VanXY C , a bifunctional enzyme from VanC-phenotype Enterococcus gallinarum BM4174 that catalyses D,D-peptidase and D,D-carboxypeptidase activities, was purified as the native protein, as a maltose-binding protein fusion and with an N-terminal tag containing six histidine residues. The kinetic parameters of His 6 -VanXY C were measured for a variety of precursors of peptidoglycan synthesis involved in resistance: for D-Ala-D-Ala, the K m was 3.6 mM and k cat , 2.5 s, K m was 18.8 mM and k cat 6.2 s )1 ; for D-Ala-D-Ser, K m was 15.5 mM and k cat 0.35 s )1 . His 6 -VanXY C was inactive against the peptido- [7,8]. The VanA and VanB gene clusters are contained on transposons that are integrated either into self-transferable plasmids or the host chromosome [8,9].VanC-type resistance is defined as intrinsic low-level resistance to vancomycin (2-32 lgAEmL )1 ), but not to teicoplanin [10]. It has been identified in Enterococcus gallinarum, E. casseliflavus and E. flavescens [6,17] [20,21]. In this study the substrate specificity of purified VanXY C , a cytoplasmic bifunctional D,D-peptidase and D,D-carboxypeptidase, was characterized kinetically and an investigation initiated of the role of specific residues in determining the substrate selectivity. E X P E R I M E N T A L P R O C E D U R E S Strains, plasmids and growth conditionsEscherichia coli strains were grown in Luria-Bertani medium, and maintained on Luria-Bertani agar (1.5%), with the exception of E. coli JM83 [22] containing deriva-

The combination of potent chemical moieties with biologically active proteins is key to some of today's most innovative therapeutic drugs. In order to obviate any chemical modification of the proteins, we present a novel and powerful strategy for the selective conjugation of recombinant protein domains with synthetically derived peptides via a cucurbit[8]uril host-guest chemistry approach.

Subcutaneous injection of a low volume (<2 mL) high concentration (>100 mg/mL) formulation is an attractive administration strategy for monoclonal antibodies (mAbs) and other biopharmaceutical proteins. Using concentrated solutions may also be beneficial at various stages of bioprocessing. However, concentrating proteins by conventional techniques, such as ultrafiltration, can be time consuming and challenging. Isolation of the dense fraction produced by macroscopic liquid-liquid phase separation (LLPS) has been suggested as a means to produce high-concentration solutions, but practicality of this method, and the stability of the resulting protein solution have not previously been demonstrated. In this proof-of-concept study, we demonstrate that LLPS can be used to concentrate a mAb solution to >170 mg/mL. We show that the structure of the mAb is not altered by LLPS, and unperturbed mAb is recoverable following dilution of the dense fraction, as judged by 1 H nuclear magnetic resonance spectroscopy. Finally, we show that the physical properties and stability of a model high concentration protein formulation obtained from the dense fraction can be improved, for example through the addition of the excipient arginine•glutamate. This results in a stable high-concentration protein formulation with reduced viscosity and no further macroscopic LLPS. Concentrating mAb solutions by LLPS represents a simple and effective technique to progress toward producing high-concentration protein formulations for bioprocessing or administration. AbbreviationsArginine•glutamate (Arg•Glu), Carr-Purcell-Meiboom-Gill (CPMG), critical temperature (T C ), highperformance size-exclusion chromatography (HPSEC), liquid-liquid phase separation (LLPS), monoclonal antibody (mAb), nuclear magnetic resonance (NMR), transverse relaxation rate (R 2

Proteins frequently exist as high-concentration mixtures, both in biological environments and increasingly in biopharmaceutical co-formulations. Such crowded conditions promote protein–protein interactions, potentially leading to formation of protein clusters, aggregation, and phase separation. Characterizing these interactions and processes in situ in high-concentration mixtures is challenging due to the complexity and heterogeneity of such systems. Here we demonstrate the application of the dark-state exchange saturation transfer (DEST) NMR technique to a mixture of two differentially 19F-labeled 145 kDa monoclonal antibodies (mAbs) to assess reversible temperature-dependent formation of small and large protein-specific clusters at concentrations up to 400 mg/mL. 19F DEST allowed quantitative protein-specific characterization of the cluster populations and sizes for both mAbs in the mixture under a range of conditions. Additives such as arginine glutamate and NaCl also had protein-specific effects on the dark-state populations and cluster characteristics. Notably, both mAbs appear to largely exist as separate self-associated clusters, which mechanistically respond differently to changes in solution conditions. We show that for mixtures of differentially 19F-labeled proteins DEST NMR can characterize clustering in a protein-specific manner, offering unique tracking of clustering pathways and a means to understand and control them.

Purpose Anti-drug antibodies can impair the efficacy of therapeutic proteins and, in some circumstances, induce adverse health effects. Immunogenicity can be promoted by aggregation; here we examined the ability of recombinant mouse heat shock protein 70 (rmHSP70) - a common host cell impurity - to modulate the immune responses to aggregates of two therapeutic mAbs in mice. Methods Heat and shaking stress methods were used to generate aggregates in the sub-micron size range from two human mAbs, and immunogenicity assessed by intraperitoneal exposure in BALB/c mice. Results rmHSP70 was shown to bind preferentially to aggregates of both mAbs, but not to the native, monomeric proteins. Aggregates supplemented with 0.1% rmHSP70 induced significantly enhanced IgG2a antibody responses compared with aggregates alone but the effect was not observed for monomeric mAbs. Dendritic cells pulsed with mAb aggregate showed enhanced IFNγ production on co-culture with T cells in the presence of rmHSP70. Conclusion The results indicate a Th1-skewing of the immune response by aggregates and show that murine rmHSP70 selectively modulates the immune response to mAb aggregates, but not monomer. These data suggest that heat shock protein impurities can selectively accumulate by binding to mAb aggregates and thus influence immunogenic responses to therapeutic proteins. Electronic supplementary material The online version of this article (10.1007/s11095-019-2586-7) contains supplementary material, which is available to authorized users.