The orthogonality of the <i>M. jannaschii</i> TyrRS- pair was verified on western blots probed with anti-V5 antibodies.

<p>Expression of full-length foldon was monitored when various tRNAs were introduced into the HEK 293T cells. Note that the tRNA mutants used in these experiments were slightly different from those depicted in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0011263#pone-0011263-g001" target="_blank">figure 1</a>.</p

Suppression of an amber codon inserted into the GFP-encoding gene.

<p>(A) Full-length GFP was expressed in HEK 293T cells only in the presence of a <i>M. jannaschii</i> TyrRS- pair designed to be orthogonal to mammalian cells. (B) Western blot analysis of full-length GFP probed with anti-His antibodies.</p

Structures of peptide ligands in PCC Agent cocktail.

<p>Acetylene-presenting anchor peptides (black) were derived from the immunogenic epitope of HIV-1 gp41 (residues 600–612). A22-nindG (<b>i</b>) and A21-hnpfk (<b>ii</b>) were evolved from the original epitope appended with Pra at the C-terminus whereas A22-eihny (<b>iii</b>) utilizes the “substituted” anchor where residue Leu-607 is replaced with Pra. Secondary ligand branches (colored) were identified from the <i>in situ</i> click screen of a 5-mer OBOC library presenting an azide functionality. Biligands (<b>i</b>) and (<b>ii</b>) were raised against the target anti-HIV antibody 3D6, and the biligand (<b>iii</b>) was raised against the antibody 4B3.</p

Screening strategy for selecting capture agents against anti-HIV antibodies 3D6 and 4B3.

<p>The flow chart represents the use of the A21 and A22 cyclic peptides as anchor ligands for separate in situ click screens against a large OBOC azide-presenting peptide library.</p

Part 1. High-Throughput Mass Spectrometry for Biopharma: A Universal Modality and Target Independent Analytical Method for Accurate Biomolecule Characterization

Reversed-phase liquid chromatographic mass spectrometry (rpLC-MS) is a universal, platformed, and essential analytical technique within pharmaceutical and biopharmaceutical research. Typical rpLC method gradient times can range from 5 to 20 min. As monoclonal antibody (mAb) therapies continue to evolve and bispecific antibodies (BsAbs) become more established, research stage engineering panels will clearly evolve in size. Therefore, high-throughput (HT) MS and automated deconvolution methods are key for success. Additionally, newer therapeutics such as bispecific T-cell engagers and nucleic acid-based modalities will also require MS characterization. Herein, we present a modality and target agnostic HT solid-phase extraction (SPE) MS method that affords the analysis of a 96-well plate in 41.4 min, compared to the traditional rpLC-MS method that would typically take 14.4 h. The described method can accurately determine the molecular weights for monodispersed and highly polydispersed biotherapeutic species and membrane proteins; determine levels of glycosylation, glycation, and formylation; detect levels of chain mispairing; and determine accurate drug-to-antibody ratio values