Process-related Impurities Research Articles

Preparation and characterization of polymyxin B- and histidine-coupled magnetic nanoparticles for purification of biologics from acquired endotoxin contamination

BackgroundEndotoxin is a major process-related impurity that can act as a strong immunostimulant leading to fever and hypotensive shock. Thus, the US FDA and international quality standards strictly direct the biologics manufacturers to control the endotoxin contamination during the purification process. In this work, a developed method for biologics purification from acquired endotoxin contamination is introduced. This is accomplished by the preparation of dextran-coated magnetic nanoparticles using a facile rapid co-precipitation method.ResultsThe resulting magnetic nanoparticles (MNPs) are characterized by dynamic light scattering, transmission electron microscope, Fourier transform infrared spectroscopy, X-ray diffraction, and vibrating sample magnetometry. The dextran-coated magnetic nanoparticles are further coupled to either polymyxin B or histidine to provide a positively charged ligand which enhances the affinity to the negatively charged endotoxin. Both ligands-coupled MNPs are tested for purification efficiency using the chromogenic kinetic assay. The method conditions are optimized using a two-level factorial design to achieve best purification conditions of the contaminated biologics and indicated endotoxin removal percentage 85.12% and maximum adsorption capacity of 38.5 mg/g, for histidine-coupled MNPs.ConclusionsThis developed method is introduced to serve biologics manufacturers to improve their manufacturing processes through providing a simple purifying tool for biologics from acquired endotoxin contamination.Graphical

Holistic analytical characterization and risk assessment of residual host cell protein impurities in an active pharmaceutical ingredient synthesized by biocatalysts.

Host cell proteins (HCPs) are a significant class of process-related impurities commonly associated with the manufacturing of biopharmaceuticals. However, due to the increased use of crude enzymes as biocatalysts for modern organic synthesis, HCPs can also be introduced as a new class of impurities in chemical drugs. In both cases, residual HCPs need to be adequately controlled to ensure product purity, quality, and patient safety. Although a lot of attentions have been focused on defining a universally acceptable limit for such impurities, the risks associated with residual HCPs on product quality, safety, and efficacy often need to be determined on a case-by-case basis taking into consideration the residual HCP profile in the product, the dose, dosage form, administration route, and so forth. Here we describe the unique challenges for residual HCP control presented by the biocatalytic synthesis of an investigational stimulator of interferon genes proteinagonist, MK-1454, which is a cyclic dinucleotide synthesized using Escherichia coli cell lysate overexpressing cyclic GMP-AMP synthaseas a biocatalyst. In this study, a holistic characterization of residual protein impurities using a variety of analytical tools including nanoscale liquid chromatography coupled to tandem mass spectrometry, together with in silico immunogenicity prediction of identified proteins, facilitated risk assessment and guided process development to achieve adequate removal of residual protein impurities in MK-1454 active pharmaceutical ingredient.

Computational, Spectral, and Bioavailability Studies of Venlafaxine Cyclic Impurity

Detection and qualification of process-related impurities assume importance in pharmacological drug development programmes. From time to time, preliminary investigations of such compounds might direct us to better drug options. Impurity profiling also tells us about the toxicity impacts due to the possible carry-over of such molecules into drug formulations. This paper depicts the structural and spectral investigation of 5-(4-Methoxyphenyl)-3-methyl-1-oxa-3-azaspiro[5,5]undecane (VEN-CI), an impurity associated with the anti-depressant drug Venlafaxine (VEN). The structural optimization was performed using Density Functional Theory and the computed data was then compared with experimentally derived data accessible in the literature. The chemical reactivity and stability of the title molecule were evaluated by assessing the HOMO-LUMO energies. The bioavailability and toxicity impacts were also investigated computationally to evaluate the medicinal chemistry friendliness.

Synthesis of process related Dapsone impurities : Selective diazotization of aromatic di-amine

Synthesis of process related Dapsone impurities : Selective diazotization of aromatic di-amine

Identification of Process-Related Impurities and Corresponding Control Strategy in Biocatalytic Production of 2-O-α-d-Glucopyranosyl-l-ascorbic Acid Using Sucrose Phosphorylase.

2-O-α-d-Glucopyranosyl-l-ascorbic acid (AA-2G) is an ideal substitute for l-ascorbic acid because of its remarkable stability and improved biological activity, which can be easily applied in cosmetic, food, and medicine fields. However, impurity identification and control are significant procedures during the manufacturing of AA-2G. This study assessed a manufacturing routine of AA-2G synthesized by sucrose phosphorylase (SPase). First, three unknown process-related impurities were observed, which were further identified as 3-O-α-d-glucopyranosyl- l-ascorbic acid (impurity I), 2-O-α-d-glucopyranosyl-l-dehydroascorbic acid (impurity II), and 13-O-α-d-glucopyranosyl-2-O-α-d-glucopyranosyl-l-ascorbic acid (impurity III), respectively. Second, a comprehensive formation pathway of impurities was elucidated, and specific strategies corresponding to controlling each impurity were also proposed. Specifically, the content of impurity I can be reduced by 50% by fine tuning reaction conditions. The impurity II-free purification process was also achieved by applying a low concentration of alkali. Finally, a semi-rational design was introduced, and a single mutant L343F was obtained by site-directed mutagenesis, which reduced impurities I and III by 63.9 and 100%, respectively, without affecting the transglycosylation activity. It is expected that the reported impurity identification and control strategies during the AA-2G production will facilitate its industrial production.

Development and validation of an RP-HPLC method for analysis of 2-(5-(4-chlorophenyl)-3-(ethoxycarbonyl)-2-methyl-1H-pyrrol-1-yl)propanoic acid and its impurities under different pH

A simple, fast and selective stability indicating RP-HPLC method was applied for following the degradation and appearance of impurities of previously synthesized 2-(5-(4-chlorophenyl)-3-(ethoxycarbonyl)-2-methyl-1H-pyrrol-1-yl)propanoic acid. The chromatographic separation was achieved on a C18 column (150×4 mm i.d., 5 μm) using a mobile phase consisting of Acetonitrile: Phosphate buffer, pH=3, (50:50% v/v) with isocratic elution at a flow rate of 1.0 mL min−1 and temperature of the column of 30 °C applying a UV/VIS detector at 225 nm. The method was validated according to the ICH guidelines. A process related impurity was determined at pH 9.0 corresponding to ethyl 2-acetyl-4-(4-chlorophenyl)-4-oxobutanoate. No change in the structure was detected at pH = 7.4.

Purification of therapeutic & prophylactic mRNA by affinity chromatography

In vitro transcribed mRNA is an emerging therapeutic and prophylactic modality with the potential to transform medicine. The drug platform features exceptionally rapid development and versatility of manufacturing processes. Despite the prompt advancement of mRNA from trials to market, purification challenges remain. The cell-free synthesis of mRNA is responsible for the generation of product and process-related impurities, creating the potential for immunogenic effects and decreased translatability into the clinic. Affinity chromatography presents itself as an effective primary capture step for the isolation of functional transcripts from product and some process related impurities. Developing platform processes for the affinity purification of mRNA is hindered by the varying strand lengths of non-amplifying, self-amplifying, and trans-amplifying constructs, with disparities in capacity being observed. Ligand chemistries may contribute to non-specific binding events which remain challenging to characterise. Improved elution and wash conditions may be pursued through novel ligand chemistries, enhanced density and spacing. Regardless of the size or application of the product, the impurities generated by in vitro transcription represent a significant obstacle to the safe administration and long-term storage of mRNA. Affinity chromatography is a valuable tool in overcoming these challenges, with current commercially available products relying heavily on oligo deoxythymidine ligand chemistries. Whilst affinity chromatography is highly valuable in the purification of mRNA, the inability to separate key secondary structures such as double-stranded RNA means it remains to be seen if this technology will adopt the same position as protein A does in mAb manufacture.

Determination of Purge Factors for Use in Oligonucleotide Control Strategies

The understanding, control, and removal of nonoligonucleotide process-related impurities (PRI) are of key importance for the manufacturing of therapeutic oligonucleotides as their presence in the final product is both a quality and safety concern. Regulatory agencies require manufacturers to demonstrate that PRI are under control or adequately purged during the manufacturing process. Purging depends on the physicochemical properties of the impurities and the unit operations of the manufacturing process but should be independent of oligonucleotide size or type. The purging power of unit operations relevant to oligonucleotide manufacturing (synthesis, cleavage and deprotection, chromatography, ultrafiltration/diafiltration) was measured using representative solvents and other small molecules typical for oligonucleotide synthesis. The results show that each unit operation has significant purging capability (synthesis >1000; cleavage and deprotection >100 (reactivity, when applicable); chromatography >1000; ultrafiltration/diafiltration >10) and that large overall purge factors can be obtained (≥1 × 107). Experimentally determined purge values are aligned with theoretical purge values; thus, the use of purge arguments in oligonucleotide control strategies is a sound scientific approach. Guidance on reasonable purge values for oligonucleotide unit operations is presented. Additionally, the data demonstrate that solvents and reagents typically used in oligonucleotide synthesis are robustly cleared by the process and should not require testing in the final product.

Toward optimal clearance: A universal affinity-based mass spectrometry approach for comprehensive ELISA reagent coverage evaluation and HCP hitchhiker analysis.

In the control strategy for process related impurities in biopharmaceuticals, the enzyme linked immunosorbent assay (ELISA) is the method of choice for the quantification of host cell proteins (HCPs). Besides two dimensional-western blots (2D-WB), the coverage of ELISA antibodies is increasingly evaluated by affinity purification-based liquid chromatography-tandem mass spectrometry (AP-MS) methods. However, all these methods face the problem of unspecific binding issues between antibodies and the matrix, involving the application of arbitrarily defined thresholds during data evaluation. To solve this, a new approach (optimized AP-MS) was developed in this study, for which a cleavable linker was conjugated to the ELISA antibodies enabling the subsequent isolation of specifically interacting HCPs. By comparing both approaches in terms of method variability and the number of false positive or negative hits, we could demonstrate that the optimized AP-MS method is very reproducible and superior in the identification of antibody detection gaps, while previously described strategies suffered from over- or underestimating the coverage. As only antibody associated HCPs were identified, we demonstrated that the method is beneficial for hitchhiker analysis. Overall, the method described herein has proven as a powerful tool for reliable coverage determination of ELISA antibodies, without the need to arbitrarily exclude HCPs during the coverage evaluation.

Isolation, synthesis, identification of new process-related impurities in evocalcet

Evocalcet is a calcimimetic for the treatment of secondary hyperparathyroidism. In the preparation of evocalcet, eleven process-related impurities were detected in the reaction solution of the last step at levels of 0.05%− 2.50% by a new HPLC method. Ten impurities were separated from the enriched mother liquor and synthesized directly. These impurities were characterized and identified by HRMS and NMR spectra and then coinjected with commercial products to confirm their retention times in HPLC. The possible formation pathways and synthetic methods of ten process-related impurities ware discussed in detail.

Simultaneous Determination of Prasugrel and its Process Related Impurities and Degradation Products in Bulk Drug Substances by RPLC-UV

Aims: This study aims to determine the quantitative prasugrel (PG) and its all possible process-related impurities. Background: To the best of our knowledge, very few analytical methods are available in the literature for monitoring process related impurities and degradation products of PG in bulk drug substance/ active pharmaceutical ingredient (API). Objective: The objective of this study is the separation of Prasugrel and its all possible process-related impurities viz., desacetyl prasugrel-tautomeric forms, intermediates including desacetyl impurity existing in its keto-enol form and positional tautomer impurities with degradation products. Method: A simple and robust HPLC-UV method having Zorbax XDB C18 column (15 cm x 4.6 mm) 3.5μm particle size column was used. Result: Prasugrel and its process related impurities were separated as well as analyzed in pharmaceutical samples' biological matrices. Conclusion: RP-LC method was developed for quantitative determination of PG and related substantial impurities were found to be highly specific, sensitive and precise. The major oxidative degradant was identified as PG desacetyl IMPs (keto-enol and positional tautomer) and hydroxyl IMP.

Identification, Characterization, and the Determination of Process-related Impurities in Minodronic Acid Bulk Drug

Background: Minodronic acid (MA) is a third-generation nitrogen-containing heterocyclic bisphosphonate used to treat osteoporosis. In the process of starting materials research and preparation, the key intermediate impurities and degradation impurities have a great impact on the quality control of the drug. Objective: A sensitive, reliable, high-performance liquid chromatography (HPLC) method was developed and validated for the quantitative determination of MA and its related impurities (a total of 6 compounds, including 2 new impurities). Methods: The separation was achieved on an InertSustain ODS-4 C18 (250 mm × 4.6 mm, 5 μm) column using the mixture of 0.01 mol/L sodium pyrophosphate and 1 mmol tetrabutylammonium phosphate (the mobile phase pH was adjusted to 7.80 by phosphonic acid). Results: The quantitative analytical method was fully validated with respect to linearity (r > 0.999), sensitivity (limit of detection < 35 ng/mL), precision, accuracy (the recovery was between 98.7% and 104.2%), and robustness. Six process-related impurities in Minodronic Acid (MA) bulk drug were determined by high-performance liquid chromatography (HPLC). Furthermore, except for two starting materials, other four impurities were identified and characterized as 2-(imidazo[1,2-a] pyridin-3-yl) ethyl acetate (Imp-C), 2-(imidazo [1,2-a] pyridin- 3- yl)acetic acid (Imp-D), 3-(2-hydroxy-2,2- diphosphonoethyl)-4H-imidazo [1,2-a] pyridine -4- oxide (Imp-E) and 2,5- Dihydroxy- 3,6-bis(imidazo[1,2-a] pyridine-3-yl methyl) -2,5-dioxo- 1,4,2,5- dioxoDiphosphonium-3,6-diyl) bisphosphonic acid (Imp-F) using liquid chromatograph- mass spectrometer (LC-MS), MS/MS, Infrared Radiation and Nuclear Magnetic Resonance spectroscopy (1H-NMR and 13C-NMR). To the best of our knowledge, two of them (Imp- E and Imp-F) are new compounds and have not been reported previously. Conclusion: The HPLC method was developed and optimized, which could be applied for quantitative detection of the impurities, and further quality evaluation of MA.

Eco-Friendly and Sensitive HPLC and TLC Methods Validated for the Determination of Betahistine in the Presence of Its Process-Related Impurity

Reducing the amounts consumed of organic solvents while keeping good chromatographic performance has been a significant step towards the greening of analytical methodologies. When sodium dodecyl sulfate (SDS) and Brij-35 surfactants are combined in a mobile phase, they can be used as a green alternative to organic modifiers. Surfactants have numerous advantages, including low cost and toxicity, safe environmental disposal, and unique selectivity, in addition to high solubilization capabilities. In this research, two highly selective chromatographic methods were adopted for the determination of betahistine (BHS) in the presence of its pharmacopeial impurity 2-(2-hydroxyethyl)pyridine (HEP). A solvent-free HPLC method was validated, in which the mixture was separated using a C18 column (3.5 µm, 75.0 × 4.6 mm) and a mobile phase composed of 0.01 M Brij-35, 0.12 M SDS, and 0.02 M disodium hydrogen phosphate adjusted to a pH of 5.5 using phosphoric acid. The flow rate was 1.5 mL min−1 and the resolved peaks were detected at 260 nm. Another HPTLC-densitometric method was validated using HPTLC aluminum plates coated with silica gel 60 F254 as the stationary phase and a developing system consisting of methylene chloride/methanol/ethyl acetate/ammonia (at a ratio of 5:2:2:0.2 by volume); the separated bands were scanned at 260 nm.

Use of In Vitro Human Skin Models to Assess Potential Immune Activation In Response to Biotherapeutic Attributes and Process-related Impurities

Subcutaneous (SubQ) injection is a common administration route for biotherapeutics. However, limited tools are available for understanding the dynamic relationships between drug products and resident cells following injection. Advances in tissue engineering have enabled the production of in vitro skin models that recapitulate the morphological structure and functional activity of human skin. Here we explore the use of a commercially available skin model to investigate potential immune activation in response to subcutaneously injected biotherapeutics. Exposure to high levels of a mixture of process-related impurities (that are known potent immune system activators) induced a robust immune response from the skin model, as indicated by enhanced metabolic activity and increased secretion of 19 cytokines and chemokines. The skin model also responded to aggregated antibodies (generated by extreme mechanical stirring and pH-jump stress, which resulted in orders of magnitude higher particle numbers than that found in products), as shown by the secretion of several signature cytokines (GM-CSF, RANTES, and MCP-1). However, the magnitude of the responses to the aggregates were significantly lower than the response to the impurities. These results highlight the promising utility of in vitro skin models for investigating the potential immune response to process-related impurities and biotherapeutic attributes in a subcutaneous environment. The use of skin models for assessing drug safety may provide new insights to help guide drug product and process development, and potentially mitigate the risk of injection site reactions and systemic immunogenic responses that may compromise the safety and efficacy of subcutaneously administered drugs.

Synthesis, characterization and control of eight process-related and two genotoxic fingolimod impurities by a validated RP-UPLC method.

An HPLC method has been described in the European Pharmacopoeia and United States Pharmacopeia for the determination of nine organic impurities (imp A-I) in fingolimod hydrochloride, a synthetic sphingosine-1-phosphate receptor modulator. The manufacturing process of fingolimod hydrochloride consists of multistep chemical synthesis wherein controls of precursors, intermediates and process steps should be performed to assure the final quality of the drug substance. We synthesized and isolated eight process-related impurities (FINI imp A-H) of fingolimod, which were different from the pharmacopoeial impurities. One unknown process-related impurity was found as a key intermediate (FINI) and was identified by LC-MS. Characterization of all of the impurities were done using spectroscopic techniques (1 H and 13 C NMR, FTIR, MS), and the mechanistic pathways to the formation of these impurities were also discussed. Two of these impurities were evaluated as potential genotoxic impurities owing to their alerting structures and alkylating properties (alkyl sulfonates and alkyl halides, class 3, ICH M7). We also developed and validated an RP-UPLC method in line with ICH Q2 guidelines for control these impurities (FINI imp A-H) and to assure the pharmacopoeial quality drug substance.

Analysis of fouling and breakthrough of process related impurities during depth filtration using confocal microscopy.

Titer improvement has driven process intensification in mAb manufacture. However, this has come with the drawback of high cell densities and associated process related impurities such as cell debris, host cell protein (HCP), and DNA. This affects the capacity of depth filters and can lead to carryover of impurities to protein A chromatography leading to early resin fouling. New depth filter materials provide the opportunity to remove more process related impurities at this early stage in the process. Hence, there is a need to understand the mechanism of impurity removal within these filters. In this work, the secondary depth filter Millistak+ X0HC (cellulose and diatomaceous earth) is compared with the X0SP (synthetic), by examining the breakthrough of DNA and HCP. Additionally, a novel method was developed to image the location of key impurities within the depth filter structure under a confocal microscope. Flux, tested at 75, 100, and 250 LMH was found to affect the maximal throughput based on the max pressure of 30 psi, but no significant changes were seen in the HCP and DNA breakthrough. However, a drop in cell culture viability, from 87% to 37%, lead to the DNA breakthrough at 10% decreasing from 81 to 55 L/m2 for X0HC and from 105 to 47 L/m2 for X0SP. The HCP breakthrough was not affected by cell culture viability or filter type. The X0SP filter has a 30%–50% higher max throughput depending on viability, which can be explained by the confocal imaging where the debris and DNA are distributed differently in the layers of the filter pods, with more of the second tighter layer being utilized in the X0SP.

Ultraviolet photodissociation facilitates mass spectrometry-based structure elucidation with pyrrolidine and piperidine containing compounds

In pharmaceutical development, structural elucidation of small molecules from process related impurities and degradation products is an essential component. As one of the most important methods in the toolbox, high resolution mass spectrometry (HRMS) and specifically tandem mass spectrometry (MS/MS) often provide fast and informative structural insights. However, many small molecule drugs containing certain biological relevant pharmacophores result in limited numbers of fragments when using traditional collision based fragmentation techniques, such as higher energy collisional dissociation (HCD), due to its inherent preference of cleaving the weakest bond first. As an alternative, ultraviolet photodissociation (UVPD), which irradiates the precursor ion with high energy photons, can lead to more extensive fragmentation from the readily UV absorbing small molecules. Here, we showcase the advantage of UVPD over HCD on pyrrolidine and piperidine containing molecules derivatized from a model compound, telmisartan. While HCD only yielded a single, highly abundant ion resulting from the pyrrolidine and pipieridine ring cleavage, UVPD generated rich and structurally informative fragment ions. UVPD is an attractive and powerful alternative for traditional fragmentation techniques for small molecule structural elucidation.

Simultaneous determination of ivermectin, clorsulon and their related substances in an injectable finished product by a stability-indicating RP-HPLC method

A reversed-phase high performance liquid chromatography (RP-HPLC) method has been developed for the determination of ivermectin and clorsulon, including the identification and estimation of their related impurities in an ivermectin and clorsulon injectable finished product. Chromatographic separation was achieved using a gradient elution on a Supelco Ascentis® Express C18 column (150 mm × 4.6 mm i.d., 5 µm particle size) maintained at 55 °C. Mobile phase-A is composed of water and mobile phase-B is composed of acetonitrile/methanol (65/35, v/v). Analytes were monitored by UV detection at 245 nm. The stability-indicating capability of this method has been demonstrated by the adequate separation of all the process related impurities and degradation products of ivermectin and clorsulon from the stress degraded samples of the injectable product. This method was also successfully validated as per the current ICH guidelines with respect to specificity, linearity (R2> 0.999), limit of detection (0.3 μg/mL), limit of quantitation (1.0 μg/mL), accuracy, precision, and robustness for both APIs. This proposed method can significantly benefit the end-users in QC laboratories with laboratory efficiency and throughput during routine analysis and stability monitoring of the injectable product.

An Efficient, Novel Synthetic Route to (R)-Salmeterol Xinafoate and Facile Synthetic Protocols for Preparation of its Process Related Potential Impurities

In present work, an effective, innovative enantioselective synthetic strategy for the synthesis of salmeterol xinafoate and its probable process-related impurities was reported. Seven pharmacopeia specified impurities were synthesized and confirmed by FT-IR, mass, 1H & 13C NMR spectroscopy. With respect to the corresponding retention factors, these contaminants were validated using the available RP-HPLC method. These compounds can be used as a reference standard for the active pharmaceutical ingredient manufactures and research organizations.

Identification, synthesis and characterization of avanafil process impurities and determination by UPLC.

Avanafil is a phosphodiesterase type 5 inhibitor which is used to treat erectile dysfunction in men. The process-related impurities of avanafil were investigated, and four kinds of impurities in several laboratory batches with a content of 0.29–1.63% were detected by the newly developed gradient ultra-high performance liquid chromatography (UPLC). Based on the synthesis route and UPLC-MS research, the impurities are inferred as Imp-A, Imp-B, Imp-C and Imp-D. The structures of the impurities were inferred from LC-MS studies and confirmed by synthesis, followed by spectroscopic characterization such as NMR and mass spectrometry. Especially, the synthesis of Imp-D is firstly reported. The drug-related substances can be separated well by efficient and selective ultra-high performance liquid chromatography on a Waters ACQUITY HSS C18 (50 × 2.1 mm, particle size 1.8 μm) column at 35 °C, with the mobile phase consisting of ammonium formate (20 mM) and acetonitrile, and the detection at 239 nm with a DAD detector. The method was validated in terms of specificity, linearity, precision, accuracy and sensitivity, and satisfactory results were obtained. The results indicated this developed UPLC method for avanafil and the proposed synthesis mechanism can be used for quality control purposes as required by regulatory agencies to ensure the safety and efficacy of the product.