Attribute Analytics Performance Metrics from the MAM Consortium Interlaboratory Study.

The multi-attribute method (MAM) was conceived as a single assay to potentially replace multiple single-attribute assays that have long been used in process development and quality control (QC) for protein therapeutics. MAM is rooted in traditional peptide mapping methods; it leverages mass spectrometry (MS) detection for confident identification and quantitation of many types of protein attributes that may be targeted for monitoring. While MAM has been widely explored across the industry, it has yet to gain a strong foothold within QC laboratories as a replacement method for established orthogonal platforms. Members of the MAM consortium recently undertook an interlaboratory study to evaluate the industry-wide status of MAM. Here we present the results of this study as they pertain to the targeted attribute analytics component of MAM, including investigation into the sources of variability between laboratories and comparison of MAM data to orthogonal methods. These results are made available with an eye toward aiding the community in further optimizing the method to enable its more frequent use in the QC environment.

New Peak Detection Performance Metrics from the MAM Consortium Interlaboratory Study.

The Multi-Attribute Method (MAM) Consortium was initially formed as a venue to harmonize best practices, share experiences, and generate innovative methodologies to facilitate widespread integration of the MAM platform, which is an emerging ultra-high-performance liquid chromatography-mass spectrometry application. Successful implementation of MAM as a purity-indicating assay requires new peak detection (NPD) of potential process- and/or product-related impurities. The NPD interlaboratory study described herein was carried out by the MAM Consortium to report on the industry-wide performance of NPD using predigested samples of the NISTmAb Reference Material 8671. Results from 28 participating laboratories show that the NPD parameters being utilized across the industry are representative of high-resolution MS performance capabilities. Certain elements of NPD, including common sources of variability in the number of new peaks detected, that are critical to the performance of the purity function of MAM were identified in this study and are reported here as a means to further refine the methodology and accelerate adoption into manufacturer-specific protein therapeutic product life cycles.

POSSIM: Parameterizing Complete Second-Order Polarizable Force Field for Proteins.

Previously, we reported development of a fast polarizable force field and software named POSSIM (POlarizable Simulations with Second order Interaction Model). The second-order approximation permits the speed up of the polarizable component of the calculations by ca. an order of magnitude. We have now expanded the POSSIM framework to include a complete polarizable force field for proteins. Most of the parameter fitting was done to high-level quantum mechanical data. Conformational geometries and energies for dipeptides have been reproduced within average errors of ca. 0.5 kcal/mol for energies of the conformers (for the electrostatically neutral residues) and 9.7° for key dihedral angles. We have also validated this force field by running Monte Carlo simulations of collagen-like proteins in water. The resulting geometries were within 0.94 Å root-mean-square deviation (RMSD) from the experimental data. We have performed additional validation by studying conformational properties of three oligopeptides relevant in the context of N-glycoprotein secondary structure. These systems have been previously studied with combined experimental and computational methods, and both POSSIM and benchmark OPLS-AA simulations that we carried out produced geometries within ca. 0.9 Å RMSD of the literature structures. Thus, the performance of POSSIM in reproducing the structures is comparable with that of the widely used OPLS-AA force field. Furthermore, our fitting of the force field parameters for peptides and proteins has been streamlined compared with the previous generation of the complete polarizable force field and relied more on transferability of parameters for nonbonded interactions (including the electrostatic component). The resulting deviations from the quantum mechanical data are similar to those achieved with the previous generation; thus, the technique is robust, and the parameters are transferable. At the same time, the number of parameters used in this work was noticeably smaller than that of the previous generation of our complete polarizable force field for proteins; thus, the transferability of this set can be expected to be greater, and the danger of force field fitting artifacts is lower. Therefore, we believe that this force field can be successfully applied in a wide variety of applications to proteins and protein-ligand complexes.

Polarizable simulations with second order interaction model (POSSIM) force field: developing parameters for protein side-chain analogues.

A previously introduced polarizable simulations with second-order interaction model (POSSIM) force field has been extended to include parameters for small molecules serving as models for peptide and protein side-chains. Parameters have been fitted to permit reproducing many-body energies, gas-phase dimerization energies, and geometries and liquid-phase heats of vaporization and densities. Quantum mechanical and experimental data have been used as the target for the fitting. The POSSIM framework combines accuracy of a polarizable force field and computational efficiency of the second-order approximation of the full-scale induced point dipole polarization formalism. The resulting parameters can be used for simulations of the parameterized molecules themselves or their analogues. In addition to this, these force field parameters are currently being used in further development of the POSSIM fast polarizable force field for proteins.

PbSe/CdSe and PbSe/CdSe/ZnSe hierarchical nanocrystals and their photoluminescence.

Multiple CdSe and ZnSe semiconductor shells were grown on PbSe semiconductor spherical cores with monolayer control. For CdSe shell coating, we found that there was little room to further increase the quantum yields of freshly-made high-quality PbSe nanocrystals that already owned very high initial values because of their good surface status; but there was great improvement for the PbSe nanocrystals with low initial quantum yields because of the poor surface status. Nonetheless, the quantum yield for the latter case could not reach the former's value. Additional ZnSe shells on PbSe/CdSe could further increase the quantum yield and protect the nanocrystals from air oxidation. The observed phenomena in the synthesis of the PbSe/CdSe and PbSe/CdSe/ZnSe core/shell structures were explained through the carrier wave function expansion and the surface polarization.

Formation of PbSe/CdSe Core/Shell Nanocrystals for Stable Near-Infrared High Photoluminescence Emission.

PbSe/CdSe core/shell nanocrystals with quantum yield of 70% were obtained by the "successive ion layer adsorption and reaction" technology in solution. The thickness of the CdSe shell was exactly controlled. A series of spectral red shifts with the CdSe shell growth were observed, which was attributed to the combined effect of the surface polarization and the expansion of carriers' wavefunctions. The stability of PbSe nanocrystals was tremendously improved with CdSe shells.

Stability study of PbSe semiconductor nanocrystals over concentration, size, atmosphere, and light exposure.

Infrared-emitting PbSe nanocrystals are of increasing interest in both fundamental research and technical application. However, the practical applications are greatly limited by their poor stability. In this work, absorption and photoluminescence spectra of PbSe nanocrystals were utilized to observe the stability of PbSe nanocrystals over several conventional factors, that is, particle concentration, particle size, temperature, light exposure, contacting atmosphere, and storage forms (solution or solid powder). Both absorption and luminescence spectra of PbSe nanocrystals exposed to air showed dependence on particle concentration, size, and light exposure, which caused large and quick blue-shifts in the optical spectra. This air-contacted instability arising from the destructive oxidation and subsequent collision-induced decomposition was kinetically dominated and differed from the traditional thought that smaller particles with lower concentrations shrank fast. The photoluminescence emission intensity of the PbSe nanocrystal solution under ultraviolet (UV) exposure in air increased first and then decreased slowly; without UV irradiation, the emission intensity monotonously decreased over time. However, if stored under nitrogen, no obvious changes in absorption and photoluminescence spectra of the PbSe nanocrystals were observed even under UV exposure or upon being heated up to 100 degrees C.

Size-dependent composition and molar extinction coefficient of PbSe semiconductor nanocrystals.

Atomic compositions and molar extinction coefficients of PbSe semiconductor nanocrystals were determined by atomic absorption spectrometry, UV-vis-NIR spectrophotometry, and transmission electron microscopy. The Pb/Se atomic ratio was found to be size-dependent with a systematic excess of Pb atoms in the PbSe nanocrystal system. Experimental results indicated that the individual PbSe nanocrystal was nonstoichiometric, consisting of a PbSe core and an extra layer of Pb atoms. For these nonstoichiometric PbSe semiconductor nanocrystals, we proposed a new computational approach to calculate the total number of Pb and Se atoms in different sized particles. This calculation played a key role on the accurate determination of the strongly size-dependent extinction coefficient, which followed a power law with an exponent of approximately 2.5.