Automated mass spectrometry multi-attribute method analyses for process development and characterization of mAbs.

More than 370 biotherapeutics drug products have been approved by regulatory agencies on the US and EU markets and this industry continues to expand. Process change and optimization is necessary to develop new effective biologics in a cost effective and productive way. Consequently, improvement of analytical techniques is required for better product characterization according to Quality by Design (QbD) approach recommended by regulatory agencies. Recently, multi-attribute method (MAM) has emerged to meet such demands using mass spectrometry coupled to liquid chromatography (LC-MS). However, traditional sample preparation or data processing would not be suitable to guide process development, because one of the common challenges during development of analytical platforms is instrument or method variability which can cause deviation in results. Here, we show a new automated analytical platform for MAM implemented on 3 different sites: the components of MAM platform include automated sample preparation, LC-MS based MAM, and data treatment automation. To our knowledge, this is the first study to show global harmonization on automated MAM platforms and the inter-sites comparability including the automated sample preparation and LC-MS instrument. Also, we demonstrate the applicability of MAM to support cell line development, cell culture process development and downstream process development. We expect that this MAM platform will effectively guide process development across multiple projects.

Biotransformation of guttiferones, Symphonia globulifera metabolites, by Bipolaris cactivora, an endophytic fungus isolated from its leaves.

The search for active microorganisms for the biotransformation of guttiferone A (1) and C (6) has been successfully undertaken from a collection of endophytic fungi of Symphonia globulifera. Of the twenty-five isolates obtained from the leaves, three are active and have been identified as Bipolaris cactivora. The products obtained are the result of xanthone cyclisation with the formation of two regioisomers among four possible and corresponding to 1,16-oxy-guttiferone and 3,16-oxy-guttiferone. The biotransformation conditions were studied. Interestingly, both oxy-guttiferones A are present in the plant, and the ratio of 3,16-oxy-guttiferone to 1,16-oxy-guttiferone is 4 : 1, very close to that observed by biotransformation (3.8 : 1). These results are consistent with the involvement of endophytes in their formation pathway from guttiferone A, in planta. Finally, biotransformation made it possible to obtain and describe for the first time oxy-guttiferones C.

Improving the analytical toolbox to investigate copurifying host cell proteins presence: N-(4)-(ß-acetylglucosaminyl)- l-asparaginase case study.

Levels of host cell proteins (HCPs) in purification intermediates and drug substances (DS) of monoclonal antibodies (mAbs) must be carefully monitored for the production of safe and efficacious biotherapeutics. During the development of mAb1, an immunoglobulin G1 product, unexpected results generated with HCP Enzyme-Linked Immunosorbent Assay (ELISA) kit triggered an investigation which led to the identification of a copurifying HCP called N-(4)-(ß-acetylglucosaminyl)-l-asparaginase (AGA, EC3.5.1.26) by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The risk assessment performed indicated a low immunogenicity risk for the copurifying HCP and an ad hoc stability study demonstrated no mAb glycan cleavage and thus no impact on product quality. Fractionation studies performed on polishing steps revealed that AGA was coeluted with the mAb. Very interestingly, the native digestion protocol implemented to go deeper in the MS-HCP profiling was found to be incompatible with correct AGA detection in last purification intermediate and DS, further suggesting a hitchhiking behavior of AGA. In silico surface characterization of AGA also supports this hypothesis. Finally, the combined support of HCP ELISA results and MS allowed process optimization and removal of this copurifying HCP.

Isolation of Guttiferones from Renewable Parts of Symphonia globulifera by Centrifugal Partition Chromatography.

The aim of this study was to investigate the species Symphonia globulifera, a source of polycyclic polyprenylated acyl phloroglucinols such as guttiferone A, which is known to exhibit a variety of biological activities including noticeable antileishmanial properties. Our goal was the identification and the quantification of guttiferone A in different renewable parts of S. globulifera and its preparative isolation. To the best of our knowledge, there is no data concerning its mechanism of action. Consequently, it is particularly interesting to isolate it in gram quantities in order to establish structure activity relationship studies. After performing high-performance liquid chromatography profiles detecting the presence of guttiferone A and proceeding to its quantification, a centrifugal partition chromatography methodology using a two-phase solvent system of cyclohexane/ethyl acetate/methanol/water (20 :  1 :  20 : 1, v/v/v/v) was applied to each extract. In conclusion, a centrifugal partition chromatography system has been developed to ensure a fast, reliable, and scalable way to isolate, with a high level of purity, guttiferone A from five renewable parts of S. globulifera. Moreover, this methodology can be extended to the isolation of other polycyclic polyprenylated acyl phloroglucinols such as guttiferones B, C, and D.

Symphonia globulifera, a widespread source of complex metabolites with potent biological activities.

Symphonia globulifera has been widely used in traditional medicine and has therefore been subjected to several phytochemical studies in the American and African continents. Interestingly, some disparities have been observed concerning its metabolic profile. Several phytochemical studies of S. globulifera have led to the identification of more than 40 compounds, including several polycyclic polyprenylated acylphloroglucinols. Biological evaluations have pointed out the promising biological activities of these secondary metabolites, mostly as antiparasitic or antimicrobial, confirming the traditional use of this plant. The purpose of this review is to describe the natural occurrence, botanical aspects, ethnomedicinal use, structure, and biogenesis, as well as biological activities of compounds isolated from this species according to their provenance.

Comparative LC-MS-based metabolite profiling of the ancient tropical rainforest tree Symphonia globulifera.

In the last few years, several phytochemical studies have been undertaken on the tropical tree Symphonia globulifera leading to the isolation and characterisation of several compounds exhibiting antiparasitic activities against Plasmodium falciparum, Trypanosoma brucei and Leishmania donovani. The comparative LC-MS based metabolite profiling study conducted on the tree led to the identification of compounds originating from specific tissues. The results showed that renewable organs/tissues can be used as the starting material for the production of polycyclic poly-prenylated-acylphloroglucinols, therefore reducing impacts on biodiversity. This study also underlined the lack of knowledge on the secondary metabolites produced by S. globulifera since only a small number of the total detected features were putatively identified using the database of known compounds for the species.

Synthesis of novel guttiferone A derivatives: in-vitro evaluation toward Plasmodium falciparum, Trypanosoma brucei and Leishmania donovani.

The catechol pharmacomodulation of the natural product guttiferone A, isolated from the Symphonia globulifera tree, led to the semisynthesis of a collection of twenty derivatives. The ester and ether derivatives of guttiferone A were evaluated for their anti-plasmodial, trypanocidal and anti-leishmanial activities. Some compounds described below have shown potent antiparasitic activity against Plasmodium falciparum, Trypanosoma brucei and Leishmania donovani in a range from 1 to 5 µM. The evaluation of guttiferone A derivatives against VERO cells highlighted catechol modulations as an interesting tool to decrease the toxicity and keep the activity of this natural compound. The current study revealed new molecules as promising new antiparasitic drug candidates.

Yeast-mediated xanthone synthesis through oxidative intramolecular cyclization.

Benzoylphloroglucinol derivatives are natural products showing diverse biological activities that could be modulated by structural modifications. For this purpose, we studied the biotransformation of guttiferone A and of maclurin using a combinatorial approach for screening active microorganism strains. We found a novel and unexpected yeast-catalyzed oxidation that has selectively given a new oxy-guttiferone A and norathyriol.

Acantholactone, a new manzamine related alkaloid with an unprecedented δ-lactone and ε-lactam ring system.

A new manzamine-related alkaloid with unprecedented δ-lactone and ε-lactam rings called acantholactone (2), was isolated from the Indonesian sponge Acanthostrongylophora sp. The relative configuration of the two new ring systems was established through detailed analysis of NOESY correlations combined with molecular modeling studies. The absolute configuration of 2 was determined as 12S, 24R, 25R, 26R by comparing the computed electronic circular dichroism (ECD) spectra with experimental values.