Ambient analysis of leachable compounds from single-use bioreactors with desorption electrospray ionization time-of-flight mass spectrometry.

RATIONALE: Trace levels of bis(2,4-di-tert-butylphenyl)phosphate (BdtbPP) leaching from single-use bioreactors (SUBs) were recently found to be highly detrimental to mammalian cell growth. The traditional approach to detect the leachable requires time-consuming solvent extraction of SUBs. To assist the qualification of SUBs and/or their manufacturing raw materials in biopharmaceutical development and manufacturing, it is essential to develop a rapid and sensitive analytical approach for detecting this leachable and related compounds, which is described in this study. METHODS: Native films from several commercially available SUBs were directly examined by desorption electrospray ionization (DESI) time-of-flight mass spectrometry (TOFMS) without sample preparation. As a comparison, the same SUBs were also analyzed by high-performance liquid chromatography (HPLC)/ultraviolet (UV) following the solvent extraction. RESULTS: With a suitable spray solvent selected in this study, DESI-TOFMS enabled rapid and sensitive detection of leachable compounds directly from SUBs. Accurate mass measurement from TOFMS allowed ready identification of BdtbPP, its parent analog compound, and other polymer components in the SUBs from their protonated surrogates. The relative abundances of BdtbPP in different SUBs measured by DESI-TOFMS exhibited good correlation with those from the traditional extraction-based approach with HPLC/UV. CONCLUSIONS: A rapid and sensitive approach was developed for direct detection of BdtbPP and other leachables from SUBs using DESI-TOFMS. The results are in high accordance with those from conventional approaches, which indicates the usefulness of the proposed method as a qualification tool for SUBs in biopharmaceutical development and also its great potential in the analysis of extractables/leachables in a wide variety of materials, process components, devices and containers used in the pharmaceutical industry.

Application of two-dimensional selective-TOCSY HMBC for structure elucidation of impurities in mixture without separation.

In the pharmaceutical industry, regulatory expectations driven by patient safety considerations make structure elucidation of impurities at levels greater than 0.1% in the active pharmaceutical ingredient (API) of primary interest. Impurities can be generated from isomers in starting materials, or produced from different process steps toward the final API. Proton peaks belonging to different impurities could be potentially identified in the one-dimensional (1)H NMR spectrum, when evaluated in combination with two-dimensional (2-D) COSY and HSQC data. However, in 2-D HMBC data, correlation responses from different impurities may overlap with those from the major component, causing uncertainty of long-range proton to carbon correlations and quaternary carbon assignments. This observation prompts us to design the 2-D selective-TOCSY HMBC experiment to distinguish responses from different impurities in mixtures to obtain 2-D NMR data for each impurity, thus eliminating the use of a chromatographic isolation step to obtain material for NMR analysis. This methodology is demonstrated for structure elucidation of impurities ranging from 8.2% in the raw material to 0.4% in the API in this study, and would be particularly useful for industrial samples in which the solubility and availability of material are not an issue.

Recommendation of Single Time Point Leachables Testing for Lyophilized Biotechnology Products Stored in Rubber Stoppered Glass Vial Systems.

As a popular format of primary container closure systems, rubber stoppered glass vials are often used in storing and delivering lyophilized and liquid formulated therapeutic protein products. Assessing extractables and leachables from rubber stoppered glass vial systems is required to ensure drug product quality and patient safety. Lyophilized biopharmaceutical drug products are generally considered as less impacted by leachables during storage and transportation than the liquid formulated drug products. Single time point leachables testing for lyophilized biopharmaceutic drug products is recommended. The recommendation is based on our published comprehensive leachable data collected at multiple time points for five lyophilized drug products stored in different rubber stoppered glass vial systems with additional supporting comprehensive leachable data collected for nineteen liquid formulated drug products stored in different syringe and vial systems, which is statistically and scientifically sound. The leachable data evaluated herein were generated based on a holistic approach which ensured successful qualification of different vial systems as primary containers and delivery systems for various biotherapeutic products. The organic and elemental impurities of the leachable profiles of all the twenty-four drug product samples were below the limit of detection at all the time points. For lyophilized drug products, product surface interaction during storage time and shipping is unlikely. Timing of single time point leachables testing can be flexible. Performing leachables testing at one-year time point is recommended as it allows for enough time for chemicals to leach out from product contact surfaces into drug products and thus provides the earliest opportunity for mitigation of unpredicted leachables of concern, if any. However, testing at other stability time points can also be considered depending on the development strategy of the sponsor. Therefore, recommendation of single time point leachables testing for lyophilized drug products stored in rubber stopped glass vials at an appropriate time point is a scientifically sound approach.

A Holistic Approach of Extractables and Leachables Assessment of Rubber Stoppered Glass Vial Systems for Biotechnology Products.

Rubber stoppered glass vial systems are widely used as primary containers for storing and delivering therapeutic protein products to patients. Addressing concerns and regulatory expectations related to the risk to biologic drug product quality and patient safety from rubber stoppered glass vial systems requires implementation of an extractable and leachable evaluation program based on material understanding, risk assessment, literature review, and a comprehensive scientifically sound analytical testing methodology. The extractable and leachable study design consisted of twelve drug products filled in twelve different size glass vials capped with laminated and nonlaminated rubber stoppers made from three different rubber formulations. Design of the model solvents was successful as they had little to no analytical interference and mimicked the formulation conditions and generated representative extractables capable of predicting leachables. The extraction conditions of time and temperature were appropriate as not to degrade the test materials or the extractable compounds, and yet generated significant quantities for identification of the extractable compounds with confidence. The extractables testing results were capable of predicting the leachable profiles of the twelve drug products. In each case, the leachable profile was a subset of the extractable profile. The organic and elemental impurities of the leachable profiles of drug products were the end-to-end verification of the quality of the glass vials, rubber stoppers and drug product lifecycles. Overall, the holistic approach was fully successful in the qualification of different vial systems as primary containers and delivery systems for different biotherapeutic products to ensure product quality and patient safety.

Observation and Mitigation of Leachables from Non-Product Contact Materials in Electromechanical Delivery Devices for Biotechnology Products.

Battery-powered drug delivery devices are widely used as primary containers for storing and delivering therapeutic protein products to improve patient compliance and quality of life. Compared to conventional delivery approaches such as pre-filled syringes, battery-powered devices are more complex in design requiring new materials/components for proper functionality, which could cause potential product safety and quality concerns from the extractable and leachables (E&L) of the new materials/components. In this study, E&L assessments were performed on a battery-powered delivery device during the development and qualification of the device, where novel compound 2­hydroxy-2-methylpropiophenone (HMPP) and related compounds were observed in both E&L. The source of the HMPP and related compounds was identified to be the nonproduct contact device batteries, in which HMPP photo-initiator was used as a curing agent in the battery sealant to prevent leakage of the battery electrolytes. Toxicology assessment was performed, which showed the levels of HMPP observed in the device lots were acceptable relative to the permitted daily exposure. A drug product HMPP spike study was also performed, where no product impact was observed. Based on these assessments, an action threshold and specification limits could be established as a control strategy, if needed, to mitigate the potential risks associate with the observed leachables. As a full resolution, seven battery candidates from different suppliers were screened and one new battery was successfully qualified for the delivery devices. Overall, the holistic E&L approach was fully successful in the development and qualification of the battery-powered devices for biotherapeutic products delivery ensuring product quality and patient safety. Non-product contact materials are commonly rated as low or no risk and typically considered as out of scope of E&L activities for delivery systems following industry benchmark and regulatory agency guidance. This case study is novel as it brings into attention the materials that might not normally be in consideration during the development process. It is highly recommended to understand materials in the context of intended use on a case-by-case basis and not to generalize to ensure successful development and qualification.

Holistic Extractables and Leachables Program: Evaluations of Prefilled Syringe Systems for Biotechnology Products.

Prefilled syringes (PFS) are a container and delivery device of choice for storing and administering therapeutic protein products to patients. Addressing concerns and regulatory expectations related to the risk to biologic drug product quality and patient safety from PFS requires implementation of an extractable and leachable program based on understanding of materials, risk assessment, review of existing literature, and testing supported by a sound scientific foundation. Extractables and leachables data generated as part of a thorough and holistic program are presented for five PFS systems, including glass and plastic syringes filled with 12 biologic drug products encompassing the implementation of traditional and single-use biotechnology manufacturing processes. The comprehensive extractables and leachables data presented demonstrate and substantiate a holistic extractable and leachable program designed to ensure product quality and patient safety.

A Risk-Based Approach to Evaluate and Control Elemental Impurities in Therapeutic Proteins.

Control of elemental impurities in the drug products evolved from the generic visual testing of heavy metals as their sulfides to specific elements of toxicological concern in the final drug products by instrumental analysis. The International Council for Harmonisation (ICH) Q3D (R1) guideline for elemental impurities describes a risk-based approach to identify, assess, and control the potential elemental impurities in drug products within the established permitted daily exposures (PDE). Challenges to this approach include how to assess the risks associated with contributing sources such as utilities, manufacturing equipment, container-closure systems, and excipients. Defining at what stage of development that such assessment should be performed to identify the risk levels can be equally challenging. In this article, we report an approach to control elemental impurities of toxicological concern, compliant to the Q3D (R1) guideline, and a summary of results obtained on multiple protein therapeutic products. This approach follows the elements of Process Validation, i.e., Design, Qualification, and Continuous Verification. The design includes the selection of excipients and their suppliers that meet the Option 1 requirement of Q3D (R1). It also comprises the selection of manufacturing equipment, container-closure systems, and utilities. The qualification includes the testing of the potential sources of elemental impurities, i.e., excipients, utilities, and leachables/extractables from the manufacturing equipment and container-closure systems. The Continuous Verification comes from the testing of representative batches at the initiation of stability studies of clinical or commercial drug product batches and at the end of shelf-life expiry of the drug product, and when changes are made to the manufacturing equipment, sources of excipients and container closure systems, and any formulation changes. Our experience shows that the risk associated with the impurity levels of the ten elements of toxicological concern in the therapeutic protein drug products, parenterally administered, is well below the control threshold (30% PDE) in the drug product recommended by the ICH Guideline. Although our focus is on the injectable therapeutic proteins, this approach can be applied to the products administered via other routes as well.

Toward top-down determination of PEGylation site using MALDI in-source decay MS analysis.

A novel matrix assisted laser desorption/ionization (MALDI)-based mass spectrometric approach has been evaluated to rapidly analyze a custom designed PEGylated peptide that is 31 residues long and conjugated with 20 kDa linear polyethylene glycol (PEG) at the side chain of Lys. MALDI-TOF MS provided sufficiently high resolution to allow observation of each of the oligomers of the heterogeneous PEGylated peptide (m/Deltam of ca. 500), while a typical ESI-MS spectrum of this molecule was extremely complex and unresolved. Reflector in-source decay (reISD) analysis using MALDI-TOF MS was attempted to identify the PEGylation site at intact molecular level without any sample treatment. An reISD spectrum of the free peptide was observed with abundant c-, y-, and [z + 2]-fragment ion series, whereas, in the fragmented PEGylated peptide, the fragment ion series were truncated at the residue where PEG was attached. Therefore, a direct comparison of these top-down reISD spectra suggested the location of the PEGylation site. Results from this study demonstrate a clear analytical utility of the ISD technique to characterize structural aspects of heterogeneous biomolecules.

Determination of interactions between antibody biotherapeutics and copper by size exclusion chromatography (SEC) coupled with inductively coupled plasma mass spectrometry (ICP/MS).

The concept of coupling of size-exclusion HPLC with ICP/MS (SEC-ICP/MS) was first applied in this work as a novel approach in the biotechnology field to assess metal binding to Immunoglobulin G (IgG) mAbs. This method can be used to probe the mechanism and biophysical properties of metal-protein interactions to gain a deeper understanding of the potential impact of metals during drug product manufacturing. Two IgG1s and one IgG2 drugs were investigated. Cu2+ was selected as the metal of interest due to its known ability to form strong complexes with organic molecules and to bind and enhance the degradation of mAbs. Instrument and separation conditions (interface, columns, and mobile phase) were studied for the separation of the protein-metal bound and unbound fractions of a bovine superoxide dismutase (SOD) standard prior to on-line detection of the mAb-metal (Cu) binding. The SEC-ICP/MS method was used to show copper binding by biotherapeutics by comparing the retention times of the protein by SEC and the metal by ICP/MS, to see if they co-elute at the same time. The approach developed offers considerable advantages over methods based on ultrafiltration followed by off-line metal determination in terms of speed, simplicity, precision and selectivity regarding the molecular weight of the complexes involved. In conjunction with other techniques, this method may provide in-depth knowledge of metal-induced mAb degradation mechanisms in biologics process development, be used as an analytical tool for mAb manufacturing in the cell culture process, and be applied during various stages of drug product manufacturing to gain a deeper understanding of the potential impact of metals during biotherapeutic development.

Characterization of Protein Aggregating Tungstates: Electrospray Mass Spectrometry Analysis of Extracts from Prefilled Syringes and from Tungsten Pins Used in the Manufacture of Syringes.

UNLABELLED: Glass prefilled syringes are increasingly becoming a container of choice for storing and administering therapeutic protein products to patients. Tungsten leaching from a PFS is known to induce protein particle formation, and the source was traced to the tungsten pins used in the manufacturing process of the syringe barrels. Study of the tungstates present in extracts from both tungsten pins used in the syringe manufacturing process and from single syringes from various suppliers was undertaken. Electrospray mass spectrometry was chosen as a technique with the sensitivity to characterize tungstates at levels (∼1 ppm of elemental tungsten) observed in single syringes. Extraction solvents were chosen to simulate the range (pH 4.0-7.0) typically used for therapeutic protein formulation. A commercial product formulation buffer was also used as an extraction solution to characterize tungstate species used for tungsten spiking studies of protein. All pin and syringe extracts from various manufacturers were similar in regards to containing stable Na/K containing lacunary polytungstate ([W11O39](7-)) species, which were the main species present in syringe extracts and are different than the metatungstate ([W12O39](6-)) species identified in commercially available sodium polytungstate and as the main species in pin extracts. These stable Na/K containing polytungstates species present in pin and syringe extracts are likely formed during the glass manufacturing process at >400 °C and may have the capability to subsequently form larger polytungstate complexes. LAY ABSTRACT: Glass prefilled syringes are a type of container used for storing and administering biotechnology medicines to patients. The manufacturing process for the syringes may lead to very low levels of the metal tungsten being present in the syringes, and thus in the medicine stored in the syringes. The presence of tungsten in certain biotechnology medicines has been shown to cause changes to the medicine. Understanding something that can cause a medicine to change is an important part of producing safe and effective medicines for patients. The study described in this article sought to increase understanding by characterizing the form of tungsten observed in syringes from a number of vendors. Study of the tungsten present in syringes from four vendors indicates the same form of tungsten is observed regardless of the vendor. The study also found that the form of tungsten differed from that expected.

Extractables analysis of single-use flexible plastic biocontainers.

UNLABELLED: Studies of the extractable profiles of bioprocessing components have become an integral part of drug development efforts to minimize possible compromise in process performance, decrease in drug product quality, and potential safety risk to patients due to the possibility of small molecules leaching out from the components. In this study, an effective extraction solvent system was developed to evaluate the organic extractable profiles of single-use bioprocess equipment, which has been gaining increasing popularity in the biopharmaceutical industry because of the many advantages over the traditional stainless steel-based bioreactors and other fluid mixing and storage vessels. The chosen extraction conditions were intended to represent aggressive conditions relative to the application of single-use bags in biopharmaceutical manufacture, in which aqueous based systems are largely utilized. Those extraction conditions, along with a non-targeted analytical strategy, allowed for the generation and identification of an array of extractable compounds; a total of 53 organic compounds were identified from four types of commercially available single-use bags, the majority of which are degradation products of polymer additives. The success of this overall extractables analysis strategy was reflected partially by the effectiveness in the extraction and identification of a compound that was later found to be highly detrimental to mammalian cell growth. LAY ABSTRACT: The usage of single-use bioreactors has been increasing in biopharmaceutical industry because of the appealing advantages that it promises regarding to the cleaning, sterilization, operational flexibility, and so on, during manufacturing of biologics. However, compared to its conventional counterparts based mainly on stainless steel, single-use bioreactors are more susceptible to potential problems associated with compound leaching into the bioprocessing fluid. As a result, extractable profiling of the single-use system has become essential in the qualification of such systems for its use in drug manufacturing. The aim of this study is to evaluate the effectiveness of an extraction solvent system developed to study the extraction profile of single-use bioreactors in which aqueous-based systems are largely used. The results showed that with a non-targeted analytical approach, the extraction solvent allowed the generation and identification of an array of extractable compounds from four commercially available single-use bioreactors. Most of extractables are degradation products of polymer additives, among which was a compound that was later found to be highly detrimental to mammalian cell growth.

Highly enantioselective hydrogenation of styrenes directed by 2'-hydroxyl groups.

A new synthetic strategy that turns styrene-type olefins into excellent substrates for Rh-catalyzed asymmetric hydrogenation by installing a 2'-hydroxyl substituent is described. This methodology accommodates trisubstituted olefinic substrates in various E/Z mixtures, leading to valuable benzylic chiral compounds including (R)-tolterodine. It is also demonstrated that the 2'-hydroxyl groups could be readily removed in high yield without loss of ee from the products. Thus, this technology represents an attractive alternative to the Ir(P-N) catalyst system for the asymmetric hydrogenation of unfunctionalized olefins.

A Multidisciplinary Investigation to Determine the Structure and Source of Dimeric Impurities in AMG 517 Drug Substance.

In the initial scale-up batches of the experimental drug substance AMG 517, a pair of unexpected impurities was observed by HPLC. Analysis of data from initial LC-MS experiments indicated the presence of two dimer-like molecules. One impurity had an additional sulfur atom incorporated into its structure relative to the other impurity. Isolation of the impurities was performed, and further structural elucidation experiments were conducted with high-resolution LC-MS and 2D NMR. The dimeric structures were confirmed, with one of the impurities having an unexpected C-S-C linkage. Based on the synthetic route of AMG 517, it was unlikely that these impurities were generated during the last two steps of the process. Stress studies on the enriched impurities were carried out to further confirm the existence of the C-S-C linkage in the benzothiazole portion of AMG 517. Further investigation revealed that these two dimeric impurities originated from existing impurities in the AMG 517 starting material, N-acetyl benzothiazole. The characterization of these two dimeric impurities allowed for better quality control of new batches of the N-acetyl benzothiazole starting material. As a result, subsequent batches of AMG 517 contained no reportable levels of these two impurities.