Table 3.

Patent position and pros and cons of analytical tools that generics can utilize for profiling generic/biosimilar LMWH drug products

Technique	Advantages	Challenges	Patent	Inventor(s)	Current assignee	Patent expiry
RP-LC–MS	Capability to perform intact chain mapping analysis; offer rich information about chain distribution and composition of LMWHs; can identify trace amounts of unusual structures or impurities, can characterize by-products, and degradation products	Insufficient separation resolution, detection sensitivity or specificity; restricted ability to identify different disaccharide building blocks of LMWHs (12 to 17)	US7329353	Thomas Dillon et al.	Amgen Inc	06–03-2026
CE-MS	Capability to perform intact chain mapping analysis; offer rich information about chain distribution and composition of LMWHs; can identify trace amounts of unusual structures or impurities rapid, economic, ultra-sensitive	Insufficient separation resolution, detection sensitivity or specificity; restricted ability to identify different disaccharide building blocks of LMWHs (12 to 17)	WO2015121366	Johann FAR et al.	Analis S.A	N/A
LIT-MS/MS/MS (or MS3)	Better in-trap fragmentation, high ion trap sensitivity, and high selectivity	Difficult high selection resolution	US9852895	Daniel James Kenny	Micromass UK Ltd	20–01-2030
MSI-CE-MS	High-throughput screening of ions within a single capillary; enhanced data processing with quality assurance	Low mechanical robustness, poor reproducibility	US9490110	Phillip Britz-McKibbin	McMaster University	14–05-2034
CE-ESI–MS	Simple, inexpensive, good in resolution, easy capillary replacement, highly compatible with MS	Insufficient capacity, low selectivity, not suitable for high MW analysis	EP2250490B1	Elizabeth Jane Maxwell, Xuefei Zhong, Hong Zhang, David Da Yong Chen	University of British Columbia	06–03-2029
ESI FT-ICR MS	Ultra high spectral resolution and mass accuracy; facilitate application of top-down MS strategies	High cost, requires large space, long signal acquisition time, not suitable for clinical laboratory applications	US8530834	Alan G. Marshall et al.	Florida State University Research Foundation, Inc	05–02-2030
DE-MALDI-TOF MS	High spectral resolution and mass accuracy, obviating the need for MS tuning during single sample signal acquisition	Presents different results under similar sample preparation using similar conditions	US9536726B2	James VanGordon, Bradford Clay	Biomerieux, Inc	27–08-2035
SEC-UPLC/Q-TOF HRMS	Simple; robust; capacity to reflect process-based structural changes; potential to reveal contribution of each individual components to the overall bioactivity of LMWHs; capacity to identify oligosaccharide with sizes upto dp30.* [37]	Cost, insufficient storage capacity	US202002000719A1	Navya Sama et al.	Alembic Pharmaceuticals Ltd	Pending (05–09-2038, if granted)
IP-HILIC LC–ESI–MS	Simple, effective for analysis of –vely charged saccharides, suitable for coupling with MS, low IP concentration increases resolution and strength of MS, highly sensitive, low contamination rate, useful for quality control of LMWHs	Sensitivity fluctuates during runs, low separation efficiency for polar compounds	WO2016051170A1	Lingzhi Gong	Isis Innovation Limited	N/A
HILIC-UPLC-CAD/MS	Capacity to separate highly sulfated and very polar heparin oligosaccharides; universal adaptability to any MS instrument; capacity to identify oligosaccharides with sizes up to dp28; robust, reproducible and easy-to-use, offers impressive increase in sensitivity when compared with RP-LC–MS	High reliance on aprotic solvent acetonitrile, less flexible	US9441053B2	ChungYao Wang, Imin Huang, Chia Yen Wu, YungTe Chiang Helen Chao	ScinoPharm Taiwan Ltd	20–02-2035
SEC-MS/RPIP-HPLC–MS	Fast, capacity to disclose size distributions and sequences; better in oligosaccharide resolution, provide interesting information about oligosaccharide structure, chain length, and chemical modifications	Expensive, low tolerance to contaminants	WO2013139478A1	Giangiacomo Torri, Antonella Bisio	Instituto di Ricerche Chimiche e Biochimiche “G. Ronzoni”	N/A
SAX-HPLC or RP-HPLC ESI–MS	Capacity to profile disaccharide building blocks; offers better resolution; signal enhancement, effective in costs	Signal suppression, low tolerance to contaminants	US9139876	Zachary Shriver, Naveen Bhatnagar, Nur Sibel Gunay, Jennifer Ozug, Elaine Y. Sun	Momenta Pharmaceuticals, Inc	23–08-2031
Orbitrap MS/MS	High resolution/ionization efficiency; high mass accuracy; miniature design, offers identification and characterization of all known disaccharide building blocks	Lower sensitivity, false negatives, low-abundance peaks	US7728290B2	Alexander Makarov	Thermo Finnigan LLC	22–10-2024
HPLC FTICR + LTQ-FTMS	Posttranslational modification analysis of complex molecules	Low-abundance peaks, limited mass accuracy and resolution	WO2006026569A2	Shiaw-Lin Wu et al.	Northeastern University	N/A
LC–MS/MS	Capable of sequencing short oligosaccharide mixture with saccharides as large as dodecasaccharides; highly sensitive, specific and rapid in detection	Higher operational cost; more limited sample throughput; less favorable concentration sensitivity	US8945933	Anthony Le, Tina Cowan	The Board of Trustees of the Leland Stanford Junior University	20–10-2032
FT-ICR MS	Incomparable high mass resolution, mass measurement accuracy; small, simple	Relatively slow acquisition rate	US5886346	Alexander Alekseevich Makarov	Thermo Finnigan LLC	29–03-2016
IMS-MS	Simple, selective, sensitive, inexpensive,	Low-resolution, limited dynamic response range, low tolerance to contamination	US9607820B2	Robert Harold Bateman et al	Micromass UK Ltd	21–05-2030
ECD-CID MS/MS	Simple, low costs, selectively cleaves c-z site on the amino acid backbone; suitable for analyzing post-translational modifications	Low in fragmentation efficiency; requires high signal-to-noise ratio for precursor ions	US8080786B2	Takashi Baba, Hiroyuki Satake, Izumi Waki	Hitachi High Tech Corp	10–07-2026
CID MS/MS	Creates MS spectra with high accuracy and efficiency; produce useful fragmentation	Reduces multiplicity of bond cleavages; Ineffective for large molecule samples; Easily detached functional groups cause loss of fragment-derived information on location and provide limited structural information	US8269166	Daisuke Okumura	Shimadzu Corp	05–02-2029
IRMPD MS/MS	Fast, free from blind spots, no degradation of high vacuum; allows product ion formation on axis	Fails to conduct complete structural analysis; easily loses side chains involves in post translational modifications of biomolecules; allows secondary fragmentation	US6717137B2	Steven A. Hofstadler, Jared J. Drader	Ibis Biosciences Inc	11–06-2022
ETD MS/MS	Cleaves main amino acid residues sequence chain while preserving the post-translationally modified site; allows more complete sequence information; works well with partially ionized precursor ion; can be practiced on a variety of other analytical tools	Not feasible for large-scale analysis, high in cost; technically complex	US8692187	Donald F. Hunt et al.	University of Virginia Patent Foundation	19–04-2025
ECD MS/MS	Efficient, suitable for analyzing complex mixtures and large biomolecules, produce more structurally important cleavages than CID and IRMPD; produce simple/predictable fragmentation pattern	Can be employed in Penning cell ion cyclotron resonance mass spectrometers	US6958472	Roman Zubarev	Syddansk Universitet	22–03-2022
EID MS/MS	Suitable for dissociating high-mass, even-electron ions produced by thermo spray and other soft ionization tools; yields extensive and reproducible fragmentation characteristics; can analyze qualitatively and quantitatively greater quantity of samples; can be easily implemented on a variety of analytical tools	Need electron sourcing and singly charged precursor ions	US4731533	Marvin L. Vestal	Applied Biosystems LLC	15–10-2006
IT-MS	Selectively trap multivalent ions having variety of charge to mass ratio	Can cause outgassing, Need for large radio frequency (RF) potentials can aggravate RF breakdown	US6847037	Yoshikatsu Umemura	Shimadzu Corp	19–05-2023
2D-Tandem MS	Much faster, offers large increase in peak capacity, can characterize intact chains or originally stable non- fragmenting ions	High cost	US6770871	Houle Wang, Kerry D. Nugnet	Bruker Corp Bruker Scientific LLC	01–06-2022
Chip cube Nano-LC–ESI–MS/MS ion-trap system	Easy, reliable, efficient, highly sensitive, can investigate a wide range of biomolecules	Reproducibility, sensitivity dependency on shape of the capillary tip and cone distance	US20160305919A1	Gregory Staples	Agilent Technologies Inc	Abandoned

RP-LC–MS reverse phase-liquid chromatography-mass spectrometry, CE capillary electrophoresis, LIT linear ion trap, FT-MS Fourier transform mass spectrometry, FT­ICR Fourier transform ion cyclotron resonance, MSI multi-segment injection, DE delayed extraction, MALDI matrix-assisted laser desorption ionization, ESI electrospray ionization, IT ion trap, CID collision-induced dissociation, IRMPD infrared multiphoton dissociation, HPLC high-performance liquid chromatography, HILIC hydrophilic interaction chromatography, Q-TOF quadruple time-of-flight, RPIP reversed phase ion pairing chromatography, SEC size exclusion chromatography, UPSEC ultra-performance size exclusion chromatography, SAX strong anion exchange chromatography, CTA-SAX cetyltrimethylammonium-coated SAX, UPLC ultra-performance liquid chromatography, HRMS high-resolution-MS, UVPD ultra-violet photodissociation, EDD electron detachment dissociation, EID electron-induced dissociation, NETD negative electron transfer dissociation

Sources: (i) For patents: www.patents.google.com, www.epo.org, www.wipo.org, www.uspto.com, www.drugfuture.com; (ii) For advantages and disadvantages of techniques: Lauren E. Pepi, Patience Sanderson, Morgan Stickney, I. Jonathan Amster, Developments in Mass Spectrometry for Glycosaminoglycan Analysis: A Review, Molecular & Cellular Proteomics, Volume 20, 2021, 100,025, ISSN 1535–9476, https://doi.org/10.1074/mcp.R120.002267; (iii) Zhangjie Wang, Lianli Chi. Recent advances in mass spectrometry analysis of low molecular weight heparins [J]. Chin. Chem. Lett., 2018, 29(1): 11–18