RESUMEN
The heterogeneity inherent in today's biotherapeutics, especially as a result of heavy glycosylation, can affect a molecule's safety and efficacy. Characterizing this heterogeneity is crucial for drug development and quality assessment, but existing methods are limited in their ability to analyze intact glycoproteins or other heterogeneous biotherapeutics. Here, we present an approach to the molecular assessment of biotherapeutics that uses proton-transfer charge-reduction with gas-phase fractionation to analyze intact heterogeneous and/or glycosylated proteins by mass spectrometry. The method provides a detailed landscape of the intact molecular weights present in biotherapeutic protein preparations in a single experiment. For glycoproteins in particular, the method may offer insights into glycan composition when coupled with a suitable bioinformatic strategy. We tested the approach on various biotherapeutic molecules, including Fc-fusion, VHH-fusion, and peptide-bound MHC class II complexes to demonstrate efficacy in measuring the proteoform-level diversity of biotherapeutics. Notably, we inferred the glycoform distribution for hundreds of molecular weights for the eight-times glycosylated fusion drug IL22-Fc, enabling correlations between glycoform sub-populations and the drug's pharmacological properties. Our method is broadly applicable and provides a powerful tool to assess the molecular heterogeneity of emerging biotherapeutics.
Asunto(s)
Glicoproteínas , Polisacáridos , Glicosilación , Glicoproteínas/metabolismo , Espectrometría de Masas/métodos , Polisacáridos/metabolismoRESUMEN
Intact protein mass spectrometry (MS) coupled with liquid chromatography was applied to characterize the pharmacokinetics and stability profiles of therapeutic proteins. However, limitations from chromatography, including throughput and carryover, result in challenges with handling large sample numbers. Here, we combined intact protein MS with multiple front-end separations, including affinity capture, SampleStream, and high-field asymmetric waveform ion mobility spectrometry (FAIMS), to perform high-throughput and specific mass measurements of a multivalent antibody with one antigen-binding fragment (Fab) fused to an immunoglobulin G1 (IgG1) antibody. Generic affinity capture ensures the retention of both intact species 1Fab-IgG1 and the tentative degradation product IgG1. Subsequently, the analytes were directly loaded into SampleStream, where each injection occurs within â¼30 s. By separating ions prior to MS detection, FAIMS further offered improvement in signal-overnoise by â¼30% for denatured protein MS via employing compensation voltages that were optimized for different antibody species. When enhanced FAIMS transmission of 1Fab-IgG1 was employed, a qualified assay was established for spiked-in serum samples between 0.1 and 25 µg/mL, resulting in â¼10% accuracy bias and precision coefficient of variation. Selective FAIMS transmission of IgG1 as the degradation surrogate product enabled more sensitive detection of clipped species for intact 1Fab-IgG1 at 5 µg/mL in serum, generating an assay to measure 1Fab-IgG1 truncation between 2.5 and 50% with accuracy and precision below 20% bias and coefficient of variation. Our results revealed that the SampleStream-FAIMS-MS platform affords high throughput, selectivity, and sensitivity for characterizing therapeutic antibodies from complex biomatrices qualitatively and quantitatively.
Asunto(s)
Inmunoglobulina G , Espectrometría de Movilidad Iónica , Espectrometría de Movilidad Iónica/métodos , Espectrometría de Masas/métodos , Cromatografía Liquida , Iones/químicaRESUMEN
Engineered cytochrome P450s are emerging as powerful synthetic tools due to their ability catalyze non-native metallocarbenoid and -nitrenoid insertion reactions. P450-mediated cyclopropanation has garnered particular interest due to the high selectivity demonstrated by engineered scaffolds and their application towards the synthesis of therapeutic agents. We previously reported that mutation of a conserved, first-shell heme-ligating Cys to Ser led to significant improvements in cyclopropanation activity in a model enzyme, P450BM3h . Here, we demonstrate that mutation of a ubiquitously conserved second-shell Phe (F393) to His or Ala, provides complementary increases in the P450 heme reduction potential and conversion to cyclopropanation products when compared to first-shell Cys to Ser mutations. Furthermore, we show that these mutations confer improved non-natural catalysis in 4 diverse P450 scaffolds.
Asunto(s)
Sistema Enzimático del Citocromo P-450/genética , Sistema Enzimático del Citocromo P-450/metabolismo , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Mutación Missense , Ciclopropanos/metabolismo , Hemo/metabolismo , Oxidación-Reducción , Ingeniería de Proteínas , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMEN
Thiopeptides are a growing class of ribosomally synthesized and post-translationally modified peptide (RiPP) natural products. Many biosynthetic enzymes for RiPPs, especially thiopeptides, are promiscuous and can accept a wide range of peptide substrates with different amino acid sequences; thus, these enzymes have been used as tools to generate new natural product derivatives. Here, we explore an alternative route to molecular complexity by engineering thiopeptide tailoring enzymes to do new or non-native chemistry. We explore cytochrome P450 enzymes as biocatalysts for cyclopropanation of dehydroalanines, chemical motifs found widely in thiopeptides and other RiPP-based natural products. We find that P450TbtJ1 and P450TbtJ2 selectively cyclopropanate dehydroalanines in a number of complex thiopeptide-based substrates and convert them into 1-amino-2-cyclopropane carboxylic acids (ACCAs), which are important pharmacophores. This chemistry takes advantage of the innate affinity of these biosynthetic enzymes for their substrates and enables incorporation of new pharmacophores into thiopeptide architectures. This work also presents a strategy for diversification of natural products through rationally repurposing biosynthetic enzymes as non-natural biocatalysts.
Asunto(s)
Alanina/análogos & derivados , Sistema Enzimático del Citocromo P-450/metabolismo , Péptidos/metabolismo , Propano/metabolismo , Compuestos de Sulfhidrilo/química , Alanina/química , Sistema Enzimático del Citocromo P-450/química , Sistema Enzimático del Citocromo P-450/genética , Péptidos/química , Propano/química , Procesamiento Proteico-Postraduccional , Ribosomas/metabolismoRESUMEN
Despite increasing interest in using enzymes as tools for synthesis, many reactions discovered through the creativity of synthetic chemists remain beyond the scope of biocatalysis. This vacancy in the field has compelled researchers to develop strategies to adapt protein scaffolds for new reactivity. Heme proteins have recently been shown to activate synthetic precursors to generate reactive metallocarbenoid and metallonitrenoid species that enable the biosynthetic construction of novel C-C, C-N, and other bonds using mechanisms not previously explored by Nature. By interrogating heme proteins with synthetic, non-natural reagents, scientists are merging the reaction space traditionally dominated by organocatalysis and transition metal catalysis with the mild reaction conditions, selectivity, and adaptability imparted by native protein scaffolds.
Asunto(s)
Hemoproteínas/metabolismo , Alquenos/metabolismo , Aziridinas/metabolismo , Catálisis , Iminas/metabolismo , Metano/análogos & derivados , Metano/metabolismoRESUMEN
We introduce a strategy that expands the functionality of hemoproteins through orthogonal enzyme/heme pairs. By exploiting the ability of a natural heme transport protein, ChuA, to promiscuously import heme derivatives, we have evolved a cytochrome P450 (P450BM3) that selectively incorporates a nonproteinogenic cofactor, iron deuteroporphyrin IX (Fe-DPIX), even in the presence of endogenous heme. Crystal structures show that selectivity gains are due to mutations that introduce steric clash with the heme vinyl groups while providing a complementary binding surface for the smaller Fe-DPIX cofactor. Furthermore, the evolved orthogonal enzyme/cofactor pair is active in non-natural carbenoid-mediated olefin cyclopropanation. This methodology for the generation of orthogonal enzyme/cofactor pairs promises to expand cofactor diversity in artificial metalloenzymes.
Asunto(s)
Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Sistema Enzimático del Citocromo P-450/química , Sistema Enzimático del Citocromo P-450/metabolismo , Hierro/química , Metaloporfirinas/química , NADPH-Ferrihemoproteína Reductasa/química , NADPH-Ferrihemoproteína Reductasa/metabolismo , Dominio Catalítico , Coenzimas , Evolución Molecular Dirigida , Metaloporfirinas/metabolismo , Modelos Moleculares , Estructura Molecular , Mutación , Oxidación-ReducciónRESUMEN
Cytochrome P450s and other heme-containing proteins have recently been shown to have promiscuous activity for the cyclopropanation of olefins using diazoacetate reagents. Despite the progress made thus far, engineering selective catalysts for all possible stereoisomers for the cyclopropanation reaction remains a considerable challenge. Previous investigations of a model P450 (P450BM3 ) revealed that mutation of a conserved active site threonine (Thr268) to alanine transformed the enzyme into a highly active and selective cyclopropanation catalyst. By incorporating this mutation into a diverse panel of P450 scaffolds, we were able to quickly identify enantioselective catalysts for all possible diastereomers in the model reaction of styrene with ethyl diazoacetate. Some alanine variants exhibited selectivities that were markedly different from the wild-type enzyme, with a few possessing moderate to high diastereoselectivity and enantioselectivities up to 97 % for synthetically challenging cis-cyclopropane diastereomers.