A potential generic downstream process using Cibracon Blue resin at very high loading capacity produces a highly purified monoclonal antibody preparation from cell culture harvest.

The use of a dye-ligand chromatography for the purification of monoclonal antibody (MAb) from cell culture and other feed streams has been largely overlooked in large scale production. Cibracon Blue dye (CB), a polycyclic anionic ligand, interacts with protein through a specific interaction between the dye, acting as a mimic of NAD+ and NADP+, or through non-specific electrostatic, hydrophobic, and other forces. In this paper, a CB resin was used to effectively and efficiently separate an IgG4 MAb from host and process impurities following the capture of the MAb on a Protein-A (PA) column. The CB unit operation, challenged at 99% by reducing SDS-PAGE). A facile three column scalable production scheme, employing CB as the second column in the process was used to generate highly purified MAb from cell culture harvest derived from two media of very different compositions. Free CB dye was

Performance comparison of protein A affinity resins for the purification of monoclonal antibodies.

During the selection of protein A affinity resin for the purification of monoclonal antibodies, dynamic binding capacity (Q(dyn10%)), volumetric production rate (Pr(vol)) and 'process robustness' are essential parameters to be evaluated. In this article, empirical mathematical models describe these parameters as a function of antibody concentration in load (C0), load flow rate (u(load)) and bed height (L). These models allow us to select optimal process conditions for each of the evaluated protein A affinity resins. C0, u(load) and L largely affect dynamic binding capacity (Q(dyn10%)) and volumetric production rate (Pr(vol)). Maximum Q(dyn10%) is generally obtained at high C0 and at low u(load). Maximum Pr(vol) is obtained at high C0 and at lowest L, run at high u(load). All evaluated resins have a relatively high robustness against variations in C0. |DeltaQ(dyn10%)/deltaC0| ranges from 0.0 to 7.8. It is clear that Q(dyn10%), Pr(vol) and 'process robustness' cannot be maximized all at the same time. Furthermore, some other aspects like IgG recovery, protein A leaching, easiness to pack, easiness to clean, number of re-uses and cost of production might be important to be taken into the equation. Certain evaluation parameters may be more important than others, depending on the specific situation. Therefore, a case-by-case evaluation is recommended.

Interpreting microbiopsies in cervical smears. A cytohistologic approach.

OBJECTIVE: To assess interobserver variation in the diagnosis of thick tissue specimens (microbiopsies) in cytology smears and histologic sections taken from them, to evaluate the applicability of MIB-1 in histologic sections from microbiopsies and to evaluate whether processing microbiopsies in inconclusive smears has additional diagnostic value. STUDY DESIGN: Cytologic smears were selected in which there were diagnostic disagreements between pathologists and cytologists and microbiopsies were present. Interobserver variation among three pathologists and three cytologists in the diagnosis of these microbiopsies was investigated. The smears were processed for histologic sections, and interobserver variation between pathologist diagnoses were analyzed. An additional histologic slide stained for MIB-1 was used for consensus diagnosis. The consensus diagnosis was compared with available follow-up and its sensitivity and specificity determined. The value of applying the microbiopsy technique in slides diagnosed as inadequate or atypical squamous cells of undetermined significance (ASCUS) was analysed. RESULTS: From a series of 62,334 cervical smears, 49 with microbiopsies were selected. It was possible to derive histologic slides from 38 cases. Interobserver variability in the diagnosis of microbiopsies and histologic sections from them was moderate--kappa = .44 (SE = .06) and kappa = .44 (SE = .09), respectively. In the consensus meeting for all cases, a conclusive diagnosis was reached. The Pearson correlation coefficient between the consensus diagnosis and MIB-1 staining was r = .62. The sensitivity of the consensus diagnosis for the follow-up diagnosis was 71% and the specificity 60%. Diagnosis on approximately 50% of slides diagnosed as inadequate or ASCUS could be made. CONCLUSION: The histotechnical workup of microbiopsies is not difficult; however, their diagnosis can be a problem. Adequate diagnostic criteria are not available. Aided by MIB-1 staining, histologic sections from microbiopsies can be diagnosed, and the diagnoses correlated with follow-up in most cases. Processing of microbiopsies in smears with an inconclusive cytologic diagnosis or a diagnosis of ASCUS allowed correct diagnosis in 50% of cases in this study.

Antibacterial activity of four cephalosporins in an experimental infection in relation to in vitro effect and pharmacokinetics.

The in vitro activity of four cephalosporins was compared with their effects in an experimental thigh infection (cefuroxime and cefamandole against Escherichia coli and cefamandole, ceftriaxone, and ceftazidime against Klebsiella pneumoniae) in granulocytopenic mice. The effect in vitro (ER) was defined as the difference between the growth rate without antibiotic and the growth rate at the steepest part of a 3-h growth curve in the presence of an antibiotic. The relation between concentration and ER was described with the Hill equation. Using pharmacokinetic parameters of the plasma concentrations in vivo and those of the Hill equation the corresponding time course of ER was calculated and by integration with respect to time (0tERdt), an estimate was obtained of the effect on bacteria. For all four antibiotics this estimate was significantly correlated with the actual values of the effect in vivo (EN), defined as the difference in numbers of bacteria between controls and antibiotic-treated animals at 4 h.

Comparison of in vivo and in vitro activities of antibiotics with different modes of action against a tolerant and a non-tolerant Staphylococcus aureus strain.

The antibacterial efficacies of 4 antibiotics with different modes of action against a penicillin-tolerant and a non-tolerant strain of Staphylococcus aureus were investigated. For the in vitro studies the minimum inhibitory concentration (MIC) and the minimum bacterial concentration (MBC) were determined and short-term growth experiments at different antibiotic concentrations were performed. For the in vivo studies, antibacterial efficacy in an experimental infection in normal and granulocytopenic mice was evaluated. For erythromycin, rifampicin and ciprofloxacin, there was no difference in the MIC and MBC values for the 2 strains. Benzylpenicillin had an MBC value for the tolerant strains which was 256 times higher than the MIC; with the non-tolerant strain there was no difference. EC50 values, calculated from the in vitro short-term growth curves, gave similar results. Only benzylpenicillin exhibited a difference in activity against the tolerant strain, as reflected by the EC50 that was 290 times the EC50 for the non-tolerant strain. Studies in normal and granulocytopenic mice gave similar results: benzylpenicillin was 268 times less active against the tolerant strain than against the non-tolerant strain. Erythromycin, rifampicin and ciprofloxacin were 2-3 times less active against the tolerant strain than against the non-tolerant strain. The presence of granulocytes is important for the antibacterial effect of all antibiotics studied, since in the absence of granulocytes higher doses of the antibiotics are needed in order to obtain the same antibacterial effect as when granulocytes are present.

The efficacy of rifampicin against Staphylococcus aureus in vitro and in an experimental infection in normal and granulocytopenic mice.

The effect of rifampicin on Staphylococcus aureus in vitro was assessed as the difference between the logarithms of the numbers of colony forming units (CFU) with and without 3 h of exposure to the drug. The efficacy was expressed as the EC50, i.e. the concentration at which 50% of the maximal effect was obtained, calculated according to the Hill equation. The value found for the EC50 was 3.8 micrograms/l and the mean maximal effect was a log ratio of 5.03 (SEM 0.33). In vivo experiments were performed in normal mice and in mice made granulocytopenic by irradiation. The effect of rifampicin was assessed as the CFU count 5 h after the injection of a suspension of bacteria into the thigh muscle and 4 h after the administration of rifampicin. The efficacy was expressed as the ED50, i.e. the dose at which 50% of the maximal effect is obtained. This value was 0.18 mg/kg for the normal mice and 0.15 mg/kg for the granulocytopenic mice. The corresponding mean plasma concentrations of non-protein-bound drug were 28 and 24 mg/l, respectively. Thus, the EC50 was found to be much higher in vivo than that in vitro. This difference should be taken into account when parameters of in-vitro efficacy are applied to establish dosage schedules.

Quantitative effect of granulocytes on antibiotic treatment of experimental staphylococcal infection.

The quantitative relation between granulocytopenia and antibiotic treatment was established for a short-term Staphylococcus aureus infection in the thighs of mice, using rifampin, benzylpenicillin, and erythromycin. Granulocytopenia was induced by total-body irradiation; the number of granulocytes decreased gradually during the first 5 days after irradiation to 10% of the number in normal mice. Experimental infections were established on each of the 5 days after irradiation. In animals not treated with antibiotics, the number of granulocytes in peripheral blood and the number of CFU at the site of infection exhibited a strong negative correlation. The influence of granulocytes on the effect of antibiotics on the number of CFU differed for the three antibiotics. For erythromycin the slope of the dose-effect relation was rather flat, but a decrease in the number of granulocytes caused a significant, nearly parallel, shift in the dose-effect relation, resulting in an increase in the number of CFU. For benzylpenicillin the slope of the dose-effect relation for normal mice was also flat, but as the number of granulocytes decreased the slope became significantly steeper, resulting in a diminishing influence of granulocytes at higher dosages. For rifampin the slope of the dose-effect relation, which was already steep for nonirradiated animals, increased significantly. Here too the effect of granulocytes decreased as the dose increased.