Regeneron Pharmaceuticals, Inc.

Regeneran Pharmaceuticals, Inc.

Haibo Qiu

Ning Li

Hongxia Wang

Xiaobin Xu

Jikang Wu

Sook Yen E

Kenneth S Graham

Douglas Kamen

Yu Huang

Rosalynn Molden

David Bramhall

Xiaoxiao Huang

Mengqi Hu

Xueqing Zhao

Ming Huang

Teng-Chieh Yang

Embodiments provide for methods of predicting glycation percentage of an amino acid in a therapeutic biomolecule. In one example, a method of predicting a glycation percentage of an amino acid in a biomolecule includes determining a first set of rates for a de-glycation reaction for a first set of temperatures, inferring a second set of one or more rate(s) for the de-glycation reaction for a second set of temperatures, and using the second set of one or more rate(s) to predict the glycation percentage at any temperature corresponding to the second set of temperatures and over any time duration. Also provided are methods for maintaining a glycation percentage of an amino acid within a predetermined glycation percentage range over a shelf-life of a therapeutic biomolecule, and methods for either reducing or increasing a potency of a therapeutic biomolecule in a subject at a time of administration.

Methods for predicting and modulating glycation of a protein

The present invention provides methods and systems for isotyping and quantification of antibodies based on immunocapture and/or Liquid Chromatography-Mass Spectrometry (LC-MS) analysis. These antibodies are induced by the administration of pharmaceutical products. The immunocapture method comprises contacting samples with a solid support, wherein the pharmaceutical product has been cross-linked directly to the solid support. The MS analysis includes conducting peptide mapping, selecting unique peptides and fragment ions to generate MRM (multiple reaction monitoring) transitions, optimizing collision energy, and determining a LLOQ (lower limit of quantification).

LC-MS methods for antibody isotyping and quantification

Disclosed are methods for improving compound detection and characterization. Methods for characterizing a sample are disclosed. The methods can include providing a sample to a liquid chromatography system capable of sample separation to generate sample components; analyzing sample components by multiplexed targeted selected ion monitoring (SIM) to generate an inclusion list; and performing iterative mass spectral data-dependent acquisition (DDA) from the inclusion list, to identify individual sample components thereby characterizing the sample. In one example, multiplexed targeted SIMs and iterative MS2 DDA acquisition is used to increase robust compound identification for cell culture medium analysis.

High confidence compound identification by liquid chromatography-mass spectrometry

The present disclosure provides methods and compositions for the determining the abundance and/or concentration of protein biomarkers in a biological sample.

Use of LC-MS/MS to quantitate protein biomarkers

The present invention provides methods to quickly and efficiently assess the effect of antibody variants, including PTMs, e.g., glycosylation, oxidation, and deamidation variants, on the binding between the antibody and FcRn. In particular, the present invention is based, at least in part, on the development of an online, two dimension liquid chromatography (2D-LC) method for assessing binding between antibody variants and FcRn. In one aspect, the online 2D-LC is coupled with mass spectrometry (MS). The present invention allows for differentiation of antibody variants by peak pattern and retention time profile by affinity chromatography, and identification of these variants by mass spectrometry analysis.

Methods for assessing binding affinity of an antibody variant to the neonatal Fc receptor

The present application provides methods and systems for identifying host cell protein impurities which have enzymatic activities in biopharmaceutical products and in samples during manufacturing processes. The present application provides various activity-based probes to characterize different enzyme classes of host cell protein impurities including probes containing functionalized molecules with reporter or affinity-based tags.

Activity based host cell protein profiling

Methods for detecting and/or discriminating between post-translational modification variants of an antibody of interest in a sample. The methods including: contacting a sample comprising one or more antibodies of interest with a protease to digest the sample into antibody fragments; separating antibody fragments by molecular weight and/or charge in one or more capillaries using capillary electrophoresis; eluting separated antibody fragments from the one or more capillaries; and determining the mass of the eluted antibody fragments by mass spec analysis, thereby detecting and/or discriminating between post-translational modification variants of the antibody of interest.

Native microfluidic CE-MS analysis of antibody charge heterogeneity

The present disclosure generally pertains to methods of identifying and quantitating host cell proteins (HCPs) in therapeutic protein development. In particular, the present invention generally pertains to methods of liquid chromatography-tandem mass spectrometry (LC-MS/MS) for unbiased identification and sensitive quantitation of HCPs in therapeutic protein development.

Unbiased and high-throughput identification and quantification of host cell protein impurities by automated iterative LC-MS/MS (HCP-AIMS) for therapeutic protein development

The disclosure provides methods of characterizing a sample of oligonucleotides of interest using liquid chromatography and mass spectrometry.

Use of liquid chromatography and mass spectrometry to characterize oligonucleotides

Compositions and methods for identifying free thiols in protein are provided. An exemplary method labeling peptides with a tag to identify free thiols and a tag to identify native disulfide bonds and analyzing the tags using targeted MS. In one embodiment, the method provides complete coverage of all 32 cysteine residues in an IgG molecule. In other embodiments the method covers the 16 cysteine residues on the heavy and light chains in an IgG molecule. In another embodiment, the method covers the 5 cysteine residues on each light chain of an IgG molecule. In another embodiment, the method covers the 11 cysteine residues on each heavy chain of an IgG molecule.

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Hartsdale, NY, United States of America

Haibo Qiu