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Article ; Online: Exploring the Higher Order Structure and Conformational Transitions in Insulin Microcrystalline Biopharmaceuticals by Proton-Detected Solid-State Nuclear Magnetic Resonance at Natural Abundance.

Pujahari, Soumya Ranjan / Purusottam, Rudra N / Mali, Pramod S / Sarkar, Sambeda / Khaneja, Navin / Vajpai, Navratna / Kumar, Ashutosh

Analytical chemistry

2024 Volume 96, Issue 12, Page(s) 4756–4763

Abstract: The integrity of a higher order structure (HOS) is an essential requirement to ensure the efficacy, stability, and safety of protein therapeutics. Solution-state nuclear magnetic resonance (NMR) occupies a unique niche as one of the most promising ... ...

Abstract	The integrity of a higher order structure (HOS) is an essential requirement to ensure the efficacy, stability, and safety of protein therapeutics. Solution-state nuclear magnetic resonance (NMR) occupies a unique niche as one of the most promising methods to access atomic-level structural information on soluble biopharmaceutical formulations. Another major class of drugs is poorly soluble, such as microcrystalline suspensions, which poses significant challenges for the characterization of the active ingredient in its native state. Here, we have demonstrated a solid-state NMR method for HOS characterization of biopharmaceutical suspensions employing a selective excitation scheme under fast magic angle spinning (MAS). The applicability of the method is shown on commercial insulin suspensions at natural isotopic abundance. Selective excitation aided with proton detection and non-uniform sampling (NUS) provides improved sensitivity and resolution. The enhanced resolution enabled us to demonstrate the first experimental evidence of a phenol-escaping pathway in insulin, leading to conformational transitions to different hexameric states. This approach has the potential to serve as a valuable means for meticulously examining microcrystalline biopharmaceutical suspensions, which was previously not attainable in their native formulation states and can be seamlessly extended to other classes of biopharmaceuticals such as mAbs and other microcrystalline proteins.
MeSH term(s)	Insulin ; Protons ; Biological Products ; Nuclear Magnetic Resonance, Biomolecular/methods ; Proteins/chemistry
Chemical Substances	Insulin ; Protons ; Biological Products ; Proteins
Language	English
Publishing date	2024-02-07
Publishing country	United States
Document type	Journal Article
ZDB-ID	1508-8
ISSN	1520-6882 ; 0003-2700
ISSN (online)	1520-6882
ISSN	0003-2700
DOI	10.1021/acs.analchem.3c04040
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Article ; Online: Simultaneous characterization of insulin HMWP and protamine sulphate in complex formulations through SEC-coupled mass spectrometry.

Srivatsa, Koduru / Gokhale, Yatika / Chakrabarti, Partha Pratim / Kulshrestha, Abhishek / Vajpai, Navratna

Journal of pharmaceutical and biomedical analysis

2021 Volume 203, Page(s) 114188

Abstract: High molecular weight protein aggregates present in a recombinant human insulin and analogs are conventionally quantified by SEC-HPLC and identified by SEC-MALS as oligomeric population which lacks precise identification of species. The limitation of ... ...

Abstract	High molecular weight protein aggregates present in a recombinant human insulin and analogs are conventionally quantified by SEC-HPLC and identified by SEC-MALS as oligomeric population which lacks precise identification of species. The limitation of these two techniques is vanquished though simultaneous separation and identification by SEC coupled with MS. The identification was established with organic solvent based isocratic elution followed by MS for parallel separation and identification of HMWP species. The developed SEC-MS method is validated to establish the method capability and variability. Further investigations under stress conditions of Insulin analogues revealed the capability of the method to separate higher order oligomeric (Trimeric, and Tetrameric) species alongside covalent dimeric species. Additionally, the method holds good in separating and sequencing protamine peptides used in suspension (Neutral Protamine Hagedorn) and biphasic/mixed (70/30) formulations of Human insulin using ETD-MSMS. The data presented here shows insight towards utilization of state-of-the-art SEC-MS technique in the biopharmaceutical field as a tool to establish the structural comparability of higher order species in biosimilars and to investigate the lot to lot batch variability for protamine sulphate in-terms of sequence confirmation.
MeSH term(s)	Biosimilar Pharmaceuticals ; Humans ; Insulin ; Mass Spectrometry ; Protamines ; Proteins
Chemical Substances	Biosimilar Pharmaceuticals ; Insulin ; Protamines ; Proteins
Language	English
Publishing date	2021-06-03
Publishing country	England
Document type	Journal Article
ZDB-ID	604917-5
ISSN	1873-264X ; 0731-7085
ISSN (online)	1873-264X
ISSN	0731-7085
DOI	10.1016/j.jpba.2021.114188
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Article ; Online: Mapping of Methyl Epitopes of a Peptide-Drug with Its Receptor by 2D STDD-Methyl TROSY NMR Spectroscopy.

Dey, Anomitra / Mitra, Debarghya / Rachineni, Kavitha / Khatri, Lakshya Raj / Paithankar, Harshad / Vajpai, Navratna / Kumar, Ashutosh

Chembiochem : a European journal of chemical biology

2022 Volume 23, Issue 23, Page(s) e202200489

Abstract: The current trend in the biopharmaceutical market has boosted the development and production of biological drugs with high efficacy and fidelity for receptor binding. While high-resolution structural insights into binding epitopes of the receptor are ... ...

Abstract	The current trend in the biopharmaceutical market has boosted the development and production of biological drugs with high efficacy and fidelity for receptor binding. While high-resolution structural insights into binding epitopes of the receptor are indispensable for better therapeutic design, it is tedious and costly. In this work, we develop a protocol by integrating two well-known NMR-based solution-state methods. Saturation transfer double-difference with methyl-TROSY (STDD-Methyl TROSY NMR) was used to probe methyl binding epitopes of the ligand in a label-free environment. This study was carried out with Human insulin as a model peptide drug, with the insulin growth factor receptor (IGFR), which is an off-target receptor for insulin. Methyl epitopes identified from STDD-Methyl TROSY NMR spectroscopy were validated through the HADDOCK platform to generate a drug-receptor model. Since this method can be applied at natural abundance, it has the potential to screen a large set of peptide-drug interactions for optimum receptor binding. Thus, we propose STDD-Methyl TROSY NMR spectroscopy as a technique for rapid screening of biologics for the development of optimized biopharmaceutics.
MeSH term(s)	Humans ; Epitopes ; Magnetic Resonance Spectroscopy/methods ; Ligands ; Peptides ; Insulins ; Nuclear Magnetic Resonance, Biomolecular/methods
Chemical Substances	Epitopes ; Ligands ; Peptides ; Insulins
Language	English
Publishing date	2022-11-10
Publishing country	Germany
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	2020469-3
ISSN	1439-7633 ; 1439-4227
ISSN (online)	1439-7633
ISSN	1439-4227
DOI	10.1002/cbic.202200489
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Article ; Online: Large scale purification and characterization of A21 deamidated variant-most prominent post translational modification (PTM) for insulins which is also widely observed in insulins pharmaceutical manufacturing and storage.

Shukla, Vibhava / Srivatsa, Koduru / Madhu Kumar, M S / Vajpai, Navratna / Agarwal, Neha / Nethra, S / Somesh, B P / Kulshrestha, Abhishek / Hazra, Partha

Protein expression and purification

2021 Volume 185, Page(s) 105895

Abstract: Biopharmaceutical development demands appropriate understanding of product related variants, which are formed due to post-translational modification and during downstream processing. These variants can lead to low yield, reduced biological activity, and ... ...

Abstract	Biopharmaceutical development demands appropriate understanding of product related variants, which are formed due to post-translational modification and during downstream processing. These variants can lead to low yield, reduced biological activity, and suboptimal product quality. In addition, these variants may undergo immune reactions, henceforth need to be appropriately controlled to ensure consistent product quality and patient safety. Deamidation of insulin is the most common post-translational modification occurring in insulin and insulin analogues. Asn
MeSH term(s)	Chromatography, Ion Exchange ; Chromatography, Reverse-Phase ; Freeze Drying/methods ; Humans ; Insulin/analogs & derivatives ; Insulin/biosynthesis ; Insulin/isolation & purification ; Pharmaceutical Preparations ; Protein Processing, Post-Translational ; Recombinant Proteins/isolation & purification
Chemical Substances	Insulin ; Pharmaceutical Preparations ; Recombinant Proteins ; insulin, desamido-
Language	English
Publishing date	2021-05-03
Publishing country	United States
Document type	Journal Article
ZDB-ID	1055455-5
ISSN	1096-0279 ; 1046-5928
ISSN (online)	1096-0279
ISSN	1046-5928
DOI	10.1016/j.pep.2021.105895
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Zs.A 3084: Show issues

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Article ; Online: Dynamic studies of H-Ras•GTPγS interactions with nucleotide exchange factor Sos reveal a transient ternary complex formation in solution.

Vo, Uybach / Vajpai, Navratna / Embrey, Kevin J / Golovanov, Alexander P

Scientific reports

2016 Volume 6, Page(s) 29706

Abstract: The cycling between GDP- and GTP- bound forms of the Ras protein is partly regulated by the binding of Sos. The structural/dynamic behavior of the complex formed between activated Sos and Ras at the point of the functional cycle where the nucleotide ... ...

Abstract	The cycling between GDP- and GTP- bound forms of the Ras protein is partly regulated by the binding of Sos. The structural/dynamic behavior of the complex formed between activated Sos and Ras at the point of the functional cycle where the nucleotide exchange is completed has not been described to date. Here we show that solution NMR spectra of H-Ras∙GTPγS mixed with a functional fragment of Sos (Sos(Cat)) at a 2:1 ratio are consistent with the formation of a rather dynamic assembly. H-Ras∙GTPγS binding was in fast exchange on the NMR timescale and retained a significant degree of molecular tumbling independent of Sos(Cat), while Sos(Cat) also tumbled largely independently of H-Ras. Estimates of apparent molecular weight from both NMR data and SEC-MALS revealed that, at most, only one H-Ras∙GTPγS molecule appears stably bound to Sos. The weak transient interaction between Sos and the second H-Ras∙GTPγS may provide a necessary mechanism for complex dissociation upon the completion of the native GDP → GTP exchange reaction, but also explains measurable GTP → GTP exchange activity of Sos routinely observed in in vitro assays that use fluorescently-labelled analogs of GTP. Overall, the data presents the first dynamic snapshot of Ras functional cycle as controlled by Sos.
Language	English
Publishing date	2016-07-14
Publishing country	England
Document type	Journal Article
ZDB-ID	2615211-3
ISSN	2045-2322 ; 2045-2322
ISSN (online)	2045-2322
ISSN	2045-2322
DOI	10.1038/srep29706
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Article ; Online: High-pressure NMR reveals close similarity between cold and alcohol protein denaturation in ubiquitin.

Vajpai, Navratna / Nisius, Lydia / Wiktor, Maciej / Grzesiek, Stephan

Proceedings of the National Academy of Sciences of the United States of America

2013 Volume 110, Issue 5, Page(s) E368–76

Abstract: Proteins denature not only at high, but also at low temperature as well as high pressure. These denatured states are not easily accessible for experiment, because usually heat denaturation causes aggregation, whereas cold or pressure denaturation occurs ... ...

Abstract	Proteins denature not only at high, but also at low temperature as well as high pressure. These denatured states are not easily accessible for experiment, because usually heat denaturation causes aggregation, whereas cold or pressure denaturation occurs at temperatures well below the freezing point of water or pressures above 5 kbar, respectively. Here we have obtained atomic details of the pressure-assisted, cold-denatured state of ubiquitin at 2,500 bar and 258 K by high-resolution NMR techniques. Under these conditions, a folded, native-like and a disordered state exist in slow exchange. Secondary chemical shifts show that the disordered state has structural propensities for a native-like N-terminal β-hairpin and α-helix and a nonnative C-terminal α-helix. These propensities are very similar to the previously described alcohol-denatured (A-)state. Similar to the A-state, (15)N relaxation data indicate that the secondary structure elements move as independent segments. The close similarity of pressure-assisted, cold-denatured, and alcohol-denatured states with native and nonnative secondary elements supports a hierarchical mechanism of folding and supports the notion that similar to alcohol, pressure and cold reduce the hydrophobic effect. Indeed, at nondenaturing concentrations of methanol, a complete transition from the native to the A-state can be achieved at ambient temperature by varying the pressure from 1 to 2,500 bar. The methanol-assisted pressure transition is completely reversible and can also be induced in protein G. This method should allow highly detailed studies of protein-folding transitions in a continuous and reversible manner.
MeSH term(s)	Cold Temperature ; Ethanol/chemistry ; Humans ; Kinetics ; Magnetic Resonance Spectroscopy/methods ; Methanol/chemistry ; Models, Molecular ; Pressure ; Protein Denaturation ; Protein Structure, Secondary ; Protein Structure, Tertiary ; Protein Unfolding ; Thermodynamics ; Ubiquitin/chemistry
Chemical Substances	Ubiquitin ; Ethanol (3K9958V90M) ; Methanol (Y4S76JWI15)
Language	English
Publishing date	2013-01-02
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	209104-5
ISSN	1091-6490 ; 0027-8424
ISSN (online)	1091-6490
ISSN	0027-8424
DOI	10.1073/pnas.1212222110
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Zs.A 1148: Show issues

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Article ; Online: NMR backbone assignments of the tyrosine kinase domain of human fibroblast growth factor receptor 1.

Vajpai, Navratna / Schott, Anne-Kathrin / Vogtherr, Martin / Breeze, Alexander L

Biomolecular NMR assignments

2014 Volume 8, Issue 1, Page(s) 85–88

Abstract: Members of the fibroblast growth factor receptor tyrosine kinase family (FGFR1-4) play an important role in many signalling cascades. Although tightly regulated, aberrant activity of these enzymes may lead to, or become features of, disease pathologies ... ...

Abstract	Members of the fibroblast growth factor receptor tyrosine kinase family (FGFR1-4) play an important role in many signalling cascades. Although tightly regulated, aberrant activity of these enzymes may lead to, or become features of, disease pathologies including cancer. FGFR isoforms have been the subject of drug discovery programmes, with a number of kinase-domain inhibitors in pre-clinical and clinical development. Here, we present the first (83% complete) backbone resonance assignments of apo-FGFR1 kinase.
MeSH term(s)	Amino Acid Sequence ; Humans ; Nuclear Magnetic Resonance, Biomolecular ; Protein Structure, Tertiary ; Receptor, Fibroblast Growth Factor, Type 1/chemistry
Chemical Substances	Receptor, Fibroblast Growth Factor, Type 1 (EC 2.7.10.1)
Language	English
Publishing date	2014-04
Publishing country	Netherlands
Document type	Journal Article
ZDB-ID	2388861-1
ISSN	1874-270X ; 1874-2718
ISSN (online)	1874-270X
ISSN	1874-2718
DOI	10.1007/s12104-013-9458-6
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Article ; Online: Kinase in motion: insights into the dynamic nature of p38α by high-pressure NMR spectroscopic studies.

Nielsen, Gerd / Jonker, Hendrik R A / Vajpai, Navratna / Grzesiek, Stephan / Schwalbe, Harald

Chembiochem : a European journal of chemical biology

2013 Volume 14, Issue 14, Page(s) 1799–1806

Abstract: Protein kinases are highly dynamic and complex molecules. Here we present high-pressure and relaxation studies of the activated p38α mitogen-activated protein kinase (MAPK). p38α plays a central role in inflammatory diseases such as rheumatoid arthritis ... ...

Abstract	Protein kinases are highly dynamic and complex molecules. Here we present high-pressure and relaxation studies of the activated p38α mitogen-activated protein kinase (MAPK). p38α plays a central role in inflammatory diseases such as rheumatoid arthritis and is therefore a highly attractive pharmaceutical target. The combination of high pressure and NMR spectroscopy allowed for a detailed per-residue based assessment of the structural plasticity of p38α and the accessibility of low-lying excited-energy conformations throughout the kinase structure. Such information is uniquely accessible through the combination of liquid-state NMR and high pressure and is of considerable value for the drug discovery process. The interactions of p38α and DFG-in and DFG-out ligands were studied under the application of high pressure, and we demonstrate how we can alter kinase dynamics by pressure in a similar way to what has previously only been observed by ligand binding. Pressure is shown to be a mild and efficient tool for manipulation of intermediate-timescale dynamics.
MeSH term(s)	Animals ; Mice ; Mitogen-Activated Protein Kinase 14/chemistry ; Mitogen-Activated Protein Kinase 14/genetics ; Mitogen-Activated Protein Kinase 14/metabolism ; Nuclear Magnetic Resonance, Biomolecular ; Pressure ; Protein Structure, Secondary ; Protein Structure, Tertiary ; Recombinant Proteins/biosynthesis ; Recombinant Proteins/chemistry ; Recombinant Proteins/genetics ; Temperature
Chemical Substances	Recombinant Proteins ; Mitogen-Activated Protein Kinase 14 (EC 2.7.11.24)
Language	English
Publishing date	2013-09-23
Publishing country	Germany
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	2020469-3
ISSN	1439-7633 ; 1439-4227
ISSN (online)	1439-7633
ISSN	1439-4227
DOI	10.1002/cbic.201300170
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Article ; Online: Physicochemical and functional characterization of MYL-1501D, a proposed biosimilar to insulin glargine.

Goyal, Parag / Pai, Harish Venkatraman / Kodali, Phanichand / Vats, Bhavesh / Vajpai, Navratna / Annegowda, Shankara / Mane, Krishnappa / Mohan, Shamini / Saxena, Shruti / Veerabhadraia, Anil Bangalore / Palande, Milee / Nair, Preethy Sasankan / More, Digvijay Chandrashekar / Karudumpa, Umamaheshwara Rao / Jyothirmai, Kunala / Bhattacharya, Adroha / Almeida, Frida / Khyade, Santosh Gulab / Gouda, Shankara /

Ranayhossaini, Daniel J / Moole, Praveen Reddy / Smith, Jeffrey P / Barve, Abhijit / Melarkode, Ramakrishnan / Ullanat, Rajesh

PloS one

2021 Volume 16, Issue 6, Page(s) e0253168

Abstract: Insulin glargine is a long-acting analogue of human insulin that has been used to manage hyperglycemia in patients with diabetes mellitus (DM) for nearly 20 years. Insulin glargine has a relatively constant concentration-time profile that mimics basal ... ...

Abstract	Insulin glargine is a long-acting analogue of human insulin that has been used to manage hyperglycemia in patients with diabetes mellitus (DM) for nearly 20 years. Insulin glargine has a relatively constant concentration-time profile that mimics basal levels of insulin and allows for once-daily administration. MYL-1501D is a biosimilar insulin glargine designed to offer greater access of insulin glargine to patients, with comparable efficacy and safety to the marketed reference product. We conducted a comprehensive panel of studies based on a formal analysis of critical quality attributes to characterize the structural and functional properties of MYL-1501D and reference insulin glargine products available in the United States and European Union. MYL-1501D was comprehensively shown to have high similarity to the reference products in terms of protein structure, metabolic activity (both in vitro cell-based assays and in vivo rabbit bioassays), and in vitro cell-based assays for mitogenic activity. The structural analyses demonstrated that the primary protein sequence was identical, and secondary and tertiary structures are similar between the proposed biosimilar and the reference products. Insulin receptor binding affinity and phosphorylation studies also established analytical similarity. MYL-1501D demonstrated high similarity in different metabolic assays of glucose uptake, adipogenesis activity, and inhibition of stimulated lipolysis. Rabbit bioassay studies showed MYL-1501D and EU-approved insulin glargine are highly similar to US-licensed insulin glargine. These product quality studies show high similarity between MYL-1501D and licensed or approved insulin glargine products and suggest the potential of MYL-1501D as an alternative cost-effective treatment option for patients and clinicians.
MeSH term(s)	3T3 Cells ; Adipogenesis/drug effects ; Amino Acid Sequence ; Animals ; Biosimilar Pharmaceuticals/chemistry ; Biosimilar Pharmaceuticals/pharmacology ; CHO Cells ; Circular Dichroism ; Cricetulus ; Gas Chromatography-Mass Spectrometry ; Glucose/metabolism ; Humans ; Insulin Glargine/chemistry ; Insulin Glargine/pharmacology ; Lipolysis/drug effects ; Magnetic Resonance Spectroscopy ; Mice ; Protein Structure, Secondary ; Protein Structure, Tertiary ; Rabbits ; Spectroscopy, Fourier Transform Infrared
Chemical Substances	Biosimilar Pharmaceuticals ; MYL-1501D ; Insulin Glargine (2ZM8CX04RZ) ; Glucose (IY9XDZ35W2)
Language	English
Publishing date	2021-06-16
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ISSN	1932-6203
ISSN (online)	1932-6203
DOI	10.1371/journal.pone.0253168
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Article ; Online: Monitoring Ras Interactions with the Nucleotide Exchange Factor Son of Sevenless (Sos) Using Site-specific NMR Reporter Signals and Intrinsic Fluorescence.

Vo, Uybach / Vajpai, Navratna / Flavell, Liz / Bobby, Romel / Breeze, Alexander L / Embrey, Kevin J / Golovanov, Alexander P

The Journal of biological chemistry

2015 Volume 291, Issue 4, Page(s) 1703–1718

Abstract: The activity of Ras is controlled by the interconversion between GTP- and GDP-bound forms partly regulated by the binding of the guanine nucleotide exchange factor Son of Sevenless (Sos). The details of Sos binding, leading to nucleotide exchange and ... ...

Abstract	The activity of Ras is controlled by the interconversion between GTP- and GDP-bound forms partly regulated by the binding of the guanine nucleotide exchange factor Son of Sevenless (Sos). The details of Sos binding, leading to nucleotide exchange and subsequent dissociation of the complex, are not completely understood. Here, we used uniformly (15)N-labeled Ras as well as [(13)C]methyl-Met,Ile-labeled Sos for observing site-specific details of Ras-Sos interactions in solution. Binding of various forms of Ras (loaded with GDP and mimics of GTP or nucleotide-free) at the allosteric and catalytic sites of Sos was comprehensively characterized by monitoring signal perturbations in the NMR spectra. The overall affinity of binding between these protein variants as well as their selected functional mutants was also investigated using intrinsic fluorescence. The data support a positive feedback activation of Sos by Ras·GTP with Ras·GTP binding as a substrate for the catalytic site of activated Sos more weakly than Ras·GDP, suggesting that Sos should actively promote unidirectional GDP → GTP exchange on Ras in preference of passive homonucleotide exchange. Ras·GDP weakly binds to the catalytic but not to the allosteric site of Sos. This confirms that Ras·GDP cannot properly activate Sos at the allosteric site. The novel site-specific assay described may be useful for design of drugs aimed at perturbing Ras-Sos interactions.
MeSH term(s)	Allosteric Site ; Catalytic Domain ; Fluorescence ; Guanosine Diphosphate/metabolism ; Guanosine Triphosphate/metabolism ; Humans ; Magnetic Resonance Spectroscopy ; Protein Binding ; Proto-Oncogene Proteins p21(ras)/chemistry ; Proto-Oncogene Proteins p21(ras)/genetics ; Proto-Oncogene Proteins p21(ras)/metabolism ; Son of Sevenless Protein, Drosophila/chemistry ; Son of Sevenless Protein, Drosophila/genetics ; Son of Sevenless Protein, Drosophila/metabolism
Chemical Substances	KRAS protein, human ; Son of Sevenless Protein, Drosophila ; Guanosine Diphosphate (146-91-8) ; Guanosine Triphosphate (86-01-1) ; HRAS protein, human (EC 3.6.5.2) ; Proto-Oncogene Proteins p21(ras) (EC 3.6.5.2)
Language	English
Publishing date	2015-11-12
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	2997-x
ISSN	1083-351X ; 0021-9258
ISSN (online)	1083-351X
ISSN	0021-9258
DOI	10.1074/jbc.M115.691238
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