Chromatographic approaches for the characterization and quality control of therapeutic oligonucleotide impurities

Zahar, Noha M. El; Magdy, Nancy; El-Kosasy, Amira M.; Bartlett, Michael G.

doi:10.1002/bmc.4088

Cited by 46 publications

(47 citation statements)

References 157 publications

(392 reference statements)

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“…Chromatography technology plays a major role in downstream protein processing as the most fundamental step in biopharmaceutical purification. Animal venom Hyal is structurally comparable with that from the sperm acrosome membrane (El Zahar, Magdy, El‐Kosasy, & Bartlett, , Sutti, Tamascia, & Hyslop, ). Hyal produced is limited if produced by the animal, but unlimited if generated by microorganisms.…”

Section: Chromatographic Methods For Purification Of Hyalmentioning

confidence: 99%

Purification of hyaluronidase as an anticancer agent inhibiting CD44

Shakouri

Adljouy

Abdolalizadeh

2019

Biomedical Chromatography

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Hyaluronidase (Hyal) can be employed to accomplish a diversity of complications related to hyaluronic acid (HA). Hyal contains some classes of catalysts that cleave HA. This enzyme is detected in several human tissues as well as in animal venoms, pathogenic organisms and cancers. Destructive cancer cells regularly increase the CD44 receptor existing in a cell membrane. This receptor acts as an exact receptor for HA, and HA is recognized to motivate the migration, spread, attack and metastasis of cancer cells. Nearly all of the methods used to purify Hyal are highly costly and not proper for industrial applications. This survey aims to review different methods of Hyal purification, which acts as an anticancer agent by degrading HA in tissues and thus inhibiting the CD44–HA interaction. Hyal can be successfully employed in the management of cancer, which is associated with HA–CD44. This review has described different methods for Hyal purification to prepare an origin to develop a novel purification technique for this highly appreciated protein. Using multiple columns is not applicable for the purification of Hyal and thus cannot be used at the industrial level. It is better to use affinity chromatography of anti‐Hyal for Hyal with one‐step purification.

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Section: Chromatographic Methods For Purification Of Hyalmentioning

confidence: 99%

Purification of hyaluronidase as an anticancer agent inhibiting CD44

Shakouri

Adljouy

Abdolalizadeh

2019

Biomedical Chromatography

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“…The stationary phase particles used in this study (2.5 μm particles) are in a size between modern HPLC and UHPLC and could be used for small-scale purification (mg scale) but are not recommended for large-scale preparative separations due to the high pressure drop and to difficulties packing homogenous beds in large diameter columns [10]. The loss of resolution expected when increasing particle size can be compensated by increasing the length of the column to maintain a constant column length over particle diameter ratio.…”

Section: Mass-overloaded Injections Of Ps-modified T16mentioning

confidence: 99%

“…With regards to effects of changing particle size on productivity, each separation problem needs to be individually optimized depending on the constraints (pressure, flow, and column dimensions) of the system [22,24,25]. Maintaining a constant particle size squared over column length ratio is a good rule of thumb applicable for binary and isocratic separation problems [10], but its applicability for gradient separation needs to be investigated. Fig.…”

Section: Mass-overloaded Injections Of Ps-modified T16mentioning

confidence: 99%

“…From the perspective of both analysis and purification, selectivity for the (n − 1) and (n + 1) impurities poses a great challenge, becoming increasingly difficult with increasing ON length [9]. ONs are expensive to synthesize compared with small molecules, so the chromatographic methods used for preparative isolation must be optimized for both productivity and yield which due to the non-linearity of "overloaded" elution profiles poses a greater challenge than optimizing analytical chromatography conditions [10].…”

Section: Introductionmentioning

confidence: 99%

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Analytical and preparative separation of phosphorothioated oligonucleotides: columns and ion-pair reagents

et al. 2019

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Oligonucleotide drugs represent an emerging area in the pharmaceutical industry. Solid-phase synthesis generates many structurally closely related impurities, making efficient separation systems for purification and analysis a key challenge during pharmaceutical drug development. To increase the fundamental understanding of the important preparative separation step, mass-overloaded injections of a fully phosphorothioated 16mer, i.e., deoxythymidine oligonucleotide, were performed on a C18 and a phenyl column. The narrowest elution profiles were obtained using the phenyl column, and the 16mer could be collected with high purity and yield on both columns. The most likely contribution to the successful purification was the quantifiable displacement of the early-eluting shortmers on both columns. In addition, the phenyl column displayed better separation of later-eluting impurities, such as the 17mer impurity. The massoverloaded injections resulted in classical Langmuirian elution profiles on all columns, provided the concentration of the ion-pairing reagent in the eluent was sufficiently high. Two additional column chemistries, C4 and C8, were also investigated in terms of their selectivity and elution profile characteristics for the separation of 5-20mers fully phosphorothioated deoxythymidine oligonucleotides. When using triethylamine as ion-pairing reagent to separate phosphorothioated oligonucleotides, we observed peak broadening caused by the partial separation of diastereomers, predominantly seen on the C4 and C18 columns. When using the ion-pair reagent tributylamine, to suppress diastereomer separation, the greatest selectivity was found using the phenyl column followed by C18. The present results will be useful when designing and optimizing efficient preparative separations of synthetic oligonucleotides.

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“…While the nature of the quality control testing varies to some extent with the type of drug and mechanism of delivery, all drug specifications must include a measurement of concentration for the active pharmaceutical ingredient (API), which is referred to as label claim, weight‐based assay, or simply assay (ICH Q6A). Several analytical methods have been used to determine assay for oligonucleotide drugs, including capillary electrophoresis, strong anion‐exchange chromatography, and ion‐pair reversed‐phase high‐performance liquid chromatography coupled to UV detection plus electrospray ionization mass spectrometry (IP‐HPLC‐UV/MS) . Regardless of the approach, it is important that the assay methodology be specific for the API and provide a high level of accuracy and precision.…”

Section: Introductionmentioning

confidence: 99%

Assay determination by mass spectrometry for oligonucleotide therapeutics

Madsen

Roussis

Schniepp

et al. 2019

Rapid Comm Mass Spectrometry

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Phosphorothioate oligonucleotide drugs typically contain product-related impurities that are difficult to resolve chromatographically from the parent oligonucleotide due to the size of these compounds and the large number of stereoisomers that comprise the parent. The presence of co-eluting impurities hinders the process of determining assay based on chromatographic separation alone. A mass spectrometry-based purity assessment of the main chromatography peak can be used to quantify co-eluting impurities and enable the accurate determination of assay, but a more direct measure of assay was desired due to the complexity of measuring all co-eluting impurities by mass spectrometry. Therefore, we developed an assay method that utilizes the specificity of mass spectrometry to measure the amount of active pharmaceutical ingredient in a sample, which eliminates the need for chromatographic separation of impurities from the product. This procedure uses a single quadrupole mass spectrometer and incorporates an internal standard that is co-sprayed with the analyte to compensate for the drift commonly associated with mass spectrometry-based quantitation. Using the mass spectrometry response ratio for sample to internal standard enables the method to achieve excellent linearity (R 2 = 0.998), repeatability (relative standard deviation = 0.5%), intermediate precision (0.6%), and accuracy, with measured assay values consistently within 2.0% of expected. The results indicate the method possesses the accuracy and precision required for measuring assay in clinical and commercial stage pharmaceutical products. Since the method is based on the specificity of the mass spectrometer, and does not rely on chromatographic separation of impurities, the procedure should be applicable to a wide variety of oligonucleotide therapeutics regardless of sequence or chemical modifications.

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Chromatographic approaches for the characterization and quality control of therapeutic oligonucleotide impurities

Cited by 46 publications

References 157 publications

Purification of hyaluronidase as an anticancer agent inhibiting CD44

Purification of hyaluronidase as an anticancer agent inhibiting CD44

Analytical and preparative separation of phosphorothioated oligonucleotides: columns and ion-pair reagents

Assay determination by mass spectrometry for oligonucleotide therapeutics

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