Compliance and regulatory considerations for the implementation of the multi-attribute-method by mass spectrometry in a quality control laboratory.

Multi-attribute methods employing mass spectrometry are applied throughout the biopharmaceutical industry for product and process characterization purposes but are not yet widely accepted as a method for batch release and stability testing under the good manufacturing practice (GMP) regime, due to limited experience and level of comfort with the technical, compliance and regulatory aspects of its implementation at quality control (QC) laboratories. This article is the second part of a two-tiered publication aiming at providing guidance for implementation of the multi-attribute method by peptide mapping liquid chromatography mass spectrometry (MAM) in a QC laboratory. The first part [1] focuses on technical considerations, while this second part provides considerations related to GMP compliance and regulatory aspects. This publication has been prepared by a group of industry experts representing 14 globally acting major biotechnology companies under the umbrella of the European Federation of Pharmaceutical Industries and Associations (EFPIA) Manufacturing & Quality Expert Group (MQEG).

Development of UPLC-UV-ELSD Method for Fatty Acid Profiling in Polysorbate 80 and Confirmation of the Presence of Conjugated Fatty Acids by Mass Spectrometry, UV Absorbance and Proton Nuclear Magnetic Resonance Spectroscopy.

Polysorbate 80 (PS80), a chemical substance composed of sorbitol, ethylene glycol, and fatty acids, is commonly used in pharmaceutical drug products to stabilize formulations. However, recent studies have demonstrated that PS80 may hydrolyze over time and the released free fatty acids (FFAs) may lead to particle formation. Naming conventions of fatty acids in current pharmacopeia and in products' certificates of analysis (CoA) of PS80 do not typically distinguish between isomeric species of fatty acids in PS80. Thus, methods to fully characterize the fatty acid species present in PS80 raw materials are needed to enhance quality control strategies of pharmaceuticals using PS80. Here, extended effort is taken to characterize fatty acids in hydrolyzed PS80 raw materials and elucidate the identities of isomeric fatty acid species. In this work, a method was developed and optimized for separation and detection of fatty acids in alkaline hydrolyzed PS80 raw materials using ultra performance liquid chromatography (UPLC) with ultra-violet (UV) detection and evaporative light scattering detection (ELSD). Fatty acids not specified in the current pharmacopeias were detected in PS80 raw material by the developed LC-UV-ELSD method including conjugated forms of linoleic and linolenic fatty acid species. Their identities were orthogonally confirmed by retention time agreement with analytical standards, accurate mass by high resolution mass spectrometry, UV absorbance, and proton nuclear magnetic resonance spectroscopy. The detected conjugated fatty acids are theoretically more hydrophobic and less soluble than their unconjugated counterparts and may increase the propensity of PS80 to form particles upon hydrolysis. This work highlights the need for better quality control of PS80 raw material, as it may eventually play a critical role in product quality of therapeutic proteins.

Technical considerations for the implementation of the multi-attribute-method by mass spectrometry in a quality control laboratory.

Multi-attribute methods employing mass spectrometry are applied throughout the biopharmaceutical industry for product and process characterization purposes but are not yet widely accepted as a method for batch release and stability testing under good manufacturing practice (GMP) due to limited experience and level of comfort with the technical, compliance and regulatory aspects of its implementation at quality control (QC) laboratories. Here, current literature related to the development and application of the multi-attribute method by peptide mapping liquid chromatography mass spectrometry (MAM) is compiled with the aim of providing guidance for the implementation of MAM in a QC laboratory. This article, focusing on technical considerations, is the first part of a two-tiered publication, whereby the second part will focus on GMP compliance and regulatory aspects. This publication has been prepared by a group of industry experts representing 14 globally acting major biotechnology companies under the umbrella of the European Federation of Pharmaceutical Industries and Associations (EFPIA) Manufacturing & Quality Expert Group (MQEG).

Microchip electrophoresis separation coupled to mass spectrometry (MCE-MS) for the rapid monitoring of multiple quality attributes of monoclonal antibodies.

Therapeutic monoclonal antibodies (mAbs) are highly heterogeneous as a result of posttranslational modifications (PTMs) during bioprocessing and storage. The modifications that impact mAb product quality are regarded as critical quality attributes and require monitoring. The conventional LC-mass spectrometer (MS) method used for product quality monitoring may require protein A purification prior to analysis. In this paper, we present a high-throughput microchip electrophoresis (<4 min) in-line with MS (MCE-MS) that enables baseline separation and characterization of Fc, Fd', and light chain (LC) domains of IdeS-treated mAb sample directly from bioreactor. The NISTmAb was used to optimize the MCE separation and to assess its capability of multiple attribute monitoring. The MCE-MS can uniquely separate and characterize deamidated species at domain level compared to LC-MS method. Two case studies were followed to demonstrate the method capability of monitoring product quality of mAb samples from stability studies or directly from bioreactors.

Industry Perspective on the Use and Characterization of Polysorbates for Biopharmaceutical Products Part 2: Survey Report on Control Strategy Preparing for the Future.

Polysorbate (PS) 20 and 80 are the main surfactants used to stabilize biopharmaceutical products. Industry practices on various aspects of PS based on a confidential survey and following discussions by 16 globally acting major biotechnology companies is presented in two publications. Part 1 summarizes the current practice and use of PS during manufacture in addition to aspects like current understanding of the (in)stability of PS, the routine QC testing and control of PS, and selected regulatory aspects of PS.1 The current part 2 of the survey focusses on understanding, monitoring, prediction, and mitigation of PS degradation pathways in order to propose an effective control strategy. The results of the survey and extensive cross-company discussions are put into relation with currently available scientific literature.

Industry Perspective on the use and Characterization of Polysorbates for Biopharmaceutical Products Part 1: Survey Report on Current State and Common Practices for Handling and Control of Polysorbates.

Polysorbates (PS) are widely used as a stabilizer in biopharmaceutical products. Industry practices on various aspects of PS are presented in this part 1 survey report based on a confidential survey and following discussions by 16 globally acting major biotechnology companies. The current practice and use of PS during manufacture across their global manufacturing sites are covered in addition to aspects like current understanding of the (in)stability of PS, the routine QC testing and control of PS, and selected regulatory aspects of PS. The results of the survey and extensive cross-company discussions are put into relation with currently available scientific literature. Part 2 of the survey report (upcoming) will focus on understanding, monitoring, prediction, and mitigation of PS degradation pathways to develop an effective control strategy.

Characterization of Polysorbate 80 by Liquid Chromatography-Mass Spectrometry to Understand Its Susceptibility to Degradation and Its Oxidative Degradation Pathway.

A liquid chromatography-mass spectrometry (LC-MS) method was developed to provide a fingerprint of polysorbate 80 (PS80) subspecies that enables identification of PS80 degradation pathway. The developed method demonstrates unique monoester peak profile of PS80 from different vendors, attributed by differences in relative abundance of the fatty acid monoesters. The LC-MS method was also applied to examine the susceptibility of PS80, at different grades, to auto-oxidation and hydrolysis. PS80 oxidative degradation induced by iron or occurred in open bottle without nitrogen overlay was found to follow the same pathway, but at a much faster rate in the former scenario. The oxidation preferentially occurs at the double bond of fatty acid chains, thus providing explanation on the faster degradation observed in PS80 at Chinese Pharmacopia (ChP) grade than at multi-compendial (MC) grade. In contrast, the difference in susceptibility of MC and ChP grade PS80 against esterase-induced hydrolysis in placebo was not pronounced. The method was also able to provide a fingerprint to identify both PS80 hydrolysis and oxidation in mAb drug product stability samples, but it required a solid phase extraction step to remove protein prior to the analysis.

Discovery and Characterization of Histidine Oxidation Initiated Cross-links in an IgG1 Monoclonal Antibody.

Novel cross-links between an oxidized histidine and intact histidine, lysine, or cysteine residues were discovered and characterized from high-molecular weight (HMW) fractions of an IgG1 monoclonal antibody (mAb). The mAb HMW fractions were collected using preparative size-exclusion chromatography (SEC) and extensively characterized to understand the mechanism of formation of the nonreducible and covalently linked portion of the HMWs. The HMW fractions were IdeS digested, reduced, and analyzed by size-exclusion chromatography coupled with mass spectrometry (SEC-MS). The nonreducible cross-links were found to be enriched in the fragment crystallizable (Fc) region of the heavy chain, with a net mass increase of 14 Da. Detailed peptide mapping revealed as many as seven covalent cross-links in the HMW fractions, where oxidized histidines react with intact histidine, lysine, and free cysteine to form cross-links. It is the first time that histidine-cysteine (His-Cys) and histidine-lysine (His-Lys) in addition to histidine-histidine (His-His) cross-links were discovered in monoclonal antibody HMW species. The histidine oxidation hot spots were identified, which include conserved histidine residues His292 and His440 in the Fc region and His231 in the hinge region of the IgG1 mAb heavy chain. Their cross-linking partners include His231, His292, His440, and Cys233 in the hinge region and Lys297 in the Fc region. A cross-linking mechanism has been proposed that involves nucleophilic addition by histidine, cysteine, or lysine residues to the carbonyl-containing histidine oxidation intermediates to form the cross-links.

What does fetal movement predict about behavior during the first two years of life?

This study evaluated whether motor activity prior to birth is predictive of motor behavior and temperament in neonates, infants, and toddlers. Three measures of fetal motor activity (activity level, amplitude, and number of movements) were collected at 24, 30, and 36 weeks of gestation in 52 healthy fetuses using Doppler-based actography. Postnatal data collection included a neurobehavioral assessment at 2-weeks postpartum (n = 41), and laboratory-based behavioral observations at 1 and 2 years of age (ns = 35). Individual stability in motor activity was present during gestation. Predictive relations between fetal movement and neonatal behavior were inconsistent; significant but small positive associations were detected between motor behavior at 36 weeks and neonatal irritability and motor development. Fetal activity level at 36 weeks was positively associated with observed 1-year activity level for boys (but inversely related for girls) and maternal report of activity level at 2 years. Fetal movement was consistently and negatively predictive of distress to limitations at 1 year and behavioral inhibition at 2 years, accounting for 21 to 43% of the variance in these measures. Intrafetal variability in motor behavior makes this a relatively unstable metric for prediction to neonatal maturational outcomes, which are relatively constrained, but fetal motor activity appears to predict temperament attributes related to regulatory behaviors in early childhood.