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AAPS PharmSci (1)
AAPS PharmSciTech (1)
Kalonia, Devendra S. (2)
Sharma, Vikas K. (2)
Lazar, Kristi L (1)
Patapoff, Thomas W (1)
Sharma, Vikas K (1)
Year of Publication
Cold denaturation of monoclonal antibodies
Lazar, Kristi L
Patapoff, Thomas W
The susceptibility of monoclonal antibodies (mAbs) to undergo cold denaturation remains unexplored. In this study, the phenomenon of cold denaturation was investigated for a mAb, mAb1, through thermodynamic and spectroscopic analyses. tryptophan fluorescence and circular dichroism (CD) spectra were recorded for the guanidine hydrochloride (GuHCl)-induced unfolding of mAb1 at pH 6.3 at temperatures ranging from −5 to 50°C. A three-state unfolding model incorporating the linear extrapolation method was fit to the fluorescence data to obtain an apparent free energy of unfolding, ΔGu, at each temperature. CD studies revealed that mAb1 exhibited polyproline II helical structure at low temperatures and at high GuHCl concentrations. the Gibbs-Helmholtz expression fit to the ΔGu versus temperature data from fluorescence gave a ΔCp of 8.0 kcal mol−1 K−1, a maximum apparent stability of 23.7 kcal mol−1 at 18°C, and an apparent cold denaturation temperature (TCD) of −23°C. ΔGu values for another mAb (mAb2) with a similar framework exhibited less stability at low temperatures, suggesting a depressed protein stability curve and a higher relative TCD. Direct experimental evidence of the susceptibility of mAb1 and mAb2 to undergo cold denaturation in the absence of denaturant was confirmed at pH 2.5. thus, mAbs have a potential to undergo cold denaturation at storage temperatures near −20°C (pH 6.3), and this potential needs to be evaluated independently for individual mAbs.
monoclonal antibodies; thermodynamic stability; cold denaturation; free energy; fluorescence
Effect of vacuum drying on protein-mannitol interactions: The physical state of mannitol and protein structure in the dried state
Kalonia, Devendra S.
The purpose of the present studies was to systematically investigate protein-mannitol interactions using vacuum drying, to obtain a better understanding of the effect of protein/mannitol wt/wt ratios on the physical state of mannitol and protein secondary structure in the dried state. Solutions containing β-lactoglobulin (βLg):mannitol (1∶1–1∶15 wt/wt) were vacuum dried at 5°C under 3000 mTorr of pressure. The physical state of mannitol was studied using x-ray powder physical state of mannitol was studied using x-ray powder diffractometry (XRPD), polarized light microscopy (PLM), Fourier-transform infrared (FTIR) spectroscopy, and modulated differential scanning calorimetry (MDSC). XRPD studies indicated that mannitol remained amorphous up to 1∶5 wt/wt βLg:mannitol ratio, whereas PLM showed the presence of crystals of mannitol in all dried samples except for the 1∶1 wt/wt βLg:mannitol dried sample. FITR studies indicated that a small proportion of crystalline mannitol was present along with the amorphous mannitol in dried samples at lower (less than 1∶5 wt/wt) βLg:mannitol ratios. The Tg of the dried 1∶1 wt/wt βLg:mannitol sample was observed at 33.4°C in MDSC studies, which indicated that at least a part of mannitol co-existed with protein in a single amorphous phase. Evaluation of the crystallization exotherms indicated that irrespective of the βLg:protein wt/wt ratio in the initial sample, the protein to amorphous mannitol ratio was below 1∶1 wt/wt in all dried samples. Second-derivative FTTR studies on dried βLg and recombinant human interferon α-2a samples showed that mannitol affected protein secondary structure to a varying degree depending on the overall mannitol content in the dried sample and the type of protein.
mannitol; proteins; vacuum drying; amorphous; protein structure
Polyethylene glycol-induced precipitation of interferon alpha-2a followed by vacuum drying: Development of a novel process for obtaining a dry, stable powder
Kalonia, Devendra S.
Feasibility studies were performed on the development of a novel process based on polyethylene glycol (PEG)-induced precipitation of proteins followed by vacuum drying in the presence of sugars to obtain dry protein powders. Apparent solubility of interferon alpha-2a (IFNα2a) was determined in the presence of various PEGs and the effect of solution pH, ionic strength, and temperature was investigated. IFNα2a precipitate was dried at a shelf temperature of 25°C at 100 mTorr either as it is or in the presence of mannitol and/or trehalose. The dried IFNα2a formulations were subjected to accelerated stability studies at 40°C (3 months), and the stability was compared with that of a similar lyophilized formulation. The results indicated that more than 90% of the protein could be precipitated using 10% wt/vol PEG the protein could be precipitated using 10% wt/vol PEG 1450 at pH 6.5 at a solution ionic strength of 71 mM. Vacuum drying of the precipitate only resulted in the formation of insoluble aggregates of IFNα2a; however, this was prevented by the addition of either mannitol or trehalose. The addition of excess mannitol resulted in low residual moisture content and better handling of the final dried product. Accelerated storage stability did not show any aggregation and showed less than 5% formation of oxidized IFNα2a in the dried formulation containing IFNα2a: trehalose: mannitol in a 1∶10∶100 wt/wt ratio upon storage at 40°C for 3 months. The stability of this vacuum dried formulation was comparable with that of a similar lyophilized formulation.
protein formulation; polyethylene glycol (PEG); precipitation; vacuum drying; protein powders
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