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• An AEX method was developed for sgRNA as an alternative to IP-RPLC and HILIC. • Temperature-dependent AEX reveals the complex higher-order structure (HOS) of sgRNAs. • The HOS of sgRNA differs from other nucleic acids, providing additional chemical stability for sgRNA. Large synthetic oligonucleotides such as guide ribonucleic acid (gRNA), a critical reagent in clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 genome editing, have complex higher order structures (HOS) inherent in their design. In this study, we first developed a generic anion exchange chromatography (AEX) method for the comprehensive analysis of a 100mer single guide ribonucleic acid (sgRNA) impurity profiling. AEX demonstrated superior resolution compared to other common chromatographic methods employed for sgRNA analysis, such as Ion-Pairing Reversed Phase Liquid Chromatography (IP-RPLC) and Hydrophilic Interaction Chromatography (HILIC). Moreover, we discovered AEX's potential in probing the HOS of RNAs by adjusting the temperature and using organic additives. Our study also highlighted that sgRNA possesses a unique HOS distinctly different from other therapeutic nucleic acids, such as antisense oligonucleotides and messenger RNAs. [ABSTRACT FROM AUTHOR]

• Higher order structure characterization is critical to protein therapeutics development. • Recent advances in mass spectrometry have provided means to characterize HOS. • The utility of native MS, HDX-MS and FPOP-MS is presented in this review. • Future trend in MS applications to HOS characterization is described. Molecular characterization of higher order structure (HOS) in protein therapeutics is crucial to the selection of candidate molecules, understanding of structure–function relationships, formulation development, stability assessment, and comparability studies. Recent advances in mass spectrometry (MS), including native MS, hydrogen/deuterium exchange (HDX)-MS, and fast photochemical oxidation of proteins (FPOP) coupled with MS, have provided orthogonal ways to characterize HOS of protein therapeutics. In this review, we present the utility of native MS, HDX-MS and FPOP-MS in protein therapeutics discovery and development, with a focus on epitope mapping, aggregation assessment, and comparability studies. We also discuss future trends in the application of these MS methods to HOS characterization. [ABSTRACT FROM AUTHOR]

Tryptophan (Trp) oxidation in proteins leads to a number of events, including changes in color, higher order structure (HOS), and biological activity. We describe here a number of new findings through a comprehensive characterization of 6 monoclonal antibodies (mAbs) following selective oxidation of Trp residues by 2,2'-azobis(2-amidinopropane) dihydrochloride. Fluorescence spectroscopy, in combination with second derivative analysis, demonstrates that the loss of Trp fluorescence intensity is a sensitive indicator of Trp oxidation in mAbs. Size-exclusion chromatography with UV and intrinsic Trp fluorescence detection was demonstrated to be a useful method to monitor Trp oxidation levels in mAbs. Furthermore, the Trp oxidation levels measured by size-exclusion chromatography with UV and intrinsic Trp fluorescence detection were found to be in agreement with the values obtained from tryptic peptide mapping by liquid chromatography with mass spectrometric detection and correlate with the total solvent accessible surface area of the exposed Trp residues from in silico modeling. Finally, near-UV circular dichroism and Raman spectroscopy were used to evaluate the impact of Trp oxidation on HOS and identify specific oxidation products, respectively. This work demonstrates that protein HOS is altered on Trp oxidation in mAbs and multiple spectroscopic markers can be used to monitor the molecule-dependent Trp oxidation behavior. [ABSTRACT FROM AUTHOR]

Biotherapeutics and their biosimilar versions have been flourishing in the biopharmaceutical market for several years. Structural and functional characterization is needed to achieve analytical biosimilarity through the assessment of critical quality attributes as required by regulatory authorities. The role of analytical strategies, particularly mass spectrometry-based methods, is pivotal to gathering valuable information for the in-depth characterization of biotherapeutics and biosimilarity assessment. Structural mass spectrometry methods (native MS, HDX-MS, top-down MS, etc.) provide information ranging from primary sequence assessment to higher order structure evaluation. This review focuses on recent developments and applications in structural mass spectrometry for biotherapeutic and biosimilar characterization. • The structural MS toolbox affords interesting methods for biosimilarity assessment. • MS approaches provides detection of small differences in biosimilar/originator mAbs. • Native MS coupled to size exclusion chromatography allows size variants evaluation. • Ion mobility spectrometry rapidly detects subtle changes in protein conformation. • HDX-MS is a key technique for higher order structure comparison of biosimilars. [ABSTRACT FROM AUTHOR]

Nuclear magnetic resonance (NMR) spectroscopy has a unique capability to probe the primary and higher order molecular structure and the structural dynamics of biomolecules at an atomic resolution, and this capability has been greatly fortified over the last five decades by an astonishing NMR instrumental and methodological development. Because of these factors, NMR has become a primary tool for the structure investigation of biomolecules, spawning a whole scientific subfield dedicated to the subject. This role of NMR is by now well established and broadly appreciated, especially in the context of academic research dealing with proteins that are purified and isotope-labeled in order to facilitate the necessary sophisticated multidimensional NMR measurements. However, the more recent industrial development, manufacturing, and quality control of biopharmaceuticals provide a different framework for NMR. For example, protein drug substances are not isotope-labeled and are present in a medium of excipients, which make structural NMR measurements much more difficult. On the other hand, biotechnology involves many other analytical requirements that can be efficiently addressed by NMR. In this respect the scope and limitations of NMR are less well understood. Having the non-expert reader in mind, herein we wish to highlight the ways in which modern NMR can effectively support biotechnological developments. Our focus will be on biosimilar proteins, pointing out certain cases where its use is probably essential. Based partly on literature data, and partly on our own hands-on experience, this paper is intended to be a guide for choosing the proper NMR approach for analytical questions concerning the structural comparability of therapeutic proteins, monitoring technology-related impurities, protein quantification, analysis of spent media, identification of extractable and leachable components, etc. Also, we focus on critical considerations, particularly those coming from drug authority guidelines, which limit the use of the well-established NMR tools in everyday practice. [ABSTRACT FROM AUTHOR]

Novel poly(thiophenyleneethynylenephenylene)s having optically inactive/active amide groups were synthesized by the Sonoghashira-Hagihara coupling polymerization of 3-substituted 2,5-dibromothiophene 1 with 1-substituted 3,5-diethynylbenzenes 2N , 2R , 2S derived from glycine and d -/ʟ-alanines. Poly( 1 – 2S ) exhibited an intense Cotton effect based on negative exciton chirality at 405 and 366 nm. On the other hand, poly( 1–2N ) exhibited a Cotton effect similar to that of poly( 1 – 2S ) but weaker, and poly( 1 – 2R ) exhibited a weak Cotton effect based on positive exciton chirality. The chiral secondary structures of the polymers were dependent on chirality cooperation and chirality competition of the side chains. The trend of the circular dichroism (CD) signs and intensities of the polymers can be explained by the concepts of “chiral cooperation” and “chiral competition” between the optically active thiophene unit and optically inactive/active phenylene unit. These polymers formed aggregate structures with increasing MeOH content of CHCl 3 /MeOH mixed solvents, with accompanying decrease of the UV–vis absorptions and photoluminescence (PL) intensities. [ABSTRACT FROM AUTHOR]

The higher order structure (HOS) of biotherapeutics is a critical quality attribute that can be evaluated by nuclear magnetic resonance (NMR) spectroscopy at atomic resolution. NMR spectral mapping of HOS can be used to establish HOS consistency of a biologic across manufacturing changes or to compare a biosimilar to an innovator reference product. A previous inter-laboratory study performed using filgrastim drug products demonstrated that two-dimensional (2D)-NMR 1 H N - 15 N H heteronuclear correlation spectroscopy is a highly robust and precise method for mapping the HOS of biologic drugs at natural abundance using high sensitivity NMR ‘cold probes.’ Here, the applicability of the 2D-NMR method to fingerprint the HOS of filgrastim products is demonstrated using lower sensitivity, room temperature NMR probes. Combined chemical shift deviation and principal component analysis are used to illustrate the performance and inter-laboratory precision of the 2D-NMR method when implemented on room temperature probes. [ABSTRACT FROM AUTHOR]

A number of recombinant protein therapeutic products, such as filgrastim (methionyl granulocyte colony stimulating factor [Met-GCSF] used to boost the immune system in chemotherapy treated cancer patients), and interferon alpha-2 (used for the treatment of various viral infections), have been chemically modified with the addition of a polyethylene glycol (PEG) chain. This modification prolongs residency of the drug in the body and reduces metabolic degradation, which allows less frequent administration of the products. Here we show how NMR spectroscopy methods can assess the higher order structure (HOS) of pegylated-filgrastim (Neulasta®), pegylated interferon-α2a (Pegasys®) pegylated interferon-α2b (PEG-Intron®) purchased from the marketplace. The addition of the PEG moiety effectively doubles the molecular weight of the three products. This presents a significant challenge for the application of NMR techniques. Nevertheless, the results showed that high-resolution spectra could be recorded for two of the three products. Comparison of the spectra of the pegylated protein and the non-pegylated protein shows that the chemical modification did not alter the HOS of these proteins. [ABSTRACT FROM AUTHOR]

Characterization of the higher order structure (HOS) of protein-based biopharmaceutical products is an important aspect of their development. Opinions vary about how best to apply biophysical methods, in which contexts to use these methods, and how to use the resulting data to make technical decisions as drug candidates are commercialized [Gabrielson JP, Weiss WF IV. J Pharm Sci . 2015;104(4):1240-1245]. The aim of this commentary is to provide guidance for the development and implementation of a robust and comprehensive HOS characterization strategy. We first consider important concepts involved in developing a strategy that is appropriately suited to a particular biologic, and then discuss ways industry can partner with academia, technology companies, government laboratories, and regulatory agencies to improve the consistency with which HOS characterization is applied across the biopharmaceutical industry. [ABSTRACT FROM AUTHOR]

We have investigated the structure and activity of chicken egg-white lysozyme in aqueous solutions of two typical ionic liquids, 1-butyl-3-methylimidazolium chloride and ethylammonium nitrate, at cryogenic temperature. An increase in structural disorder due to the unfolding and a decrease in the α-helical structure of lysozyme were noticeable upon glass formation. However, a decrease in the structural stability after cooling was less than that before cooling. The secondary and tertiary structures showed good reversibility upon cooling to 77 K and then reverting back to ambient temperature. We discussed an influence of a cooling upon the structure in aqueous ionic liquid solutions. [ABSTRACT FROM AUTHOR]

Antibody aggregate is a common issue in therapeutic antibodies, which may compromise product efficacy and cause adverse effects. Antibody aggregate level is normally controlled in bioprocessing by polishing steps after Protein A capture. This paper studied the Higher Order Structures (HOS) of antibody aggregates (dimer H1 and H2) and their adsorption on Protein A resin and thus elucidated the mechanism using Protein A capture for enhanced aggregate removal. The HOS of antibody aggregates and their complex with Protein A were characterized using HDX-MS combined with SEC-MALS, Protein Conformational Array (PCA), and molecular modeling. The aggregate size and Protein A binding ratio suggested that H2 has much more compact structure than H1. HDX-MS and PCA further revealed that H1 was formed by single Fab-Fab interaction while H2 formed by Fab-Fab and likely Fc-Fc interaction. On Protein A resin, both the molar binding ratio and the correlation between protein size and ligand distance support that each monomer can only bind one Protein A ligand, while each dimer can bind two ligands, thus resulting in stronger resin binding. Furthermore, dimer H2 binds stronger than dimer H1 due to its compact structure. By integrating biophysical analysis and molecular modeling with process development, this study revealed the antibody aggregate structures and the mechanism of aggregate removal using Protein A chromatography. It also provided a general strategy for in-depth product and process understanding in antibody and other biologics development. [ABSTRACT FROM AUTHOR]

The peptide teriparatide, also known as parathyroid hormone (1-34), PTH(1-34), was developed for intranasal delivery, requiring extended stability of the reconstituted product for up to four weeks at room temperature. Lyophilized formulations of PTH(1-34), containing glycine and trehalose and using lactate as the buffer, are stable for months upon storage. However, the physical stability of the peptide after reconstitution unexpectedly varied considerably, depending on peptide concentration and storage temperature, with precipitation seen within two to four weeks in some samples. By comparison, equivalent samples that did not undergo lyophilization did not display any precipitation upon storage in the liquid state for as long as twelve weeks. PTH(1-34) appears to adopt a higher order structure that is perturbed by the combined stresses of freezing and drying, leading to greater propensity to aggregate, which is accentuated at higher peptide concentrations and at higher temperatures. The precipitation seems to be correlated with increased amounts of subvisible particles. This study shows the importance of peptide conformation in long-term stability and illustrates the ability of lyophilization to cause increased propensity to aggregate, even in a peptide. [ABSTRACT FROM AUTHOR]

This review highlights recent advancements in using high resolution nuclear magnetic resonance (NMR) spectroscopy as a characterization tool to expedite biologics formulation development, meeting a current need in the biopharmaceutical industry. Conformational changes of protein therapeutics during formulation development can result in various protein-protein and protein-excipient interactions, which can lead to physical aggregation and/or chemical degradation. Innovative analytical techniques that allow studying protein integrity with high specificity during formulation development are urgently needed in order to assess protein formulation stability and mitigate product quality risks. Solution NMR spectroscopy is emerging as a powerful analytical tool for biophysical characterization of protein therapeutics. For instance, one-dimensional (1D) NMR has been employed in high sensitivity monitoring of monoclonal antibody (mAb) structural changes and protein-excipient interactions in parenteral formulations, demonstrating it as a potential tool for formulation screening. 2D NMR, such as ALSOFAST-[1H–13C]-HMQC experiments, on the other hand, offer superior capability to detect higher order structural (HOS) changes of mAbs in formulated solutions and their interactions with excipients. These determinations need to be achieved in actual formulations, where proteins of natural abundance are typically at low concentrations depending on the actual dose regimen. Studying proteins with natural abundance in the presence of hundredfold more concentrated excipients makes NMR studies of proteins in formulations extremely difficult considering the sample matrix interferences. Thus, successfully suppressing buffer signals while enhancing the protein signals of interest by optimizing the instrument specific parameters is critically important. Given the large size of typical mAbs, with a molecular weight (MW) ranging from 100 to 240 kDa, coupled with low protein concentrations, data collection becomes a demanding task in terms of NMR instrument time. As such, the biopharmaceutical industry is facing the common challenge of developing innovative NMR approaches to enhance signal detection (sensitivity and selectivity) and reduce experimental/instrument time. XL-ALSOFAST -[1H–13C]-HMQC was recently developed for tackling high MW proteins (up to 240 kDa) with much improved sensitivity and selectivity. We at BMS have implemented the XL-ALSOFAST experiment utilizing its high sensitivity and superior artifact suppression to successfully analyze formulations of several investigational proteins. In this manuscript we will discuss the general utility of this superior tool for studying therapeutic proteins across a range of molecular sizes and buffers. We envisage that this manuscript will serve as a primer to expand the role of NMR spectroscopy as a characterization tool supporting biologics formulation development. • Recent advancements solution nuclear magnetic resonance (NMR) spectroscopy techniques useful in biologics formulation are reviewed. • Applications of NMR experiments to biologics formulation characterization are discussed. • NMR spectroscopy as a standard characterization tool for protein therapeutics is proposed. [ABSTRACT FROM AUTHOR]

Characterization of the higher order structure ( HOS) of biological products has been growing in importance in recent years. Scientists in the biopharmaceutical industry, academic researchers, and regulators are all increasingly aware of the critical role that HOS plays in maintaining the stability and intended biological function of biopharmaceutical products. We organized a consortium of scientists and researchers from industry and academic institutions to address how HOS data can be used most effectively to drive decisions during product development. In this commentary, we introduce the purpose, objectives, and scope of the consortium and then provide some brief points to consider in the context of characterizing HOS of biopharmaceutical products. Scientific advances in HOS analysis, as well as continued dialogue among academia, industry, and regulatory agencies will ensure that appropriate methodologies are used to inform technical decision-making during biopharmaceutical development. © 2015 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 104:1240-1245, 2015 [ABSTRACT FROM AUTHOR]

Protein therapeutics differ considerably from small molecule drugs because of the presence of higher order structure (HOS), post-translational modifications, inherent molecular heterogeneity, and unique stability profiles. At early stages of development, multiple molecular candidates are often produced for the same biological target. In order to select the most promising molecule for further development, studies are carried out to compare and rank order the candidates in terms of their manufacturability, purity, and stability profiles. This note reports a case study on the use of selected HOS characterization methods for candidate selection and the role of HOS data in identifying potential challenges that may be avoided by selecting the optimal molecular entity for continued development. © 2015 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 104:1533-1538, 2015 [ABSTRACT FROM AUTHOR]

With growing regulatory interest in ensuring the integrity and consistency of the higher order structure of biopharmaceutical and vaccine products, validated methods will be required that operate in quality-regulated environments and in compliance with guidelines such as those published by the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use [ICH]. Circular dichroism [CD] is a widely available, rapid and sensitive method to compare the secondary and tertiary structures of samples. For use in quality-regulated analyses multiple factors need to be considered. Firstly, a thorough understanding of the sources of experimental error is required, alongside protocols and reference standards for sample preparation, instrument calibration and performance monitoring. Secondly, validated algorithms to objectively compare spectra are required, with statistical evaluation to assess when values are significantly different and to define compliance criteria. Thirdly, regulatory authorities mandate the process of method assessment and validation to be acceptable for use in regulated environments. In this review I discuss regulatory expectation, the sources and magnitudes of instrument- and sample-derived uncertainties, the use of reference standards to define and correct instrument performance, and the algorithms used to objectively compare spectra. The ideas discussed here support the use of CD not only in quality-regulated environments but also best practice when used in a research environment. • Protein circular dichroism spectra report thigher order structure and its response to solution and structural changes. • Objective comparison of pharmaceuticals in quality regulated systems requires understanding the impact of spectral quality. • This review discusses sources of error, their magnitude and algorithms used to compare spectra from a regulatory viewpoint. [ABSTRACT FROM AUTHOR]

Abstract: The higher order structure of Mucor miehei lipase and micelle size in a cationic cetyltrimethylammonium bromide (CTAB) reverse micellar system was investigated. Circular dichroic (CD) measurement revealed that the lipase far-UV CD spectra changed markedly, going from buffer solution to the reverse micellar solution, and were very similar for any organic solvent used. The ellipticity of the solubilized lipase in the far-UV region markedly decreased with increasing water content (W 0: molar ratio of water to CTAB), indicating that the secondary structure of lipase changed with the water content. The linear correlation between the W 0 and the micelle size was obtained by measuring dynamic light scattering. From the linear correlation between the micelle size and W 0, the higher order structure of the solubilized lipase appears to be affected directly by the micellar interface. The species and concentration of alcohol as a cosurfactant had an inferior effect on lipase structure. Especially, at ratios of 1-pentanol to CTAB of less than 8, the secondary and tertiary structures of lipase were preserved in the reverse micelles. The CTAB concentration had little effect on the lipase structure in the micelles. The catalytic activity of the lipase solubilized in the CTAB reverse micelles increased with increasing the W 0. [Copyright &y& Elsevier]

Abstract: The higher order structure of proteins solubilized in an bis(2-ethylhexyl) sulfosuccinate sodium (AOT) reverse micellar system was investigated. From circular dichroic (CD) measurement, CD spectra of cytochrome c, which is solubilized at the interface of reverse micelles, markedly changed on going from buffer solution to the reverse micellar solution, and the ellipticity values in the far- and near-UV regions decreased with decreasing the water content (W0: molar ratio of water to AOT), indicating that the secondary and tertiary structures of cytochrome c changed with the water content. The ellipticity of ribonuclease A, which is solubilized in the center of micellar water pool, in the near-UV region was dependent on W0 and became minimum when W0 of ca. 8 while the ellipticity in the far-UV region was almost constant, indicating that the tertiary structure of ribonuclease A was affected by the water content, but the secondary structure was conserved. The degree of curvature of the micellar interface appears to influence the protein structure because the reverse micelle size is linearly proportional to the W0 value. As evidence of this, when the micelle size was comparable to the protein''s dimensions, the structures were more affected by the water content. Judging from the dependence of the factor influencing the protein structure on the protein species, the location of solubilized protein in reverse micelles is significantly related to whether the protein structure in the system is affected by the micellar interface. In the cases of cytochrome c and lysozyme, the ellipticity against W0 was dependent on the AOT concentration. In contrast, ribonuclease A gave very similar ellipticity values whatever the AOT concentration. In the n-hexane micellar system, cytochrome c exhibited lower ellipticity values and ribonuclease A in the lower W0 range (W0 < ca. 8) higher ellipticity values. These results indicated that the interaction between the protein and the micellar interface is a dominant factor influencing the protein structure in reverse micelles, and that it is governed by the location of solubilized proteins and the state of the micellar interface. [Copyright &y& Elsevier]

Pure bacterial homo poly(3-hydroxybutyrate) (PHB) fibers were prepared by melt spinning, followed by cold-drawing in an amorphous state at a temperature just above its glass transition temperature. Cold drawn fibers obtained were further drawn at higher temperatures, followed by annealing at various temperatures under tension. Relations among the processing conditions, higher order structures and mechanical properties were investigated using wide- and small-angle X-ray diffractions (WAXD and SAXD, respectively), birefringence, differential scanning calorimetry (DSC), and tensile measurements. PHB has two different crystalline forms, 21 helix conformation (α-form) and planar zigzag conformation (β-form). A single broad reflection of β-form was detected even in a PHB fiber drawn once at a temperature just above its Tg immediately after quenching and it tended to be stronger after 2nd drawing at higher temperatures. Annealing under low temperature and high tension facilitates the occurrence of β-form. It is suggested that the β-form crystal is formed not only from the tie chains between α-form lamella, but also from completely free amorphous chains. Changes in the amount of two types of crystals were analyzed using the WAXD integrated intensity. Birefringence of these fibers shows negative and positive values, depending on process conditions. Changes in higher order structure on the mechanical properties are also discussed. [Copyright &y& Elsevier]

• Trastuzumab and Rituximab originator and biosimilars have been assessed for comparability of conformational stability via changes in intrinsic fluorescence. • Rate of heating significantly affects melting profiles of trastuzumab and rituximab. • Reversible unfolding is observed in trastuzumab and rituximab at temperatures below first transition. • Use of nanoDSF as a rapid assessment tool for protein conformational and colloidal stability has been demonstrated. Differential scanning fluorimetry (DSF) or thermal shift has emerged in recent years as a high-throughput screening method in biotherapeutic formulation studies. The present article reports on a fast-track assessment platform for rapid investigation of therapeutic proteins such as monoclonal antibodies (mAb) with minimal sample concentration, volume, and preparation. The proposed nanoDSF platform has been demonstrated for rapid assessment of two commercial IgG 1 drug products (DP), trastuzumab and rituximab, and their biosimilars with respect to their conformational and colloidal stability. Domain specific differences for each of the IgGs have been elucidated with respect to onset of domain unfolding (T onset) and melting temperatures. These thermal unfolding and transition midpoint (T m) measurements are based on the intrinsic aromatic amino acid residue fluorescence of proteins. Moreover, to understand the possibility of nanoDSF as a predictive tool, data from nanoDSF has been correlated with accelerated stability studies. Melting temperatures across brands were found to be highly comparable to the rate of heating, thereby exhibiting a significant domain specific effect on melting temperatures for both trastuzumab and rituximab. Conservation of higher order structure (HOS) through reversible unfolding was also examined and both the mAbs were found to regain tertiary structure up till the first transition midpoint. No clear correlation was found between formation of higher molecular weight species (HMWS) and unfolding parameters (T onset and T agg) for accelerated stability studies. Finally, a discussion on the need for fast predictive assessment of conformation and colloidal stability as well as a comparison of advantages and limitations of the technique with routine/classical tools such as circular dichroism spectrophotometry and differential scanning calorimetry has been presented. [ABSTRACT FROM AUTHOR]

Protein therapeutics are vitally important clinically and commercially, with monoclonal antibody (mAb) therapeutic sales alone accounting for $115 billion in revenue for 2018.[1] In order for these therapeutics to be safe and efficacious, their protein components must maintain their high order structure (HOS), which includes retaining their three-dimensional fold and not forming aggregates. As demonstrated in the recent NISTmAb Interlaboratory nuclear magnetic resonance (NMR) Study[2], NMR spectroscopy is a robust and precise approach to address this HOS measurement need. Using the NISTmAb study data, we benchmark a procedure for automated outlier detection used to identify spectra that are not of sufficient quality for further automated analysis. When applied to a diverse collection of all 252 1H,13C gHSQC spectra from the study, a recursive version of the automated procedure performed comparably to visual analysis, and identified three outlier cases that were missed by the human analyst. In total, this method represents a distinct advance in chemometric detection of outliers due to variation in both measurement and sample. • Outlier classification for 2D NMR spectra of biopharmaceuticals is demonstrated. • Kullback-Liebler divergence is used as a measure of spectral similarity. • The procedure was demonstrated on 252 spectra from a recent international study. • The automated method identified three outliers missed by a human analyst. • The method could be re-purposed for other types of supervised classification. [ABSTRACT FROM AUTHOR]