2.2.1 AGITATION
Agitation experiments of the mAb were carried out on a “Thermomixer” or on an “Orbit 300” shaking device. Polypropylene centrifugal tubes (1.5 ml) were placed vertically onto these devices which were then shaken at a speed of 1200 rpm or 800 rpm, respectively. Rh-GCSF and rh-GH were also agitated on a Thermomixer but at 1100 rpm and 1000 rpm, respectively. By agitation the air-water interface within in the tubes was greatly increased and constantly renewed with substantial entrainment of air bubbles into the solution. The temperature was 20°C throughout all agitation experiments. The tubes were initially filled with 1ml of the respective formulation, leaving enough headspace for the formation of a large air- water interface. At certain intervals (depending on the experiment) 100 μl aliquots of the samples were drawn, centrifuged at 12100 g to remove potential precipitates before subjecting the supernatants to further analysis for remaining monomer and soluble aggregates. In addition tubes were filled with 1.5 ml formulation (leaving no headspace) as a reference in order to evaluate the effect of the absence of an air-water interface.
For the experiments at high mAb-concentration (50 mg/ml) 2R vials (Glass type I, Schott AG, Mainz, Germany) were used instead of polypropylene centrifugal tubes and the vials were fixed horizontally on a shaking device where they moved horizontally at 200 rpm. By filling the vials with 2 mL sufficient headspace for bubble entrainment and constant renewal of the air-water interface was left. After certain intervals aliquots of 100 µL were drawn from the vials which were then closed again with a stopper (FluroTec®-coating, West Pharmaceutical
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Services, Eschweiler, Germany) and crimped again until the next time point of analysis. At the start and at the end of the experiment the centrifuged supernatant was also analyzed for alterations in the mAb’s secondary and tertiary structure by IR-spectroscopy and UV second derivative spectroscopy.
2.2.2 STIRRING
The stirring stress onto the mAb-formulations was exerted at a constant stirring rate of 200 rpm by placing 6R vials vertically onto a multi-position magnetic stirring device (Variomag™ Magnetic Stirrer, Thermo Electron GmbH, Langenselbold, Germany). Washed and sterilized 6 mm × 3 mm Teflon® coated stirrer bars (VWR International GmbH, Darmstadt, Germany) were put into the vials and the vials were filled with 3 mL mAb-solution each. Temperature was kept constant at 20°C and the samples were protected from direct light. Vials were analyzed at the intervals shown in Chapter 3 by removing aliquots of 100 µL from the vials, centrifuging the aliquots at 12,000 g and subjecting the supernatants to HP-SEC analysis for remaining monomer and soluble aggregates. As usual formulations were tested in triplicates and control samples without a Teflon® stirrer bar were analyzed as well to be sure that the observed effects are due to the stirring-stress.
2.2.3 AGITATION WITH GLASS BEADS
The agitation experiment described above for the highly concentrated mAb-formulations was also carried out at the lower mAb-concentration (1.8 mg/ml) in the presence of glass beads (size 0.25-0.50 mm Carl Roth GmbH + Co. KG, Karlsruhe, Germany). The addition of 1.4 g of glass beads to each vial (filled with no headspace at all, roughly 4 mL per vial) was carried out in order to create an extensive glass-water interface to which the mAb can potentially adsorb. The vials were agitated so that constant renewal of the interface was guaranteed and to create an accelerated stability model in which desorbed and potentially structurally altered and aggregated mAb can subsequently be detected in solution.
2.2.4 FREEZE-THAW EXPERIMENTS
Samples were freeze-thawed (referred to as “FT”) by filling 1.0 mL of the respective formulation aliquot into 1.5 mL polypropylenes tubes. The tubes were then immersed into liquid nitrogen for 5 min to ensure complete freezing of the samples. To thaw the samples, the tubes were kept in a Thermomixer™ (without agitation) for 15 min at 25°C. The freeze- thaw cycles were repeated 15 times and after gentle homogenization aliquots of 100µL were drawn from the tubes after every five cycles and analyzed according to the procedure described for the agitation experiment above. The procedure was the same for all three model proteins studied in this thesis.
Chapter 2 2.2.5 INCUBATION AT ELEVATED TEMPERATURE AND LONG-TERM STORAGE
MONOCLONAL ANTIBODY
For the evaluation of mAb stability at elevated temperature, 1 ml samples were incubated in polypropylene centrifugal tubes for 8 days at 60°C. Prior to analysis, all samples were first gently homogenized and then centrifuged at 12100 g. The supernatants were analyzed for monomer and soluble aggregates by size exclusion chromatography.
For the mAb-long term incubation study the formulations were stored in cleaned and sterilized 2R-vials (Glass type I, Schott AG, Mainz, Germany) that were sealed with Teflon®- coated rubber stoppers (FluroTec®-coating, West Pharmaceutical Services, Eschweiler, Germany) under a nitrogen atmosphere and subsequently crimped. Each vial was filled with 2 mL of the respective formulation and all samples were prepared in triplicates. The samples were analyzed after 0 months, 3 months and 6 months of storage. Instead of removing aliquots for analysis separate vials were prepared for each time point of analysis in order to avoid extrinsic particle contamination. Storage was carried out at 4°C, 25°C and 40°C.
For the evaluation of mAb temperature stability at higher concentrations (50 mg/ml) a storage study at 50°C was carried out. After certain intervals aliquots of 100 µL were drawn from the vials which were then again closed with a stopper (FluroTec®-coating, West Pharmaceutical Services, Eschweiler, Germany) and crimped again until the next time point of analysis. At the start and at the end of the experiment the centrifuged supernatant was also analyzed for alterations in the mAb’s secondary and tertiary structure by IR-spectroscopy and UV second derivative spectroscopy.
RECOMBINANT GRANULOCYTE-COLONY STIMULATING FACTOR
For the evaluation of rh-GCSF stability at elevated temperature, 1ml samples were incubated in polypropylene centrifugal tubes for 230 h at 50°C. After certain intervals 100 µL aliquots were removed from the incubated samples which were then centrifuged at 12000g. The supernatants were analyzed for monomer and soluble aggregates by size exclusion chromatography and at the beginning and at the end of the study for conformational changes by IR-spectroscopy and second-derivative UV-spectroscopy.
RECOMBINANT HUMAN GROWTH HORMONE
For the evaluation of rh-GH stability at elevated temperature, 1ml samples were incubated in polypropylene centrifugal tubes for one month at 50°C. After certain intervals 100 µL aliquots were removed from the incubated samples which were then centrifuged at 12000g. The supernatants were analyzed for monomer and soluble aggregates by size exclusion chromatography. In addition to that the uncentrifuged samples were analyzed for high molecular weight soluble and insoluble aggregates by asymmetric flow field flow fractionation.
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