Thermodynamics of Coupled Folding in the Interaction of Archaeal RNase P Proteins RPP21 and RPP29

We have used isothermal titration calorimetry (ITC) to identify and describe binding-coupled equilibria in the interaction between two protein subunits of archaeal ribonuclease P (RNase P). In all three domains of life, RNase P is a ribonucleoprotein complex that is primarily responsible for catalyzing the Mg²⁺-dependent cleavage of the 5′ leader sequence of precursor tRNAs during tRNA maturation. In archaea, RNase P has been shown to be composed of one catalytic RNA and up to five proteins, four of which associate in the absence of RNA as two functional heterodimers, POP5–RPP30 and RPP21–RPP29. Nuclear magnetic resonance studies of the <i>Pyrococcus furiosus</i> RPP21 and RPP29 proteins in their free and complexed states provided evidence of significant protein folding upon binding. ITC experiments were performed over a range of temperatures, ionic strengths, and pH values, in buffers with varying ionization potentials, and with a folding-deficient RPP21 point mutant. These experiments revealed a negative heat capacity change (Δ<i>C</i>_<i>p</i>), nearly twice that predicted from surface accessibility calculations, a strong salt dependence for the interaction, and proton release at neutral pH, but a small net contribution from these to the excess Δ<i>C</i>_<i>p</i>. We considered potential contributions from protein folding and burial of interfacial water molecules based on structural and spectroscopic data. We conclude that binding-coupled protein folding is likely responsible for a significant portion of the excess Δ<i>C</i>_<i>p</i>. These findings provide novel structural and thermodynamic insights into coupled equilibria that allow specificity in macromolecular assemblies

Solubility Challenges in High Concentration Monoclonal Antibody Formulations: Relationship with Amino Acid Sequence and Intermolecular Interactions

The purpose of this work was to elucidate the molecular interactions leading to monoclonal antibody self-association and precipitation and utilize biophysical measurements to predict solubility behavior at high protein concentration. Two monoclonal antibodies (mAb-G and mAb-R) binding to overlapping epitopes were investigated. Precipitation of mAb-G solutions was most prominent at high ionic strength conditions and demonstrated strong dependence on ionic strength, as well as slight dependence on solution pH. At similar conditions no precipitation was observed for mAb-R solutions. Intermolecular interactions (interaction parameter, <i>k</i>_D) related well with high concentration solubility behavior of both antibodies. Upon increasing buffer ionic strength, interactions of mAb-R tended to weaken, while those of mAb-G became more attractive. To investigate the role of amino acid sequence on precipitation behavior, mutants were designed by substituting the CDR of mAb-R into the mAb-G framework (GM-1) or deleting two hydrophobic residues in the CDR of mAb-G (GM-2). No precipitation was observed at high ionic strength for either mutant. The molecular interactions of mutants were similar in magnitude to those of mAb-R. The results suggest that presence of hydrophobic groups in the CDR of mAb-G may be responsible for compromising its solubility at high ionic strength conditions since deleting these residues mitigated the solubility issue

RP-HPLC DAR Characterization of Site-Specific Antibody Drug Conjugates Produced in a Cell-Free Expression System

Antibody drug conjugates (ADCs) harness the target specificity of a monoclonal antibody (mAb) and the high cytotoxicity of a small molecule, enabling improved delivery of a potent antitumor agent compared to traditional chemotherapy for cancer therapy. Only two ADCs have been marketed, both of which are produced via nonsite-specific conjugation of the cytotoxic drug to either interchain cysteine (Adcetris) or lysine (Kadcyla). A growing body of evidence suggests that site-specific ADCs, because of their payload homogeneity, will improve pharmacokinetics and have wider therapeutic windows when compared to heterogeneous ADCs. Previously, we have demonstrated the use of a cell free expression system (Xpress CF+) for rapid production of site-specific ADCs. Here we report the generation of a variety of ADCs via conjugation between a site-specific incorporated non-natural amino acid (nnAA), <i>para</i>-azidomethyl-l-phenylalanine (pAMF), and dibenzocyclooctyl-(polyethylene glycol)₄ (DBCO-(PEG)₄) linked payloads using this platform. We developed a reversed phase HPLC method for drug to antibody ratio (DAR) characterization, which is applicable to both reduced and intact ADCs. We demonstrate that these ADCs are of near complete conjugation and exhibit potent cell killing activity and in vitro plasma stability. Moreover, we generated an ADC conjugated at both light and heavy chains, resulting in a DAR close to 4. With the increased number of payloads, the resultant DAR 4 ADC is potentially more efficacious than its DAR 2 counterparts, which could further improve its therapeutic index. These studies have demonstrated the competency of Xpress CF+ for site-specific ADC production and improved our understanding of the site-specific ADCs in general