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Papers by Himanshu Gadgil

Research paper thumbnail of SEC-MS analysis of aggregates in protein mixtures

Lc Gc North America, 2003

Size exclusion chromatography (SEC) coupled with mass spectrometry was used to study heat-induced... more Size exclusion chromatography (SEC) coupled with mass spectrometry was used to study heat-induced aggregation in protein mixture.

Research paper thumbnail of Pre-clinical development of an anti-cancer drug ENZ302, a promising candidate for multiple cancer therapeutics

e13053 Background: Vernonia cinerea extract has been known to be used for various haematological ... more e13053 Background: Vernonia cinerea extract has been known to be used for various haematological disorders including cancer. In the current study, using state-of-the-art extraction and bioanalytical tools we have identified an active compound having anti-tumor properties from the same plant Vernonia cinerea. The crude extract of Vernonia cinerea as well as the active molecule obtained through the chemical route (ENZ302) showed anti-tumor activity against various cancer models including CML, AML, ALL, HER2+ breast cancer, colon cancer, etc. Methods: Various methods including RP-HPLC, XRD, NMR, IR, cell based assay and LC-MS were used to elucidate the molecular structure of ENZ302. MTD, DRF, PK, and Xenograft study etc were carried out in murine model during the preclinical development of ENZ302. Results: ENZ302 resulted significant cytotoxic effect within 8 hrs of treatment in TNBC (Triple Negative Breast Cancer) model, MDA-MB-468 cells when compared with a known inhibitor Lapatinib....

Research paper thumbnail of Nouveau procédé de clonage, d'expression et de purification pour la préparation de ranibizumab

Research paper thumbnail of Neuartiges klonungs-, expressions- und reinigungsverfahren zur herstellung von ranibizumab

Research paper thumbnail of Composition pharmaceutique liquide d'adalimumab

La presente invention concerne une composition pharmaceutique liquide comprenant un anticorps ant... more La presente invention concerne une composition pharmaceutique liquide comprenant un anticorps anti-TNFα, un tampon, un agent stabilisant et un agent tensio-actif.

Research paper thumbnail of Continuous process for reducing heterogeneity of therapeutic protein

Research paper thumbnail of Solid-State Protein Formulation

Research paper thumbnail of Methods for refolding of recombinant antibodies

Research paper thumbnail of Demonstration of Clinical Comparability of the Biosimilar Filgrastim to Neupogen, in Terms of Safety and Efficacy, in Healthy Volunteers and Patients Receiving Myelosuppressive Chemotherapy

European Oncology & Haematology, 2014

Aim: To demonstrate biosimilarity as evidenced by the pharmacokinetic (PK), pharmacodynamics (PD)... more Aim: To demonstrate biosimilarity as evidenced by the pharmacokinetic (PK), pharmacodynamics (PD), efficacy and safety comparability of AccofilR/GrastofilR (filgrastim), a recombinant human granulocyte colony-stimulating factor, and the reference product, NeupogenR. Patients and methods: Four phase I studies were conducted to demonstrate the comparative efficacy of Accofil/Grastofil and Neupogen. PD and PK parameters of Accofil/Grastofil (filgrastim) at and around the main clinical dose (5 μg/kg), using both the intravenous (IV) and subcutaneous (SC) routes of administration, were compared with Neupogen (EU) in healthy volunteers in three phase I clinical studies in a single-dose setting and following multiple-dose administration. An additional phase I PK/PD study was performed to compare Accofil/ Grastofil (filgrastim) to both EU-approved Neupogen and US-licensed Neupogen following the administration of a fixed SC dose of 300 μg. The efficacy and safety of Accofil/Grastofil was als...

Research paper thumbnail of Identification and Quantification of Degradants and Impurities in Antibodies

Pramanik/Impurities and Degradants Using MS, 2011

Research paper thumbnail of DNA Affinity Chromatography

Chromatographic Science Series, 2005

Research paper thumbnail of Affinity Chromatography in Molecular Biology

Chromatographic Science Series, 2005

Research paper thumbnail of Proteome of monocytes primed with lipopolysaccharide: Analysis of the abundant proteins

Research paper thumbnail of Effect of Sugar Molecules on the Viscosity of High Concentration Monoclonal Antibody Solutions

Pharmaceutical Research, 2011

To assess the effect of sugar molecules on solution viscosity at high protein concentrations. A h... more To assess the effect of sugar molecules on solution viscosity at high protein concentrations. A high throughput dynamic light scattering method was used to measure the viscosity of monoclonal antibody solutions. The effects of protein concentration, type of sugar molecule (trehalose, sucrose, sorbitol, glucose, fructose, xylose and galactose), temperature and ionic strength were evaluated. Differential scanning fluorimetry was used to reveal the effect of the same sugars on protein stability and to provide insight into the mechanism by which sugars increase viscosity. The addition of all seven types of sugar molecules studied result in a significant increase in viscosity of high concentration monoclonal antibody solutions. Similar effects of sugars were observed in the two mAbs examined; viscosity could be reduced by increasing the ionic strength or temperature. The effect by sugars was enhanced at higher protein concentrations. Disaccharides have a greater effect on the solution viscosity at high protein concentrations compared to monosaccharides. The effect may be explained by commonly accepted mechanisms of interactions between sugar and protein molecules in solution.

Research paper thumbnail of Improving mass accuracy of high performance liquid chromatography/electrospray ionization time-of-flight mass spectrometry of intact antibodies

Journal of the American Society for Mass Spectrometry, 2006

Research paper thumbnail of Comparison of LC and LC/MS methods for quantifying N-glycosylation in recombinant IgGs

Journal of the American Society for Mass Spectrometry, 2008

Research paper thumbnail of Affinity purification of DNA-binding proteins

Journal of Biochemical and Biophysical Methods, 2001

The focus of this review is on DNA affinity chromatography, which is the most powerful tool for p... more The focus of this review is on DNA affinity chromatography, which is the most powerful tool for purification of DNA binding proteins. The use of nonspecific-, sequence specific- and single stranded-DNA affinity columns in purification of various DNA binding proteins is discussed. The purification strategies for transcription factors, restriction enzymes, telomerases, DNA and RNA polymerase and DNA binding antibodies are described. Different applications of DNA affinity chromatography are presented.

Research paper thumbnail of Succinimide formation at Asn 55 in the complementarity determining region of a recombinant monoclonal antibody IgG1 heavy chain

Journal of Pharmaceutical Sciences, 2009

Research paper thumbnail of The effect of sucrose hydrolysis on the stability of protein therapeutics during accelerated formulation studies

Journal of Pharmaceutical Sciences, 2009

Stability studies of protein therapeutics are often accelerated by storing potential formulations... more Stability studies of protein therapeutics are often accelerated by storing potential formulations at elevated temperatures where the rates of various chemical and physical degradation pathways are increased. An often overlooked caveat of using these studies is the potential degradation of the formulation components themselves. In this report, we show that the monoclonal antibody MAB001 aggregated at a faster rate when formulated with sucrose compared to samples that contained sorbitol or no excipient during accelerated stability studies following an initial lag phase where the rates of aggregate formation were similar in all formulations. The duration of the lag phase was both pH and temperature dependent and a significant increase of protein glycation was noticed during this time. These observations indicate that the enhanced rate of antibody aggregation in sucrose containing formulations is likely due to protein glycation following sucrose hydrolysis under accelerated conditions. This hypothesis was confirmed by demonstrating that antibody directly glycated with glucose aggregated at a faster rate than nonglycated antibody stored in the identical formulation. These findings question the utility of using accelerated stability data for predicting protein stability in sucrose containing formulations stored at 2-8 degrees C, where no glycation or change in aggregation rate was observed.

Research paper thumbnail of Elucidation of two major aggregation pathways in an IgG2 antibody

Journal of Pharmaceutical Sciences, 2009

Two major aggregation pathways observed in an IgG2 molecule are described. Different aggregate sp... more Two major aggregation pathways observed in an IgG2 molecule are described. Different aggregate species generated by long-term incubation of the antibody at 378C were collected by a semi-preparative size exclusion chromatography method. These purified species were analyzed extensively by denaturing size-exclusion chromatography methods. The major aggregation pathway at low pH (4.0) resulted in the formation of both dimers and high molecular weight (HMW) aggregates. It was found that these dimers and HMW aggregates contain antibody molecules that have a peptide bond cleavage between an aspartic acid and proline residue in the CH2 domain. Evidence that unfolding of the CH2 domain may be driving the aggregation at low pH is presented. At higher pH (pH-6.0), formation of a dimer having approximately 75% covalent character was the major aggregation pathway while formation of higher molecular weight aggregates were largely suppressed. The covalent dimer consisted of both disulfide linked antibody molecules and another species ($26%) that was formed due to nondisulfide covalent bonds between two heavy chains. At pH-5.0, both dimer and higher molecular weight aggregates were formed and the aggregation pathway was a combination of the major pathways observed at pH-4.0 and 6.0. The dimer species formed at pH-5.0 had a larger contribution from covalent species-both disulfide and nondisulfide linked, while the HMW aggregate contained a higher percentage of molecules that had the peptide bond cleavage in the CH2 domain. The dimer formed at pH-6.0 was found to have identical secondary and tertiary structure as the intact antibody molecule. However, the dimer and higher molecular weight aggregate formed at pH-4.0 have altered secondary and tertiary structure.

Research paper thumbnail of SEC-MS analysis of aggregates in protein mixtures

Lc Gc North America, 2003

Size exclusion chromatography (SEC) coupled with mass spectrometry was used to study heat-induced... more Size exclusion chromatography (SEC) coupled with mass spectrometry was used to study heat-induced aggregation in protein mixture.

Research paper thumbnail of Pre-clinical development of an anti-cancer drug ENZ302, a promising candidate for multiple cancer therapeutics

e13053 Background: Vernonia cinerea extract has been known to be used for various haematological ... more e13053 Background: Vernonia cinerea extract has been known to be used for various haematological disorders including cancer. In the current study, using state-of-the-art extraction and bioanalytical tools we have identified an active compound having anti-tumor properties from the same plant Vernonia cinerea. The crude extract of Vernonia cinerea as well as the active molecule obtained through the chemical route (ENZ302) showed anti-tumor activity against various cancer models including CML, AML, ALL, HER2+ breast cancer, colon cancer, etc. Methods: Various methods including RP-HPLC, XRD, NMR, IR, cell based assay and LC-MS were used to elucidate the molecular structure of ENZ302. MTD, DRF, PK, and Xenograft study etc were carried out in murine model during the preclinical development of ENZ302. Results: ENZ302 resulted significant cytotoxic effect within 8 hrs of treatment in TNBC (Triple Negative Breast Cancer) model, MDA-MB-468 cells when compared with a known inhibitor Lapatinib....

Research paper thumbnail of Nouveau procédé de clonage, d'expression et de purification pour la préparation de ranibizumab

Research paper thumbnail of Neuartiges klonungs-, expressions- und reinigungsverfahren zur herstellung von ranibizumab

Research paper thumbnail of Composition pharmaceutique liquide d'adalimumab

La presente invention concerne une composition pharmaceutique liquide comprenant un anticorps ant... more La presente invention concerne une composition pharmaceutique liquide comprenant un anticorps anti-TNFα, un tampon, un agent stabilisant et un agent tensio-actif.

Research paper thumbnail of Continuous process for reducing heterogeneity of therapeutic protein

Research paper thumbnail of Solid-State Protein Formulation

Research paper thumbnail of Methods for refolding of recombinant antibodies

Research paper thumbnail of Demonstration of Clinical Comparability of the Biosimilar Filgrastim to Neupogen, in Terms of Safety and Efficacy, in Healthy Volunteers and Patients Receiving Myelosuppressive Chemotherapy

European Oncology & Haematology, 2014

Aim: To demonstrate biosimilarity as evidenced by the pharmacokinetic (PK), pharmacodynamics (PD)... more Aim: To demonstrate biosimilarity as evidenced by the pharmacokinetic (PK), pharmacodynamics (PD), efficacy and safety comparability of AccofilR/GrastofilR (filgrastim), a recombinant human granulocyte colony-stimulating factor, and the reference product, NeupogenR. Patients and methods: Four phase I studies were conducted to demonstrate the comparative efficacy of Accofil/Grastofil and Neupogen. PD and PK parameters of Accofil/Grastofil (filgrastim) at and around the main clinical dose (5 μg/kg), using both the intravenous (IV) and subcutaneous (SC) routes of administration, were compared with Neupogen (EU) in healthy volunteers in three phase I clinical studies in a single-dose setting and following multiple-dose administration. An additional phase I PK/PD study was performed to compare Accofil/ Grastofil (filgrastim) to both EU-approved Neupogen and US-licensed Neupogen following the administration of a fixed SC dose of 300 μg. The efficacy and safety of Accofil/Grastofil was als...

Research paper thumbnail of Identification and Quantification of Degradants and Impurities in Antibodies

Pramanik/Impurities and Degradants Using MS, 2011

Research paper thumbnail of DNA Affinity Chromatography

Chromatographic Science Series, 2005

Research paper thumbnail of Affinity Chromatography in Molecular Biology

Chromatographic Science Series, 2005

Research paper thumbnail of Proteome of monocytes primed with lipopolysaccharide: Analysis of the abundant proteins

Research paper thumbnail of Effect of Sugar Molecules on the Viscosity of High Concentration Monoclonal Antibody Solutions

Pharmaceutical Research, 2011

To assess the effect of sugar molecules on solution viscosity at high protein concentrations. A h... more To assess the effect of sugar molecules on solution viscosity at high protein concentrations. A high throughput dynamic light scattering method was used to measure the viscosity of monoclonal antibody solutions. The effects of protein concentration, type of sugar molecule (trehalose, sucrose, sorbitol, glucose, fructose, xylose and galactose), temperature and ionic strength were evaluated. Differential scanning fluorimetry was used to reveal the effect of the same sugars on protein stability and to provide insight into the mechanism by which sugars increase viscosity. The addition of all seven types of sugar molecules studied result in a significant increase in viscosity of high concentration monoclonal antibody solutions. Similar effects of sugars were observed in the two mAbs examined; viscosity could be reduced by increasing the ionic strength or temperature. The effect by sugars was enhanced at higher protein concentrations. Disaccharides have a greater effect on the solution viscosity at high protein concentrations compared to monosaccharides. The effect may be explained by commonly accepted mechanisms of interactions between sugar and protein molecules in solution.

Research paper thumbnail of Improving mass accuracy of high performance liquid chromatography/electrospray ionization time-of-flight mass spectrometry of intact antibodies

Journal of the American Society for Mass Spectrometry, 2006

Research paper thumbnail of Comparison of LC and LC/MS methods for quantifying N-glycosylation in recombinant IgGs

Journal of the American Society for Mass Spectrometry, 2008

Research paper thumbnail of Affinity purification of DNA-binding proteins

Journal of Biochemical and Biophysical Methods, 2001

The focus of this review is on DNA affinity chromatography, which is the most powerful tool for p... more The focus of this review is on DNA affinity chromatography, which is the most powerful tool for purification of DNA binding proteins. The use of nonspecific-, sequence specific- and single stranded-DNA affinity columns in purification of various DNA binding proteins is discussed. The purification strategies for transcription factors, restriction enzymes, telomerases, DNA and RNA polymerase and DNA binding antibodies are described. Different applications of DNA affinity chromatography are presented.

Research paper thumbnail of Succinimide formation at Asn 55 in the complementarity determining region of a recombinant monoclonal antibody IgG1 heavy chain

Journal of Pharmaceutical Sciences, 2009

Research paper thumbnail of The effect of sucrose hydrolysis on the stability of protein therapeutics during accelerated formulation studies

Journal of Pharmaceutical Sciences, 2009

Stability studies of protein therapeutics are often accelerated by storing potential formulations... more Stability studies of protein therapeutics are often accelerated by storing potential formulations at elevated temperatures where the rates of various chemical and physical degradation pathways are increased. An often overlooked caveat of using these studies is the potential degradation of the formulation components themselves. In this report, we show that the monoclonal antibody MAB001 aggregated at a faster rate when formulated with sucrose compared to samples that contained sorbitol or no excipient during accelerated stability studies following an initial lag phase where the rates of aggregate formation were similar in all formulations. The duration of the lag phase was both pH and temperature dependent and a significant increase of protein glycation was noticed during this time. These observations indicate that the enhanced rate of antibody aggregation in sucrose containing formulations is likely due to protein glycation following sucrose hydrolysis under accelerated conditions. This hypothesis was confirmed by demonstrating that antibody directly glycated with glucose aggregated at a faster rate than nonglycated antibody stored in the identical formulation. These findings question the utility of using accelerated stability data for predicting protein stability in sucrose containing formulations stored at 2-8 degrees C, where no glycation or change in aggregation rate was observed.

Research paper thumbnail of Elucidation of two major aggregation pathways in an IgG2 antibody

Journal of Pharmaceutical Sciences, 2009

Two major aggregation pathways observed in an IgG2 molecule are described. Different aggregate sp... more Two major aggregation pathways observed in an IgG2 molecule are described. Different aggregate species generated by long-term incubation of the antibody at 378C were collected by a semi-preparative size exclusion chromatography method. These purified species were analyzed extensively by denaturing size-exclusion chromatography methods. The major aggregation pathway at low pH (4.0) resulted in the formation of both dimers and high molecular weight (HMW) aggregates. It was found that these dimers and HMW aggregates contain antibody molecules that have a peptide bond cleavage between an aspartic acid and proline residue in the CH2 domain. Evidence that unfolding of the CH2 domain may be driving the aggregation at low pH is presented. At higher pH (pH-6.0), formation of a dimer having approximately 75% covalent character was the major aggregation pathway while formation of higher molecular weight aggregates were largely suppressed. The covalent dimer consisted of both disulfide linked antibody molecules and another species ($26%) that was formed due to nondisulfide covalent bonds between two heavy chains. At pH-5.0, both dimer and higher molecular weight aggregates were formed and the aggregation pathway was a combination of the major pathways observed at pH-4.0 and 6.0. The dimer species formed at pH-5.0 had a larger contribution from covalent species-both disulfide and nondisulfide linked, while the HMW aggregate contained a higher percentage of molecules that had the peptide bond cleavage in the CH2 domain. The dimer formed at pH-6.0 was found to have identical secondary and tertiary structure as the intact antibody molecule. However, the dimer and higher molecular weight aggregate formed at pH-4.0 have altered secondary and tertiary structure.