibody.

Process development for the production of a therapeutic humanised antibody is a very complex operation. It involves recombinant genetics, verification of a strong expression system, gene amplification, characterisation of a stable host cell expression system, optimisation and design of the mammalian cell culture fermentation system and development of an efficient recovery process resulting in high yields and product quality. Rapid progress in the field and the wish of some pharmaceutical companies for outsourcing their production are the driving forces for process changes relatively late in the development phase. This literature survey is aimed at identifying the limits of acceptable process changes in up scaling of the fermentation and down stream processing of biopharmaceuticals and defining the demand in production validation to prove product equivalency and identity of the isolated, purified therapeutic antibody.

Differential effect of food on kinetics of bromocriptine in a modified release capsule and a conventional formulation.

The influence of food on release of drug from a modified release capsule of bromocriptine 5 mg (Parlodel SRO) and a conventional formulation of bromocriptine 5 mg has been studied in 8 healthy male volunteers. Both formulations produced objective and subjective effects, such as orthostatic reactions, nausea, dizziness, vomiting and nasal congestion. The modified release capsule caused fewer side-effects than the normal capsule. Both formulations had less cardiovascular effect in the fed than in the fasting state. There was no significant difference between the normal and the modified release capsules taken fasting or after a meal in terms of the AUC extrapolated to infinity. The relative bioavailability of the 5 mg modified release capsule was 84.6% of the normal capsule under fasting conditions and 107.5% after food. In contrast to the virtually unchanged extent of absorption, the rate of absorption was markedly affected by food, especially from the conventional capsule. The mean time of 50% absorption increased from 1.06 h (fasting) to 3.2 h (fed), whereas for the modified release capsule food mainly resulted in an increased lag time of absorption. The almost instantaneous dissolution of bromocriptine from the normal capsule in vitro (both in HCl and fasting human gastric juice) and the delay of absorption after a meal in vivo suggest that the rate limiting step in absorption of the normal capsules is delivery of released drug from the stomach to the small intestine, which is delayed by food. Both the modified release 5-mg capsule and the normal 5-mg capsule showed extended suppression of prolactin over 36 h, in all subjects, both fasted and after a meal.

High-performance liquid chromatographic determination of enantiomeric amino acids and amino alcohols after derivatization with o-phthaldialdehyde and various chiral mercaptans. Application to peptide hydrolysates.

o-Phthaldialdehyde in combination with a chiral mercaptan is a powerful chiral reagent for the pre-column derivatization of many enantiomeric compounds bearing primary amino groups. The diastereoisomers formed can efficiently be resolved on conventional reversed-phase columns. Simultaneous determination of the enantiomers of various amino acids, amino alcohols and biogenic amines was achieved by gradient elution and fluorescence detection. The resolution was optimized by varying the chiral mercaptan in the reagent, Boc-L-cysteine, N-acetyl-L-cysteine and N-acetyl-D-penicillamine being used for this purpose. The resolutions were calculated. Most of the enantiomers showed good resolution with each of the three chiral mercaptans, whereas some enantiomers were only separable by one or two of them. The method was applied to the analysis of peptide hydrolysates. The composition of peptides bearing L- and D-amino acids and an amino alcohol was determined.

Quantitative analysis of nonapeptides in pharmaceutical dosage forms by high-performance liquid chromatography.

A high-performance liquid chromatographic (HPLC) method is described for determining oxytocin, lypressin and other nonapeptides and their by-products in liquid and solid pharmaceutical dosage forms. The use of injection volumes up to 750 microliter permits accurate determination of, e.g., oxytocin in injection solutions containing only 1 I.U. (international unit) per ml. Reproducibilities between 1.0 and 1.5% (relative S.D) have been obtained for liquid dosage forms and up to 3% (relative S.D.) for tablets. The correlation between results obtained by bioassay and by the proposed method is highly significant and suggests the use of HPLC as an alternative technique for stability and content-uniformity tests on dosage forms and concentrates.

The separation of nonapeptides by reversed-phase high-performance liquid chromatography.

The separation properties of five nonapeptides on commercial reversed-phase materials have been investigated and the effects of pH, salt concentration and solvent composition have been studied. With appropriate variation of the pH and salt concentration in the mobile phase, it is possible to resolve all of the peptides investigated and their by-products. Mixtures of water and organic solvents (acetonitrile, dioxan, methanol and n-propanol) have been used. The choice of the organic solvent does not strongly influence the separation pattern. The simplicity, speed and quality of the separations and the favourable detection limits (ca. 30 ng) at 220 nm render this technique suitable to routine quantitative analysis.