Publications by authors named "Francis Bitsch"

16 Publications

  • Page 1 of 1

Solving Complex Biologics Truncation Problems by Top-Down Mass Spectrometry.

J Am Soc Mass Spectrom 2021 Jan 4. Epub 2021 Jan 4.

NIBR Biologics Center, Novartis Institutes for BioMedical Research, Klybeckstrasse 141, CH-4057, Basel, Switzerland.

With increasing protein therapeutics being designed as non-mAb (non-monoclonal antibody) modalities, additional efforts and resources are required to develop and characterize such therapeutic proteins. Truncation is an emerging issue for manufacturing of non-mAb drug substances and requires sophisticated methods to investigate. In this paper, we describe two cases with complex truncation problems where traditional methods such as intact mass spectrometry led to inclusive or wrong identifications. Therefore, we developed an online top-down LC-MS (liquid chromatography-mass spectrometry) based workflow to study truncated drug substances, and we successfully identified the clipping locations. Compared to other orthogonal methods, this method provides a unique capability of solving protein clipping problems. The successful identification of truncated species and the high compatibility to routine intact MS make it a very valuable tool for resolving truncation problems during protein production in the pharmaceutical industry.
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http://dx.doi.org/10.1021/jasms.0c00343DOI Listing
January 2021

A natural ligand for the orphan receptor GPR15 modulates lymphocyte recruitment to epithelia.

Sci Signal 2017 Sep 12;10(496). Epub 2017 Sep 12.

Novartis Institutes for BioMedical Research, CH-4056 Basel, Switzerland.

GPR15 is an orphan G protein-coupled receptor (GPCR) that is found in lymphocytes. It functions as a co-receptor of simian immunodeficiency virus and HIV-2 and plays a role in the trafficking of T cells to the lamina propria in the colon and to the skin. We describe the purification from porcine colonic tissue extracts of an agonistic ligand for GPR15 and its functional characterization. In humans, this ligand, which we named GPR15L, is encoded by the gene and has some features similar to the CC family of chemokines. was found in some human and mouse epithelia exposed to the environment, such as the colon and skin. In humans, was also abundant in the cervix. In skin, was readily detected after immunologic challenge and in human disease, for example, in psoriatic lesions. Allotransplantation of skin from -deficient mice onto wild-type mice resulted in substantial graft protection, suggesting nonredundant roles for GPR15 and GPR15L in the generation of effector T cell responses. Together, these data identify a receptor-ligand pair that is required for immune homeostasis at epithelia and whose modulation may represent an alternative approach to treating conditions affecting the skin such as psoriasis.
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http://dx.doi.org/10.1126/scisignal.aal0180DOI Listing
September 2017

Approaches to selective fibroblast growth factor receptor 4 inhibition through targeting the ATP-pocket middle-hinge region.

Medchemcomm 2017 Aug 8;8(8):1604-1613. Epub 2017 Jun 8.

Novartis Institutes for BioMedical Research , CH-4002 Basel , Switzerland . Email:

A diverse range of selective FGFR4 inhibitor hit series were identified using unbiased screening approaches and by the modification of known kinase inhibitor scaffolds. In each case the origin of the selectivity was consistent with an interaction with a poorly conserved cysteine residue within the middle-hinge region of the kinase domain of FGFR4, at position 552. Targeting this region identified a non-covalent diaminopyrimidine series differentiating by size, an irreversible-covalent inhibitor in which Cys552 undergoes an SNAr reaction with a 2-chloropyridine, and a reversible-covalent inhibitor series in which Cys552 forms a hemithioacetal adduct with a 2-formyl naphthalene. In addition, the introduction of an acrylamide into a known FGFR scaffold identified a pan-FGFR inhibitor which reacted with both Cys552 and a second poorly conserved cysteine on the P-loop of FGFR4 at position 477 which is present in all four FGFR family members.
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http://dx.doi.org/10.1039/c7md00213kDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6072211PMC
August 2017

Detection of dihydroxycholesterols in human plasma using HPLC-ESI-MS/MS.

Steroids 2015 Jul 12;99(Pt B):131-8. Epub 2015 Feb 12.

Analytical Sciences and Imaging, Novartis Institutes for BioMedical Research, Novartis Pharma AG, Basel, Switzerland. Electronic address:

We report a straightforward sample preparation procedure and a direct liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) method for the analysis of 7alpha,25-dihydroxycholesterol (7α25-OHC) and 7alpha,27-dihydroxycholesterol (7α27-OHC). By applying a slow protein precipitation approach using cold ethanol, we were able to detect and quantify 7α25-OHC and 7α27-OHC in a fast and reliable manner. The average concentrations from 20 healthy individuals were determined to be 0.21±0.05nM for 7α25-OHC and 3.4±0.1nM for 7α27-OHC. In addition, we are the first to report the average degrees of esterification (n=8) to be 73.8% and 82% for 7α25-OHC and 7α27-OHC, respectively. Using the established method, we achieved the sensitivity sufficient for detecting low abundant dihydroxylated oxysterols in healthy individuals. This result should enable extension of these studies towards a comprehensive analysis of oxysterol levels under disease conditions.
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http://dx.doi.org/10.1016/j.steroids.2015.02.002DOI Listing
July 2015

Critical comparison of MS and immunoassays for the bioanalysis of therapeutic antibodies.

Bioanalysis 2009 Nov;1(8):1375-88

Service de Pharmacologie et d'Immunologie, Ibitecs, Gif-sur-Yvette, France.

Therapeutic antibody assessment in biofluids requires fit-for-purpose bioanalytical methods. The reference is the immunoassay, the accuracy of which may be compromised by interference by endogenous components. Here, we report the inherent analytical problems posed by immunoassays and propose an alternative based on LC-MS that should be readily applicable to the analysis of therapeutic antibodies in biological fluids. We review problems linked to assay sensitivity, the choice of the assay format involving either immunodetection or MS, and strategies in the assessment of bound versus free forms.
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http://dx.doi.org/10.4155/bio.09.121DOI Listing
November 2009

Allosteric non-bisphosphonate FPPS inhibitors identified by fragment-based discovery.

Nat Chem Biol 2010 Sep 15;6(9):660-6. Epub 2010 Aug 15.

Center for Proteomic Chemistry and Global Discovery Chemistry, Novartis Institutes for Biomedical Research, Basel, Switzerland.

Bisphosphonates are potent inhibitors of farnesyl pyrophosphate synthase (FPPS) and are highly efficacious in the treatment of bone diseases such as osteoporosis, Paget's disease and tumor-induced osteolysis. In addition, the potential for direct antitumor effects has been postulated on the basis of in vitro and in vivo studies and has recently been demonstrated clinically in early breast cancer patients treated with the potent bisphosphonate zoledronic acid. However, the high affinity of bisphosphonates for bone mineral seems suboptimal for the direct treatment of soft-tissue tumors. Here we report the discovery of the first potent non-bisphosphonate FPPS inhibitors. These new inhibitors bind to a previously unknown allosteric site on FPPS, which was identified by fragment-based approaches using NMR and X-ray crystallography. This allosteric and druggable pocket allows the development of a new generation of FPPS inhibitors that are optimized for direct antitumor effects in soft tissue.
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http://dx.doi.org/10.1038/nchembio.421DOI Listing
September 2010

Absolute quantification of monoclonal antibodies in biofluids by liquid chromatography-tandem mass spectrometry.

Anal Chem 2008 Feb 25;80(4):1290-6. Epub 2008 Jan 25.

BioAnalytical Sciences, Discovery Technologies, Novartis Institutes for BioMedical Research, Novartis, Basel, Switzerland.

The development of a quantification method for monoclonal antibodies in serum has been accomplished by high-performance liquid chromatography multiple reactions monitoring mass spectrometry. A human monoclonal antibody (HmAb) was used as the model protein for method development and validation. A peptide from the CDR3-region of its heavy chain was selected and used for quantifying the entire mAb. This signature peptide served as a template for the internal standard. Prior to mass spectrometric analysis approximately 50% of the total serum protein content was removed by albumin depletion. The accuracy of the method ranged between 99 and 112% in cynomolgus monkey serum. The intra-assay coefficient of variation (CV) was lower than 4% at 4 microg/mL and 200 microg/mL HmAb (n = 3). The CV at 400 microg/mL corresponded to 9% (n = 3). In addition, the interassay variation was investigated in a male cynomolgus serum pool and in a female cynomolgus serum pool. The CV for the male cynomolgus pool at 4 microg/mL HmAb was 7% (n = 3). The CV obtained from the female pool was 8% (n = 3), at 4 microg/mL. The dynamic range of the method was 3 orders of magnitude. After albumin depletion of 25 microL of serum, a lowest limit of quantification of 2 microg/mL HmAb was reached in both human and cynomolgus monkey samples.
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http://dx.doi.org/10.1021/ac702115bDOI Listing
February 2008

Biophotonics applied to proteomics.

Subcell Biochem 2007 ;43:323-38

Novartis Institutes for BioMedical Research, Genome and Protein Sciences Systems Biology, Basel, Switzeland.

Since the completion of the human genome sequencing, our understanding of gene and protein function and their involvement in physiopathological states has increased dramatically, partly due to technological developments in photonics. Photonics is a very active area where new developments occur on a weekly basis, while established tools are adapted to fulfill the needs of other disciplines like genomics and proteomics. Biophotonics emerged at the interface of photonics and biology as a very straightforward and efficient approach to observe and manipulate living systems. In this chapter, we review the current applications of photonics and imaging to proteomics from 2D gels analysis to molecular imaging.
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http://dx.doi.org/10.1007/978-1-4020-5943-8_15DOI Listing
January 2008

Novel beta-lactam derivatives: potent and selective inhibitors of the chymotrypsin-like activity of the human 20S proteasome.

Bioorg Med Chem Lett 2007 Jan 24;17(2):358-62. Epub 2006 Oct 24.

Novartis Institutes for BioMedical Research, WKL-136.4.25, CH-4002 Basel, Switzerland.

A series of beta-lactam derivatives has been designed and synthesized to inhibit the chymotrypsin-like activity of the human 20S proteasome. The most potent compounds of this new structural class of beta-subunit selective 20S proteasome inhibitors exhibit IC50 values in the low-nanomolar range and show good selectivity over the trypsin-like and post-glutamyl-peptide hydrolytic activities of the enzyme.
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http://dx.doi.org/10.1016/j.bmcl.2006.10.047DOI Listing
January 2007

Structural basis for the exceptional in vivo efficacy of bisphosphonate drugs.

ChemMedChem 2006 Feb;1(2):267-73

Novartis Institutes for BioMedical Research, Discovery Technologies, 4002 Basel, Switzerland.

To understand the structural basis for bisphosphonate therapy of bone diseases, we solved the crystal structures of human farnesyl pyrophosphate synthase (FPPS) in its unliganded state, in complex with the nitrogen-containing bisphosphonate (N-BP) drugs zoledronate, pamidronate, alendronate, and ibandronate, and in the ternary complex with zoledronate and the substrate isopentenyl pyrophosphate (IPP). By revealing three structural snapshots of the enzyme catalytic cycle, each associated with a distinct conformational state, and details about the interactions with N-BPs, these structures provide a novel understanding of the mechanism of FPPS catalysis and inhibition. In particular, the accumulating substrate, IPP, was found to bind to and stabilize the FPPS-N-BP complexes rather than to compete with and displace the N-BP inhibitor. Stabilization of the FPPS-N-BP complex through IPP binding is supported by differential scanning calorimetry analyses of a set of representative N-BPs. Among other factors such as high binding affinity for bone mineral, this particular mode of FPPS inhibition contributes to the exceptional in vivo efficacy of N-BP drugs. Moreover, our data form the basis for structure-guided design of optimized N-BPs with improved pharmacological properties.
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http://dx.doi.org/10.1002/cmdc.200500059DOI Listing
February 2006

Efficient uniform isotope labeling of Abl kinase expressed in Baculovirus-infected insect cells.

J Biomol NMR 2005 Apr;31(4):343-9

Novartis Institutes for BioMedical Research, Basel, Switzerland.

This report shows for the first time the efficient uniform isotope labeling of a recombinant protein expressed using Baculovirus-infected insect cells. The recent availability of suitable media for (15)N- and (13)C/(15)N-labeling in insect cells, the high expression of Abl kinase in these labeling media and a suitable labeling protocol made it possible to obtain a (1)H-(15)N-HSQC spectrum for the catalytic domain of Abl kinase of good quality and with label incorporation rates > 90%. The presented isotope labeling method should be applicable also to further proteins where successful expression is restricted to the Baculovirus expression system.
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http://dx.doi.org/10.1007/s10858-005-2451-3DOI Listing
April 2005

Evidence for ligand-independent transcriptional activation of the human estrogen-related receptor alpha (ERRalpha): crystal structure of ERRalpha ligand binding domain in complex with peroxisome proliferator-activated receptor coactivator-1alpha.

J Biol Chem 2004 Nov 26;279(47):49330-7. Epub 2004 Aug 26.

Protein Structure Unit, Novartis Institutes for Biomedical Research, Basel, Switzerland.

The crystal structure of the ligand binding domain (LBD) of the estrogen-related receptor alpha (ERRalpha, NR3B1) complexed with a coactivator peptide from peroxisome proliferator-activated receptor coactivator-1alpha (PGC-1alpha) reveals a transcriptionally active conformation in the absence of a ligand. This is the first x-ray structure of ERRalpha LBD, solved to a resolution of 2.5 A, and the first structure of a PGC-1alpha complex. The putative ligand binding pocket (LBP) of ERRalpha is almost completely occupied by side chains, in particular with the bulky side chain of Phe328 (corresponding to Ala272 in ERRgamma and Ala350 in estrogen receptor alpha). Therefore, a ligand of a size equivalent to more than approximately 4 carbon atoms could only bind in the LBP, if ERRalpha would undergo a major conformational change (in particular the ligand would displace H12 from its agonist position). The x-ray structure thus provides strong evidence for ligand-independent transcriptional activation by ERRalpha. The interactions of PGC-1alpha with ERRalpha also reveal for the first time the atomic details of how a coactivator peptide containing an inverted LXXLL motif (namely a LLXYL motif) binds to a LBD. In addition, we show that a PGC-1alpha peptide containing this nuclear box motif from the L3 site binds ERRalpha LBD with a higher affinity than a peptide containing a steroid receptor coactivator-1 motif and that the affinity is further enhanced when all three leucine-rich regions of PGC-1alpha are present.
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http://dx.doi.org/10.1074/jbc.M407999200DOI Listing
November 2004

Crystal structure of the human RORalpha Ligand binding domain in complex with cholesterol sulfate at 2.2 A.

J Biol Chem 2004 Apr 13;279(14):14033-8. Epub 2004 Jan 13.

Discovery Technologies, Protein Structure Unit, Basel, Switzerland.

The retinoic acid-related orphan receptor alpha (RORalpha) is an orphan member of the subfamily 1 of nuclear hormone receptors. Our recent structural and functional studies have led to the hypothesis that cholesterol or a cholesterol derivative is the natural ligand of RORalpha. We have now solved the x-ray crystal structure of the ligand binding domain of RORalpha in complex with cholesterol-3-O-sulfate following a ligand exchange experiment. In contrast to the 3-hydroxyl of cholesterol, the 3-O-sulfate group makes additional direct hydrogen bonds with three residues of the RORalpha ligand binding domain, namely NH-Gln(289), NH-Tyr(290), and NH1-Arg(370). When compared with the complex with cholesterol, seven well ordered water molecules have been displaced, and the ligand is slightly shifted toward the hydrophilic part of the ligand binding pocket, which is ideally suited for interactions with a sulfate group. These additional ligand-protein interactions result in an increased affinity of cholesterol sulfate when compared with cholesterol, as shown by mass spectrometry analysis done under native conditions and differential scanning calorimetry. Moreover, mutational studies show that the higher binding affinity of cholesterol sulfate translates into an increased transcriptional activity of RORalpha. Our findings suggest that cholesterol sulfate could play a crucial role in the regulation of RORalpha in vivo.
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http://dx.doi.org/10.1074/jbc.M400302200DOI Listing
April 2004

Identification of natural ligands of retinoic acid receptor-related orphan receptor alpha ligand-binding domain expressed in Sf9 cells--a mass spectrometry approach.

Anal Biochem 2003 Dec;323(1):139-49

Central Technologies, Novartis Institutes for Biomedical Research, Lichtstrasse 35, CH-4002 Basel, Switzerland.

The ligand-binding domain (LBD) of the human retinoic acid receptor-related orphan receptor (RORalpha-LBD), expressed in Sf9 cells, was purified and analyzed by electrospray ionization-mass spectrometry (ESI-MS). ESI-MS operated under native conditions showed the presence of a fortuitous ligand with molecular weight 386. Further analysis by gas chromatography-mass spectrometry (GC-MS) allowed the identification of the ligands bound to the LBD. Cholesterol (77%) and 7-dehydrocholesterol (provitamin D(3); 18%) were shown to be the major ligands. A monohydroxylated cholesterol derivative was identified as a minor ligand. In addition, ligand exchange experiments monitored by ESI-MS showed that cholesterol sulfate has a higher affinity for RORalpha-LBD than cholesterol and 25-hydroxycholesterol. Binding of coactivator (CoA) peptide GRIP1P was shown to occur in a stoichiometric manner. Therefore, monitoring of binding of CoAs by mass spectrometry could be used for classification of the ligands as agonist or antagonist molecules.
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http://dx.doi.org/10.1016/j.ab.2003.08.029DOI Listing
December 2003

Amino-acid-type selective isotope labeling of proteins expressed in Baculovirus-infected insect cells useful for NMR studies.

J Biomol NMR 2003 Aug;26(4):367-72

Novartis Pharma AG, Central Technologies, Oncology Research, CH-4002 Basel, Switzerland.

Culture conditions for successful amino-acid-type selective isotope labeling of proteins expressed in Baculovirus-infected insect cells are described. The method was applied to the selective labeling of the catalytic domain of c-Abl kinase with (15)N-phenylalanine, (15)N-glycine, (15)N-tyrosine or (15)N-valine. For the essential amino acids phenylalanine, tyrosine and valine high (15)N-label incorporation rates of >/=90% and approximately the expected number of resonances in the HSQC spectra were observed, which was not the case for the non-essential amino acid glycine. The method should be applicable to amino-acid-type selective isotope labeling of other recombinant proteins which have not been amenable to NMR analysis.
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http://dx.doi.org/10.1023/a:1024013111478DOI Listing
August 2003

X-ray structure of the hRORalpha LBD at 1.63 A: structural and functional data that cholesterol or a cholesterol derivative is the natural ligand of RORalpha.

Structure 2002 Dec;10(12):1697-707

Central Technologies, Protein Structure Unit, Novartis Pharma AG, CH-4002 Basel, Switzerland.

The retinoic acid-related orphan receptor alpha (RORalpha) is an orphan member of the subfamily 1 of nuclear hormone receptors. No X-ray structure of RORalpha has been described so far, and no ligand has been identified. We describe the first crystal structure of the ligand binding domain (LBD) of RORalpha, at 1.63 A resolution. This structure revealed a ligand present in the ligand binding pocket (LBP), which was identified by X-ray crystallography as cholest-5-en-3beta-ol (cholesterol). Moreover, RORalpha transcriptional activity could be modulated by changes in intracellular cholesterol level or mutation of residues involved in cholesterol binding. These findings suggest that RORalpha could play a key role in the regulation of cholesterol homeostasis and thus represents an important drug target in cholesterol-related diseases.
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http://dx.doi.org/10.1016/s0969-2126(02)00912-7DOI Listing
December 2002