Mark Manning

Protein instability following transport or storage on dry ice

properties of transcription2,6 (Supplementary Note 4). To validate these methods, we first used simulations (Supplementary Note 5). For transcription sites smaller than the optical resolution, all methods yielded accurate estimates. However, for larger transcription sites, the simple methods led to gross underestimates, whereas FISH-quant gave accurate results (Fig. 1e). For elongated transcription sites, only the PSF superposition approach worked reliably. For experimental validation, we used an artificial reporter with transcription sites frequently exceeding the resolution. An RNase protection assay provided a rough, but independent, estimate of the ratio of mature versus nascent mRNA. The assay yielded ratios in the same range as the FISH-based quantifications, confirming their general validity (Supplementary Note 6 and Supplementary Methods). For a more accurate assessment of simple methods and FISH-quant, we then compared the nascent transcript counts. Much as for the large simulated transcription sites, the simple methods led to underestimated counts (Fig. 1f). Thus, FISH-quant accurately quantified nascent mRNA even when simple approaches did not. Finally, we used FISH-quant to analyze b-actin mRNA after serum induction and measured more than twice the amount of nascent mRNA than we did with simple methods, which illustrates the importance of accurate quantification even for endogenous genes (Supplementary Note 6 and Supplementary Methods). FISH-quant could also be applied to other structures with a dense accumulation of mRNA, such as processing (P)-bodies or stress granules. FISH-quant is controlled via graphical user interfaces in Matlab and requires no computational expertise. A batch mode allows users to automatically process multiple images. FISH-quant is available at http://code.google.com/p/fish-quant/ with a detailed manual and test data.

Reduction of metal carbonyls with alkali metal carbides

Abstract The reaction of Na 2 C 2 and Li 2 C 2 with Mn(CO) 5 Br yields Mn 2 (CO) 10 . The neutral complex Co 4 (CO) 12 has been reduced to the Co 6 (CO) 4− 14 cluster complex with Na 2 C 2 . A similar reaction with Fe 3 (CO) 12 yielded [N(C 2 H 5 ) 4 ][HFe 3 (CO) 11 ] upon addition of a methanol solution of [N(C 2 H 5 ) 4 ] Br. These reactions may be carried out at room temperature and in tetrahydrofuran solvent.

Stability of lyophilized sucrose formulations of an IgG1: subvisible particle formation.

Eight lyophilized formulations of a IgG1 monoclonal antibody (MAb) were prepared containing increasing levels of sucrose. In addition, three of the formulations had sorbitol added at a level of 5% w/w relative to sucrose. The samples were stored for up to 4 weeks at 40°C, which is well below the Tg. Upon reconstitution, the levels of subvisible particles were measured using microflow imaging (MFI). The formulation containing no sucrose contained exceedingly high levels of subvisible particles, accounting for as much as 25% of the weight of the protein. Addition of sucrose markedly decreased the number of subvisible particles, with the maximal sucrose:protein weight ratio being 2:1 (the highest level tested). Addition of sorbitol further decreased subvisible particle levels, even for formulations where the sucrose:protein ratio was relatively high. This suggests that even small amounts of a plasticizer like sorbitol can improve the storage stability of a lyophilized antibody formulation, probably by dampening β-relaxations within the amorphous glass.

Characterization of Protein Higher Order Structure Using Vibrational Circular Dichroism Spectroscopy

Better understanding of protein higher order structures (HOS) is of major interest to researchers in the field of biotechnology and biopharmaceutics. Monitoring a proteins HOS is crucial towards understanding the impact of molecular conformation on the biotechnological application. In addition, maintaining the HOS is critical for achieving robust processes and developing stable formulations of therapeutic proteins. Loss of HOS contributes to increased aggregation, enhanced immunogenicity and loss of function. Selecting the proper biophysical methods to monitor the secondary and tertiary structures of therapeutic proteins remains the central question in this field. In this study, both Fourier Transform Infrared (FTIR) and vibrational circular dichroism (VCD) spectroscopy are employed to characterize the secondary structures of various proteins as a function of temperature and pH. Three proteins with different secondary structures were examined, human serum albumin (HSA), myoglobin, and the monoclonal antibody, ofatumumab. This work demonstrates that VCD is useful technique for monitoring subtle secondary structure changes of protein therapeutics that may occur during processing or handling.

Use of the Amide II Infrared Band of Proteins for Secondary Structure Determination and Comparability of Higher Order Structure

Demonstrating comparability of secondary structure composition as part of higher order structure (HOS) in therapeutic proteins is a significant challenge. Previously, we showed that the variability of second derivative amide I Fourier transform infrared (FTIR) spectra were small enough that significant differences in secondary structures could be seen for a variety of model proteins. Those comparisons used spectral overlap and spectral correlation coefficients to quantify spectral differences. However, many of the excipients used in downstream purification process, drug substance, and drug product formulation, such as free amino acids and sugars, can interfere with the absorbance in the amide I region. In this study, analysis of amide II FTIR spectra is shown as an alternative to using spectral data from the amide I region to analyze protein secondary structure to assess their HOS. This research provided spectral overlap and spectral correlation coefficient mathematical approaches for analysis of amide II FTIR spectra to demonstrate comparability of protein secondary structure. Spectral overlap and spectral correlation coefficients results show strong correlations between changes in the second derivative of amide II and amide I FTIR spectra for various model proteins under different conditions, which demonstrate the applicability of using amide II FTIR spectra for the comparability of protein secondary structure. These results indicate that the analysis of the second derivative of amide II FTIR spectra may be used to monitor and demonstrate comparability of protein secondary structure during downstream process and formulation development of protein therapeutics.