Site-specific tryptophan oxidation induced by autocatalytic reaction of polysorbate 20 in protein formulation.

PURPOSE: Tryptophan (Trp) oxidation leading to atypical degradation of a protein (Fab) formulated with polysorbate 20 (PS20) was investigated. Such atypical Trp oxidation was discussed in relation to a kinetic model that involves initiation of oxidizing free radical through an autocatalytic reaction. METHODS: Ion-exchange chromatography and peptide mapping were used to determine Trp oxidation. Peroxides in PS20 and free radicals in Fab samples were detected by fluorometric assay and electron paramagnetic resonance (EPR), respectively. RESULTS: PS20 with increased peroxides level led to degradation of Fab stored at 30°C. Degradation was characterized as Trp50 oxidation, which was not observed in a Fab variant where His31 was replaced. EPR peaks related to known spin adducts of 5,5 dimethylpyrroline N-oxide were detected in Fab exhibiting Trp oxidation, indicating free radicals were present. Trp oxidation of Fab observed in several drug product lots with different degradation rates fits an autocatalytic reaction model that involves free radicals. EDTA, catalase, and free tryptophan prevented oxidation. CONCLUSIONS: A metal-binding amino acid, His31, was responsible for Trp50 oxidation of Fab induced by peroxides in PS20 present in the protein formulation. Oxidation was induced by autocatalytic degradation of PS20 and could be inhibited by antioxidants.

Raman spectroscopic characterization of drying-induced structural changes in a therapeutic antibody: correlating structural changes with long-term stability.

We characterized the secondary structure of a therapeutic recombinant humanized monoclonal antibody (rhuMAb), formulated with different concentrations of sucrose, trehalose, and histidine and in solution, lyophilized, and spray-dried states. Quantitative secondary structure estimates were obtained using amide I band Raman spectroscopy and a previously developed spectral deconvolution procedure. On lyophilization or spray drying in the absence of sugar, the antibody underwent significant structural perturbation. The beta-sheet content decreased with corresponding gain in the turn and unordered content. With increasing amount of sucrose or trehalose, the extent of structural perturbation decreased. Eventually, at sugar-to-protein molar ratios of > or =360, almost complete structural preservation was observed. Histidine also protected the antibody against lyophilization-induced structural changes. The extent of structural perturbation immediately after lyophilization or spray drying exhibited good correlation with the rate of aggregation for the antibody during long-term storage under accelerated conditions. The results demonstrate that amide I band Raman spectroscopy could be a quick and reliable way to screen excipients and their concentrations during lyophilized or spray dried formulation development.

Mechanisms of aggregate formation and carbohydrate excipient stabilization of lyophilized humanized monoclonal antibody formulations.

The purpose of this study was to evaluate the mechanisms of aggregate formation and excipient stabilization in freeze-dried formulations of a recombinant humanized monoclonal antibody. Protein degradation was measured using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE) and native size exclusion chromatography, and protein structure was studied using Fourier transform-infrared spectrometry and circular dichroism. The results showed that protein aggregates present following reconstitution were composed of native antibody structure and a reduced amount of free thiol when compared to protein monomer, which implied that intermolecular disulfides were involved in the aggregation mechanism. An excipient-free formulation resulted in reversible solid-state protein structural alteration and increased aggregation during storage. This correlated with dehydration to an extent that the amount of water was less than the estimated number of surface-accessible hydrogen-bonding sites on the protein. Improved native-like solid-state protein structure and reduced aggregation were obtained by formulation with enough carbohydrate to fulfill the hydrogen-bonding sites on the surface of the protein. Carbohydrate in excess of this concentration has less of an influence on protein aggregation. Reduced aggregation during storage was obtained by the addition of sufficient excipient to both stabilize solid-state protein structure and provide an environment that consisted of an amorphous glassy state matrix.

Determination of residual moisture in lyophilized protein pharmaceuticals using a rapid and non-invasive method: near infrared spectroscopy.

This study examined the application of near-infrared (NIR) spectroscopy to analyze residual moisture in lyophilized protein pharmaceuticals sealed in glass vials. We demonstrated that NIR was able to determine residual moisture in five marketed and clinical products with the same precision as Karl Fischer titration. We further investigated how changes in product configuration and protein formulation affected NIR measurement accuracy using a lyophilized monoclonal antibody rhuMAb E25 containing 1% to 5% residual moisture. The results indicated that the lyophilized cake porosity and dimensions had no effect on NIR measurement when the cake height and diameter exceeded the NIR penetration depth. In addition, changing the buffer and surfactant concentrations in the formulation did not affect moisture determination by NIR. However, doubling or halving the concentration of a disaccharide, which was used as a lyoprotectant, caused significant deviation between the NIR and Karl Fischer data because the NIR absorbance of the disaccharide overlapped with the moisture signal. Furthermore, complete removal of the disaccharide resulted in alteration of the protein NIR spectra, suggesting that NIR may be used to evaluate solid-state protein structure. The disaccharide concentration must be kept constant in this formulation to obtain accurate moisture results by NIR.

Application of frequency-modulated spectroscopy in vacuum seal integrity testing of lyophilized biological products.

This study examined the feasibility of applying frequency-modulated spectroscopy (FMS) to test vacuum seal integrity of lyophilized protein pharmaceuticals in glass vials. A lyophilized recombinant monoclonal antibody was used as an example to demonstrate that FMS is a non-destructive method that could accurately and quickly determine vial vacuum integrity within a pressure range of 0.04 to 0.5 atm. The coefficient of determination (R2) of a bench-top instrument was found to be >0.99. Only seconds were required to analyze each sample. The instrument sensitivity and specificity were 0.95 and >0.99, respectively, based on analysis of approximately 40,000 samples. Because of low energy input by the instrument, no adverse effect on the protein quality was found immediately after up to 1 h of continuous laser exposure. The laser-exposed samples had comparable stability to non-exposed control vials after 12 weeks of storage at 40 degrees C.

Investigating Liquid Leak from Pre-Filled Syringes upon Needle Shield Removal: Effect of Air Bubble Pressure.

This study is to investigate the effect of headspace air pressure in pre-filled syringes on liquid leak (dripping) from the syringe needle upon needle shield removal. Drip tests to measure drip quantity were performed on syringes manually filled with 0.5 or 1.0 mL of various aqueous solutions. Parameters assessed included temperature (filling and test), bulk storage conditions (tank pressure and the type of the pressurized gas), solution composition (pure water, 0.9% sodium chloride, and a monoclonal antibody formulation), and testing procedures. A headspace pressure analyzer was used to verify the drip test method. Results suggested that leakage is indeed caused by headspace pressure increase, and the temperature effect (ideal gas expansion) is a major, but not the only, factor. The dissolved gases in the liquid bulk prior to or during filling may contribute to leakage, as these gases could be released into the headspace due to solubility changes (in response to test temperature and pressure conditions) and cause pressure increase. Needle shield removal procedures were found to cause dripping, but liquid composition played little role. Overall, paying attention to the processing history (pressure and temperature) of the liquid bulk is the key to minimize leakage. The headspace pressure could be reduced by decreasing liquid bulk storage pressure, filling at a higher temperature, or employing lower solubility gas (e.g., helium) for bulk transfer and storage. Leakage could also be mitigated by simply holding the syringe needle pointing upward during needle shield removal. LAY ABSTRACT: Substantial advances in pre-filled syringe technology development, particularly in syringe filling accuracy, have been made. However, there are factors, as subtle as how the needle shield (or tip cap) is removed, that may affect dosing accuracy. We recently found that upon removal of the tip cap from a syringe held vertically with needle pointed downwards, a small amount of solution, up to 3-4% of the 1 mL filled volume or higher for filled volume of <1 mL, leaked out from the needle. This paper identified the root causes of this problem and offered solutions from the perspectives of the syringe fill process and the end user procedure. The readers will benefit from this paper by understanding how each process step prior to and during syringe filling may affect delivery performance of the pre-filled syringe device.

Root Cause Investigation of Rubber Seal Cracking in Pre-filled Cartridges: Ozone and Packaging Effects.

Pre-filled syringes/cartridges as primary packaging for parenterally delivered biopharmaceutical liquids consist of multiple components, including containers made of glass or plastic, and stoppers/plungers and disk seals (septa) made of rubber materials. Cracking of rubber components may be cosmetically unacceptable and in extreme cases may compromise enclosure integrity. The purpose of this study was to investigate the root cause of septum cracking and evaluate parameters/solutions to delay or prevent cracking from occurring. Custom-made chambers capable of tightly controlling ozone levels were assembled to deliberately create septum cracks. Cracks were qualitatively assessed by optical microscopy and quantified using image analysis by ImageJ. The results confirmed that ozone attack is the root cause of septum cracking during storage, and the stress-the result of crimping on the glass cartridge by the aluminum lined seal-made the septum particularly vulnerable to ozone attack. Ozone concentration as low as 10-40 ppb (levels routinely detected on a busy street) could crack the stressed septum in hours while days of ozone exposure at 50 ppm could not cause the unstressed septum to crack. Under ozone attack cracks initially grow in length and width uniformly across the stressed area and then stop progressing, perhaps due to residual stress release. Although the use of impermeable barriers could prevent cracking completely, this study suggested that any form of packaging barriers, including a highly permeable Tyvek® sheet, could postpone cracking by slowing down ozone diffusion and convection. We demonstrate that simple double packaging-placing the Tyvek®-lidded blister tray in a cardboard carton-could sufficiently protect the stressed septum for years in a surrounding environment with ozone at normal indoor levels (≤2 ppb). LAY ABSTRACT: Pre-filled syringes/cartridges as primary packaging for parenterally delivered biopharmaceutical liquids contain multiple components, including a disk seal (septum) made of rubber materials. Cracking of rubber components may be cosmetically unacceptable and in extreme cases may compromise enclosure integrity. The septum, if not appropriately packaged, might crack under uncontrolled storage environment. The purpose of this study was to investigate the root cause of septum cracking and evaluate parameters/solutions to delay or prevent cracking from occurring. Custom-made chambers capable of tightly controlling ozone levels were assembled to deliberately create septum cracks. The results confirmed that ozone attack is the root cause of septum cracking during storage, and the stress-the result of crimping on the glass cartridge by the aluminum lined seal-made the septum particularly vulnerable to ozone attack. Ozone concentration as low as 10-40 ppb (levels routinely detected on a busy street) could crack the stressed septum in hours. Although the use of impermeable barriers could prevent cracking completely, this study suggested that any form of packaging barriers, including a highly permeable Tyvek® sheet, could postpone cracking by slowing down ozone diffusion and convection. This investigation will raise awareness of manufacturers of pre-filled cartridge/syringe parenteral products to storage and packaging requirements for the long-term physical stability of cartridge components as small as the rubber septum.