Integration of ancient DNA with transdisciplinary dataset finds strong support for Inca resettlement in the south Peruvian coast.

Ancient DNA (aDNA) analysis provides a powerful means of investigating human migration, social organization, and a plethora of other crucial questions about humanity's past. Recently, specialists have suggested that the ideal research design involving aDNA would include multiple independent lines of evidence. In this paper, we adopt a transdisciplinary approach integrating aDNA with archaeological, biogeochemical, and historical data to investigate six individuals found in two cemeteries that date to the Late Horizon (1400 to 1532 CE) and Colonial (1532 to 1825 CE) periods in the Chincha Valley of southern Peru. Genomic analyses indicate that these individuals are genetically most similar to ancient and present-day populations from the north Peruvian coast located several hundred kilometers away. These genomic data are consistent with 16th century written records as well as ceramic, textile, and isotopic data. These results provide some of the strongest evidence yet of state-sponsored resettlement in the pre-Colonial Andes. This study highlights the power of transdisciplinary research designs when using aDNA data and sets a methodological standard for investigating ancient mobility in complex societies.

Investigation of Color in a Fusion Protein Using Advanced Analytical Techniques: Delineating Contributions from Oxidation Products and Process Related Impurities.

PURPOSE: Discoloration of protein therapeutics has drawn increased attention recently due to concerns of potential impact on quality and safety. Investigation of discoloration in protein therapeutics for comparability is particularly challenging primarily for two reasons. First, the description of color or discoloration is to certain extent a subjective characteristic rather than a quantitative attribute. Secondly, the species contributing to discoloration may arise from multiple sources and are typically present at trace levels. Our purpose is to development a systematic approach that allows effective identification of the color generating species in protein therapeutics. METHODS: A yellow-brown discoloration event observed in a therapeutic protein was investigated by optical spectroscopy, ultra-performance liquid chromatography, and mass spectrometry (MS). RESULTS: Majority of the color generating species were identified as oxidatively modified protein. The location of the oxidized amino acid residues were identified by MS/MS. In addition, the impact of process-related impurities co-purified from media on discoloration was also investigated. Finally a semi-quantitative scale to estimate the contribution of each color source is presented, which revealed oxidized peptides are the major contributors. CONCLUSIONS: A systematic approach was developed for identification of the color generating species in protein therapeutics and for estimation of the contribution of each color source.

Probing the Tryptophan Environment in Therapeutic Proteins: Implications for Higher Order Structure on Tryptophan Oxidation.

Tryptophan (Trp) oxidation in proteins leads to a number of events, including changes in color, higher order structure (HOS), and biological activity. We describe here a number of new findings through a comprehensive characterization of 6 monoclonal antibodies (mAbs) following selective oxidation of Trp residues by 2,2'-azobis(2-amidinopropane) dihydrochloride. Fluorescence spectroscopy, in combination with second derivative analysis, demonstrates that the loss of Trp fluorescence intensity is a sensitive indicator of Trp oxidation in mAbs. Size-exclusion chromatography with UV and intrinsic Trp fluorescence detection was demonstrated to be a useful method to monitor Trp oxidation levels in mAbs. Furthermore, the Trp oxidation levels measured by size-exclusion chromatography with UV and intrinsic Trp fluorescence detection were found to be in agreement with the values obtained from tryptic peptide mapping by liquid chromatography with mass spectrometric detection and correlate with the total solvent accessible surface area of the exposed Trp residues from in silico modeling. Finally, near-UV circular dichroism and Raman spectroscopy were used to evaluate the impact of Trp oxidation on HOS and identify specific oxidation products, respectively. This work demonstrates that protein HOS is altered on Trp oxidation in mAbs and multiple spectroscopic markers can be used to monitor the molecule-dependent Trp oxidation behavior.

Enhanced Precision of Circular Dichroism Spectral Measurements Permits Detection of Subtle Higher Order Structural Changes in Therapeutic Proteins.

Protein higher order structure (HOS) is an essential quality attribute to ensure protein stability and proper biological function. Protein HOS characterization is performed during comparability assessments for product consistency as well as during forced degradation studies for structural alteration upon stress. Circular dichroism (CD) spectroscopy is a widely used technique for measuring protein HOS, but it remains difficult to assess HOS with a high degree of accuracy and precision. Moreover, once spectral changes are detected, interpreting the differences in terms of specific structural attributes is challenging. Spectral normalization by the protein concentration remains one of the largest sources of error and reduces the ability to confidently detect differences in CD spectra. This work develops a simple method to enhance the precision of the CD spectral measurements through normalization of the CD spectra by the protein concentration determined directly from the CD measurement. This method is implemented to successfully detect small CD spectral changes in multiple forced degradation studies as well as comparability assessments during biologics drug development. Furthermore, the interpretation of CD spectral changes in terms of HOS differences are provided based on orthogonal data in conjunction with structural insights gained through in silico homology modeling of the protein structure.

Identification and quantification of signal peptide variants in an IgG1 monoclonal antibody produced in mammalian cell lines.

Sequence variants of a monoclonal antibody resulting from incomplete processing of signal peptide were identified and characterized using multiple mass spectrometry platforms and reverse phase chromatography. Detection and quantification of these variants by three LC/MS platforms were assessed. Quantification was also performed by mass spectrometric analysis of the subunits of the antibody generated by reduction and IdeS proteolysis. Peptide mapping with LC/MS/MS detection was used to quantify and confirm the identities of signal peptide sequence variants. Although quantification of the signal peptide variants thru mass spectrometry approaches is system dependent, our data revealed the results are close to the values determined by chromatographic separation with UV detection. Each of the methods have proven effective in demonstrating the consistency of signal peptide variants levels across the manufacture history of the antibody.

Characterization and identification of alanine to serine sequence variants in an IgG4 monoclonal antibody produced in mammalian cell lines.

Low levels of alanine to serine sequence variants were identified in an IgG4 monoclonal antibody by ultra/high performance liquid chromatography and tandem mass spectrometry. The levels of the identified sequence variants A183S and A152S, both in the light chain, have been determined to be 7.8-9.9% and 0.5-0.6%, by extracted ion currents of the tryptic peptides L16 and L14, respectively. The A183S variant was confirmed through tryptic map spiking experiments using synthetic peptide, SDYEK, which incorporated Ser at the position of native Ala in the tryptic peptide L16. Both mutations were also observed by endoproteinase Asp-N peptide mapping. The variant level of A183S was also quantified by LC-UV with detection at 280nm and fluorescence detection of tyrosine residues on the tryptic peptides. The results from LC-MS, UV, and fluorescence detection are in close agreement with each other. The levels of the sequence variants are comparable among the antibody samples manufactured at different scales as well as locations, indicating that the variants' levels are not affected by manufacture scale or locations. DNA sequencing of the master cell bank revealed the presence of mixed bases at position 183 encoding both wild and mutated populations, whereas bases encoding the minor sequence variant at position 152 were not detected. The root cause for A152S mutation is not yet clearly understood at this moment.

Characterization of glycosylation sites for a recombinant IgG1 monoclonal antibody and a CTLA4-Ig fusion protein by liquid chromatography-mass spectrometry peptide mapping.

Liquid chromatography mass spectrometry (LC-MS) peptide mapping can be a versatile technique for characterizing protein glycosylation sites without the need to remove the attached glycans as in conventional oligosaccharide mapping methods. In this way, both N-linked and O-linked sites of glycosylation can each be directly identified, characterized, and quantified by LC-MS as intact glycopeptides in a single experiment. LC-MS peptide mapping of the individual glycosylation sites avoids many of the limitations of preparing and analyzing an entire pool of released N-linked oligosaccharides from all sites mixed together. In this study, LC interfaced to a linear ion trap mass spectrometer (ESI-LIT-MS) were used to characterize the glycosylation of a recombinant IgG1 monoclonal antibody and a CTLA4-Ig fusion protein with multiple sites of N-and O-glycosylation. Samples were reduced, S-carboxyamidomethylated, and cleaved with either trypsin or endoproteinase Asp-N. Enhanced detection for minor IgG1 glycoforms (∼0.1 to 1.0 mol% level) was obtained by LC-MS of the longer 32-residue Asp-N glycopeptide (4+ protonated ion) compared to the 9-residue tryptic glycopeptide (2+ ion). LC-MS peptide mapping was run according to a general procedure: (1) Locate N-linked and/or O-linked sites of glycosylation by selected-ion-monitoring of carbohydrate oxonium fragment ions generated by ESI in-source collision-induced dissociation (CID), i.e. 204, 366, and 292 Da marker ions for HexNAc, HexNAc-Hex, and NeuAc, respectively; (2) Characterize oligosaccharides at each site via MS and MSMS. Use selected ion currents (SIC) to estimate relative amounts of each glycoform; and (3) Measure the percentage of site-occupancy by searching for any corresponding nonglycosylated peptide.