Jonathan Bones

An automated robotic platform for rapid profiling oligosaccharide analysis of monoclonal antibodies directly from cell culture

Oligosaccharides attached to Asn297 in each of the CH2 domains of monoclonal antibodies play an important role in antibody effector functions by modulating the affinity of interaction with Fc receptors displayed on cells of the innate immune system. Rapid, detailed, and quantitative N-glycan analysis is required at all stages of bioprocess development to ensure the safety and efficacy of the therapeutic. The high sample numbers generated during quality by design (QbD) and process analytical technology (PAT) create a demand for high-performance, high-throughput analytical technologies for comprehensive oligosaccharide analysis. We have developed an automated 96-well plate-based sample preparation platform for high-throughput N-glycan analysis using a liquid handling robotic system. Complete process automation includes monoclonal antibody (mAb) purification directly from bioreactor media, glycan release, fluorescent labeling, purification, and subsequent ultra-performance liquid chromatography (UPLC) analysis. The entire sample preparation and commencement of analysis is achieved within a 5-h timeframe. The automated sample preparation platform can easily be interfaced with other downstream analytical technologies, including mass spectrometry (MS) and capillary electrophoresis (CE), for rapid characterization of oligosaccharides present on therapeutic antibodies.

Glycan characterization of biopharmaceuticals: Updates and perspectives

Therapeutic proteins are rapidly becoming the most promising class of pharmaceuticals on the market due to their successful treatment of a vast array of serious diseases, such as cancers and immune disorders. Therapeutic proteins are produced using recombinant DNA technology. More than 60% of therapeutic proteins are posttranslationally modified following biosynthesis by the addition of N- or O-linked glycans. Glycosylation is the most common posttranslational modifications of proteins. However, it is also the most demanding and complex posttranslational modification from the analytical point of view. Moreover, research has shown that glycosylation significantly impacts stability, half-life, mechanism of action and safety of a therapeutic protein. Considering the exponential growth of biotherapeutics, this present review of the literature (2009-2015) focuses on the characterization of protein glycosylation, which has witnessed an improvement in methodology. Furthermore, it discusses current issues in the fields of production and characterization of therapeutic proteins. This review also highlights the problem of non-standard requirements for the approval of biosimilars with regard to their glycosylation and discusses recent developments and perspectives for improved glycan characterization.

Development and validation of an ultra-performance liquid chromatography method for the determination of bis(2,4-di-tert-butylphenyl)phosphate and related extractable compounds from single-use plastic films

A UPLC-UV method is described for the detection of bDtBPP and related breakdown compounds of Irgafos 168 from single-use bioprocessing bag films using a CSH (charged surface hybrid) fluoro phenyl column and UV detection at 220nm. The method limits of detection were between 16μgL-1 and 60μgL-1, repeatability was %RSD≤1.8 and linearity was R2≥0.9992. The method was applied to an extractables and leachables test of three single-use bioprocessing bag films. bDtBPP and oxidised Irgafos were detected from all three films in the extractables study, but not in the leachables study. A comparison of whole bag and small scale film extractions demonstrated that the small scale extractions were more suitable for finding concentration per area of film and/or estimating total load of extractables in the entire bag. The feasibility of upstream monitoring of extractable and leachable compounds with the Waters PATROL UPLC System via a Flownamics® sampling probe was also tested with cell growth medium spiked with bDtBPP. bDtBPP was only detected in at-line injections or online injections when the probe membrane was removed indicating that with the current sampling interface, monitoring of extractables and/or leachables is limited to at-line sampling.

Charge Variant Analysis of Monoclonal Antibodies Using Direct Coupled pH Gradient Cation Exchange Chromatography to High-Resolution Native Mass Spectrometry

Charge variant analysis (CVA) of monoclonal antibodies (mAbs) using cation exchange chromatography is routinely used as a fingerprint of the distribution of posttranslational modifications present on the molecule. Traditional salt or pH based eluents are not suited for direct coupling to mass spectrometry due to nonvolatility or high ionic strength. This makes further analysis complicated when an alteration in the charge variant profile or the emergence of an additional peak is encountered. Here, the use of pH gradient elution using volatile, low ionic strength buffers is reported with direct coupling to high-resolution Orbitrap mass spectrometry. The development of a universal method based on pH elution was explored using a number of mAb drug products. Optimized methods facilitated the separation and identification of charge variants including individual glycoforms of the mAbs investigated using the same buffer system but with tailored gradient slopes. The developed method represents an exciting advance for the characterization of biopharmaceuticals as intact entities through the combination of native charge variant separations with high-resolution native mass spectrometry.

Non-volatile extractable analysis of prefilled syringes for parenteral administration of drug products

Graphical abstract Figure. No caption available. HighlightsDetermination of extractables profile for prefilled syringes at several conditions.Implementation of a simulated extractable study for long‐term storage.Application of a simulated immersion study for stoppers according to ISO 10993‐12.Use of liquid–liquid extraction as sample treatment for aqueous‐based extracts.Use of UHPLC‐QToF‐MS as analytical technique for extractables identification. Abstract The determination of extractable profiles for single‐use technologies represents an important aspect of pharmaceutical production to minimize any possible compromise in drug product quality or potential risk to patients by identifying substances that may potentially leach from such devices. An approach for the extractable assessment of prefilled syringes, a promising alternative for parenteral administration of pharmaceutical products, is described herein. Four extraction solvents were selected: a mixture 2‐propanol:water (1:1), was intended to represent aggressive conditions to extract a broad spectrum of extractables, including organic additives and substances which are poorly water‐soluble. Extractions with buffers at three different working pH values spanning a range standardly used in pharmaceutical formulations were also evaluated to identify substances that require specific conditions for their extraction due to their individual chemical properties. Syringes from two different brands were analysed along with their corresponding plunger stoppers. Syringes were extracted at 40 °C for 4 days, the plunger stoppers were extracted with 2‐propanol at 70 °C for 24 h according to ISO 10993‐12:2012. Extractables were identified by UHPLC–MS on a quadrupole time of flight instrument using a non‐targeted discovery strategy. A total of 25 compounds were identified, mostly polymer additives and their degradation products. The presented methodology represents a reference point for further studies focused on the characterisation of extractables and leachables from prefilled syringes.

Evaluation of solvent systems for optimized extractables studies of single use bioprocessing solutions

Despite their advantages, there is concern that single-use systems used in biopharmaceutical manufacture might release potentially toxic substances during standard unit operations that negatively impact cell growth. Characterization of the extractables profile for single-use systems is necessary to know which compounds potentially become leachables under operational cell culture conditions. A key issue in the design of extractables studies is the composition of the model solvent, in particular its pH and polarity. In this study, a new approach, based on design of experiments (DoE), has been applied to determine the composition of the model solvent for extractable profiling of single-use bags (SUBs). Particular focus was placed on the determination of the degradation products of the antioxidant Irgafos 168®, due to evidence that some of these degradation products have cytotoxic effects on CHO cells. Results indicated that 2-propanol:water is the most appropriate solvent for the extraction of highly hydrophobic compounds with polar groups and/or acid-base properties from SUBs. The described DoE approach simplifies the number of experiments, evaluates all possible solvent water mixtures to select the best extraction solvent based on polarity, establishes the influence of each variable and provides information about variable interaction, which represents an important improvement over current best practice. The developed approach was applied to seven SUBs from different vendors and production dates facilitating the identification of potentially non-satisfactory films for cultivation of CHO cell lines under process conditions.

Containment challenges in HPAPI manufacture for ADC generation

Antibody-drug conjugates (ADCs) are emerging as an impactful class of therapeutics for the treatment of cancer because of their ability to harness the specificity of an antibody and the cytotoxic potential of the payload to target and destroy cancer cells. However, the potent nature of the cytotoxic payload creates associated manufacturing challenges for active pharmaceutical ingredient (API) manufacturers, because huge investment in containment technology is required to ensure the protection of operators and the environment. Here, we examine the differing attitudes to high-potency categorisation and levels of containment control. We also provide an overview of the most widely used containment strategies for facility design, powder handling, purification, analysis, and cleaning. Finally, we briefly consider the health and safety regulatory challenges associated with the manufacture of cytotoxic payloads for use in antibody-drug conjugates.

Large-Scale Assessment of Extractables and Leachables in Single-Use Bags for Biomanufacturing

Single-use technologies (SUTs) are widely used during biopharmaceutical manufacture as disposable bioreactors or media and buffer storage bags. Despite their advantages, the risk of release of extractable and leachable (E&Ls) substances is considered an important drawback in adopting disposables in the biomanufacturing process. E&Ls may detrimentally affect cell viability or productivity or may persist during purification and present a risk to the patient if remaining in the final drug product. In this study, 34 plastic films from single-use bags (SUBs) for cell cultivation were extracted with selected solvents that represent reasonable worst-case conditions for most typical biomanufacturing applications. SUBs were incubated at small-scale under accelerated-aging conditions that represented standard operational conditions of use. Leachables analysis was performed following dispersive liquid-liquid microextraction (DLLME) for analyte preconcentration and removal of matrix interference. Resulting extracts were characterized by GC-headspace for volatiles, high resolution GC-Orbitrap-MS/MS for semivolatiles, high resolution LC-Orbitrap-MS/MS for nonvolatiles, and ICP-MS for trace elemental analysis. Multivariate statistical analysis of the analytical data revealed significant correlations between the type and concentration of compounds and bags features including brand, manufacturing date and polymer type. The analytical data demonstrates that, over recent years, the nature of E&Ls has been altered due to the implementation of manufacturing changes and new types of polymers and may change further with the future advent of regulations that will limit or ban the use of certain raw materials and additives. The broad E&L database generated herein facilitates toxicological assessments from a biomanufacturing standpoint and provides practical guidelines for confident determination of E&Ls to enable screening and elimination of nonsatisfactory films for single use bioprocessing.

Monitoring leachables from single-use bioreactor bags for mammalian cell culture by dispersive liquid-liquid microextraction followed by ultra high performance liquid chromatography quadrupole time of flight mass spectrometry

A method for the identification of leachables in chemically defined media for CHO cell culture using dispersive liquid-liquid microextraction (DLLME) and UHPLC-MS is described. A Box-Behnken design of experiments (DoE) approach was applied to obtain the optimum extraction conditions of the target analytes. Performance of DLLME as extraction technique was studied by comparison of two commercial chemically defined media for CHO cell culture. General extraction conditions for any group of leachables, regardless of their specific chemical functionalities can be applied and similar optimum conditions were obtained with the two media. Extraction efficiency and matrix effects were determined. The method was validated using matrix-matched standard calibration followed by recovery assays with spiked samples. Finally, cell culture media was incubated in 7 single use bioreactors (SUBs) from different vendors and analysed. TBPP was not detected in any of the samples, whereas DtBP and TBPP-ox were found in all samples, with bDtBPP detected in six SUBs. This method can be used for early identification of non-satisfactory SUB films for cultivation of CHO cell lines for biopharmaceutical production.

Monoclonal antibody sequence assessment using a hybrid quadrupole-Orbitrap mass spectrometer

Primary sequence determination is a key component in the development and characterisation of biotherapeutic drug substances. Although frequently performed using peptide mapping experiments, complementary methods are necessary to confirm full sequence identification. The requirement for a multi-faceted approach to sequence verification, ensuring single amino acid variations in the primary sequence are not overlooked, is highlighted in the data presented herein. A combination of liquid chromatography-mass spectrometry (LC-MS) strategies, including bottom-up peptide mapping, middle-up mass analysis of monoclonal antibody (mAb) IdeS digested subunits and intact protein analysis, were used to assess primary sequence for anti-interleukin 8 IgG1, a recombinantly expressed mAb from Chinese hamster ovary cells. This structured approach to primary sequence evaluation provides a framework for the evaluation of mAbs and biosimilar candidates and accentuates the importance of incorporating multiple analytical methods for therapeutic protein characterisation.