Automated method for quantification of 20 amino acids in cell culture media during biopharmaceutical development.

Herein, a step-by-step protocol for simultaneous detection of 20 amino acids commonly present in cell culture media is described. The protocol facilitates detection of both primary and secondary amino acids through a two-step precolumn derivatization strategy using ortho-phthalaldehyde and 9-fluorenylmethyl chloroformate as derivatizing agents. The separation of derivatized amino acids with varying hydrophobicity is achieved through reverse-phase chromatography. The amino acids are simultaneously detected in a single workflow through the use of Variable Wavelength Detector at 338 and 262 nm. The protocol is applicable for both mammalian and bacterial cell culture matrices with an option for automation of precolumn derivatization.

Profiling Enzyme Activity of l-Asparaginase II by NMR-Based Methyl Fingerprinting at Natural Abundance.

l-asparaginase II (MW 135 kDa) from E. coli is an FDA-approved protein drug used for the treatment of childhood leukemia. Despite its long history as a chemotherapeutic, the structural basis of enzyme action, in solution, remains widely contested. In this work, methyl-based 2D [1H-13C]-heteronuclear single-quantum correlation (HSQC) NMR, at natural abundance, has been used to profile the enzymatic activity of the commercially available enzyme drug. The [1H-13C]-HSQC NMR spectra of the protein reveal the role of a flexible loop segment in the activity of the enzyme, in solution. Addition of asparagine to the protein results in distinct conformational changes of the loop that could be signatures of intermediates formed in the catalytic reaction. To this end, an isothermal titration calorimetry (ITC)-based assay has been developed to measure the enzymatic reaction enthalpy, as a marker for its activity. Combining both ITC and NMR, it was shown that the disruption of the protein conformation can result in the loss of function. The scope, robustness, and validity of the loop fingerprints in relation to enzyme activity have been tested under different solution conditions. Overall, our results indicate that 2D NMR can be used reliably to gauge the structure-function of this enzyme, bypassing the need to label the protein. Such natural abundant NMR methods can be potentially extended to probe the structure-function aspects of high-molecular-weight protein therapeutics (glycosylated protein drugs, enzymes, therapeutic monoclonal antibodies, antibody-drug conjugates, and Fc-fusion proteins), where (a) flexible loops are required for their function and (b) isotope labeling may not be straightforward.

A native multi-dimensional monitoring workflow for at-line characterization of mAb titer, size, charge, and glycoform heterogeneities in cell culture supernatant.

With growing maturity of the biopharmaceutical industry, new modalities entering the therapeutic design space and increasing complexity of formulations such as combination therapy, the demands and requirements on analytical workflows have also increased. A recent evolution in newer analytical workflows is that of multi-attribute monitoring workflows designed on chromatography-mass spectrometry (LC-MS) platform. In comparison to traditional one attribute per workflow paradigm, multi-attribute workflows are designed to monitor multiple critical quality attributes through a single workflow, thus reducing the overall time to information and increasing efficiency and throughput. While the 1st generation multi-attribute workflows focused on bottom-up characterization following peptide digestion, the more recent workflows have been focussing on characterization of intact biologics, preferably in native state. So far intact multi-attribute monitoring workflows suitable for comparability, utilizing single dimension chromatography coupled with MS have been published. In this study, we describe a native multi-dimensional multi-attribute monitoring workflow for at-line characterization of monoclonal antibody (mAb) titer, size, charge, and glycoform heterogeneities directly in cell culture supernatant. This has been achieved through coupling ProA in series with size exclusion chromatography in 1st dimension followed by cation exchange chromatography in the 2nd dimension. Intact paired glycoform characterization has been achieved through coupling 2D-LC with q-ToF-MS. The workflow with a single heart cut can be completed in 25 mins and utilizes 2D-liquid chromatography (2D-LC) to maximize separation and monitoring of titer, size as well as charge variants.

Taking the individual bias out of examining comparability of biosimilars: A case study on monoclonal antibody therapeutics.

Biosimilar manufacturers need to perform analytical and functional similarity assessments against the reference product. Successful demonstration allows for an abbreviated clinical path, thereby translating to affordable biosimilars. Current practices for regulatory concurrence on analytical similarity data are based on chart visualization and open to individual (human) bias. Here, we present a novel, chemometric approach for assessing biosimilarity that aims to simplify assessment and eliminate individual bias from decision making through application of weighted principal component analysis. Through the proposed approach, chemical information across the analytical characterization platform and drug products can be collated into a single plot for quantitative biosimilarity assessment. The proposed one-plot analysis offers a holistic visualization of 1) inter-product variability (w.r.t reference product) in cases where multiple batches per product have been investigated and 2) intra-product variability for each critical quality attribute (CQA) wherein information from orthogonal tools can be incorporated within the same plot. This allows for numerical grading of similarity for biosimilars of any given reference product. Although the proposed statistical approach is novel, it builds on standardized measures of CQA, criticality, and analytical procedures, thus making this approach easy to incorporate within the existing regulatory framework.

Ion exchange chromatography hyphenated with fluorescence detector as a sensitive alternative to UV detector: Applications in biopharmaceutical analysis.

Cation-exchange chromatography (CEX) is widely used for analysis of charge heterogeneity in monoclonal antibodies (mAbs) in the biopharmaceutical industry. Charge variant separation is typically achieved by either alteration of ionic strength or pH, and detection of the various species is performed using diode array detectors (DAD) at 280 nm. In this study, we investigate the suitability of fluorescence detector (FLD) as a sensitive alternative to traditional UV280 detection-based charge variant analysis. The objective of this work is to compare the performance of charge variants detection through cation exchange chromatography between FLD and DAD detectors. This was demonstrated for a mass spectroscopy compatible, pH based CEX method utilizing volatile ammonium acetate salt. The stability of FLD detection was tested as per method validation criteria set in the ICH Q2-(R1). LODFLD and LOQFLD were 59.07 and 59.64 times lower in comparison to LODUV and LOQUV, respectively (LODFLD: 0.13 µg, LOQFLD: 0.39 µg). The LinearityFLD was obtained in the range of 1-200 µg. Accuracy calculated as recovery value was between 93.38% and 103.35% and highest RSD value obtained for robustness was 2.54% (area under the curve). The application of FLD detection based CEX as a Process Analytical Technology (PAT) enabler in mAb biopharmaceutical analysis was demonstrated through direct charge variant profiling of CHO cell harvest supernatant (without pre purification through Pro-A). The method was also applied to and found suitable for biosimilarity assessment of drug products. While the present study is focused on analysis of trastuzumab biosimilars, the proposed method is expected to be applicable to any mAb product after suitable gradient modification.

N-Glycosylation of monoclonal antibody therapeutics: A comprehensive review on significance and characterization.

N-glycosylation of therapeutic antibodies starts as a co-translational step followed by a set of post-translational modifications and is considered as one of the critical quality attributes because of its impact on biological functions as well as therapy outcome. In addition to detailed product characterization of these glycans by the manufacturers, their comprehensive analysis is also a regulatory requirement. However, the structural complexity and heterogeneity of these N-linked carbohydrates make their characterization quite challenging. In this review, we give a comprehensive overview of N-glycosylation diversity and its functional importance for monoclonal antibody therapeutic products. A descriptive coverage is also provided for the various strategies and techniques employed for oligosaccharide characterization, that include analysis of intact-glycoproteins, their sub-units, glycopeptides as well as released glycans through chromatographic, electrophoretic and spectroscopic techniques. To assist the readers, relevant examples from the literature are cited and critically discussed for each of the strategies and techniques. To conclude, a discussion on unique challenges associated with the analysis of this important post-translational modification is presented.

NMR based quality evaluation of mAb therapeutics: A proof of concept higher order structure biosimilarity assessment of trastuzumab biosimilars.

Higher-order structural (HOS) biosimilarity assessment is a regulatory requirement for intended biosimilars, and manufacturers are required to demonstrate the biosimilarity of their product in comparison to the reference product concerning both safety and efficacy. NMR has recently emerged as a powerful technique for complex biologics such as mAbs that offers holistic Higher-order structure (HOS) assessment. In this paper, a proof-of-concept for similarity assessment for marketed biosimilars of trastuzumab has been presented using both 1Dimensional (1H NMR) and 2Dimensional 1H-13C-methyl correlated NMR techniques. Samples were prepared without sample buffer exchange in the presence of excipients and assessed at natural abundance. Data were recorded using a 750 MHz NMR spectrometer equipped with a room temperature probe. Both visual and statistical assessment was performed to test biosimilarity. 1H- NMR was useful for the assessment of overall comparability with sensitivity towards changes in the formulation components, whereas 13C- 1H methyl correlation-based assessment indicated structural biosimilarity. All marketed biosimilars were found to be within the range established by originator batches used in this study. Using 1D and 2D NMR techniques, we aim to provide a perspective on the requirements and challenges of HOS biosimilarity assessments to be conducted for the drug product as a whole, inclusive of excipients as opposed to drug substance alone in buffer exchanged conditions.

Analytical Similarity Assessment of Biosimilars: Global Regulatory Landscape, Recent Studies and Major Advancements in Orthogonal Platforms.

Biopharmaceuticals are one of the fastest-growing sectors in the biotechnology industry. Within the umbrella of biopharmaceuticals, the biosimilar segment is expanding with currently over 200 approved biosimilars, globally. The key step towards achieving a successful biosimilar approval is to establish analytical and clinical biosimilarity with the innovator. The objective of an analytical biosimilarity study is to demonstrate a highly similar profile with respect to variations in critical quality attributes (CQAs) of the biosimilar product, and these variations must lie within the range set by the innovator. This comprises a detailed comparative structural and functional characterization using appropriate, validated analytical methods to fingerprint the molecule and helps reduce the economic burden towards regulatory requirement of extensive preclinical/clinical similarity data, thus making biotechnological drugs more affordable. In the last decade, biosimilar manufacturing and associated regulations have become more established, leading to numerous approvals. Biosimilarity assessment exercises conducted towards approval are also published more frequently in the public domain. Consequently, some technical advancements in analytical sciences have also percolated to applications in analytical biosimilarity assessment. Keeping this in mind, this review aims at providing a holistic view of progresses in biosimilar analysis and approval. In this review, we have summarized the major developments in the global regulatory landscape with respect to biosimilar approvals and also catalogued biosimilarity assessment studies for recombinant DNA products available in the public domain. We have also covered recent advancements in analytical methods, orthogonal techniques, and platforms for biosimilar characterization, since 2015. The review specifically aims to serve as a comprehensive catalog for published biosimilarity assessment studies with details on analytical platform used and critical quality attributes (CQAs) covered for multiple biotherapeutic products. Through this compilation, the emergent evolution of techniques with respect to each CQA has also been charted and discussed. Lastly, the information resource of published biosimilarity assessment studies, created during literature search is anticipated to serve as a helpful reference for biopharmaceutical scientists and biosimilar developers.

Unexplored Excipients in Biotherapeutic Formulations: Natural Osmolytes as Potential Stabilizers Against Thermally Induced Aggregation of IgG1 Biotherapeutics.

Monoclonal antibodies (mAbs), while incredibly successful, are prone to a variety of degradation pathways, the most significant of which is aggregation. One of the most commonly used strategy to overcome protein aggregation is addition of excipients to the formulation. Osmolytes such as trehalose, sucrose, and glycine are widely used. In this paper, we explore potential use of naturally occurring osmolytes such as betaine, sarcosine, ectoine, and hydroxyectoine for reducing aggregation of mAb therapeutics. Experimentation has been performed on two IgG1 mAbs via accelerated stability studies. A variety of analytical tools have been used for monitoring the impact, dynamic light scattering (DLS) for colloidal stability, Fourier transform infrared (FTIR) spectroscopy and fluorescence spectroscopy for conformational stability and the higher order structure (HOS), and differential scanning calorimetry (DSC) for thermal stability. No significant impact of osmolyte addition was observed on protein structure, on comparative Fc receptor (FcRn) binding, and on biocompatibility as per our hemolytic assay. Our results rank the osmolytes' stabilizing trend to be sarcosine > betaine > hydroxyectoine > ectoine. Sarcosine emerged as the most successful osmolyte rendering highest degree of protection against aggregation. Our data support the prospect of using these osmolytes as successful excipients for mAb formulations.

Monitoring size and oligomeric-state distribution of therapeutic mAbs by NMR and DLS: Trastuzumab as a case study.

Monoclonal antibodies (mAbs) are the modalities of choice for immunotherapy. This class of products are known to exhibit considerable heterogeneity with respect to size, aggregation states, and charge. This makes it challenging for biopharmaceutical manufacturers, in particular biosimilar producers, to maintain consistency in product quality. In order to fingerprint these biotherapeutic products, multiple, high-resolution analytical tools are used to characterize the numerous critical quality attributes. Recently, there has been growing interest in enhancing adaptability of 1D and 2D NMR platforms for characterization of higher order structure with emphasis on 1D 1H, 2D 1H-15N and 1H-13C NMR experiments at natural abundance. In this communication, we report the applicability of 2D-DOSY NMR for quantification of colloidal diffusivities, namely diffusion coefficient (and associated hydrodynamic radius) for monomeric IgG1 mAb formulations at physiological conditions. Similarity assessment has been performed for trastuzumab originator (multiple batches) and marketed biosimilars to showcase the applicability of this approach. While dynamic light scattering measurements are known to be sensitive to presence of larger particles with a concentration dependence for estimation of colloidal diffusivities, size estimated by NMR experiments was found to be more in agreement with the computational hydrodynamic size estimations derived from the published crystal structures of intact mAb at formulation concentration.

Identification and characterization of carbonylation sites in trastuzumab biosimilars.

Detection of metal catalyzed carbonylation in proteins is traditionally based on derivatization followed by detection and quantification via spectroscopy or immunodetection. However, these measure only cumulative carbonylation and do not identify the specific sites of modification within the protein. Recently, fluorescein thiosemicarbazide (FTC) based semi-microplate method was adapted for high throughput monitoring of carbonyl content during mAb process development, using size-exclusion chromatography followed by ultraviolet and fluorescence detection. Here, we have examined carbonylation in originators and 4 biosimilars of an IgG1 therapeutic monoclonal antibody, trastuzumab, a first line of therapy for HER2 positive breast cancer. The hyphenated RP-ESI-MS/MS detection was able to identify the location of each of the carbonylated amino acids for all products. The result is a comprehensive map of a total of 27 unique identified carbonylation sites of trastuzumab found across multiple batches of originator as well as marketed biosimilars. Our results demonstrate that although the different carbonylation sites are spread across different domains throughout the mAb sequence, the complementarity determining regions (CDRs) are free of carbonylation and all identified sites lie within the framework region of the variable domain. Moreover, the constant- heavy domain 3 (CH3) region seems to be particularly resistant to process induced carbonylation.

Correction to: Assessment of Structural and Functional Comparability of Biosimilar Products: Trastuzumab as a Case Study.

Page 215, Section "3.1.2 Primary Structure: Peptide Mapping, Posttranslational Modification.

An application of Nano Differential Scanning Fluorimetry for Higher Order Structure assessment between mAb originator and biosimilars: Trastuzumab and Rituximab as case studies.

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 (Tonset) and melting temperatures. These thermal unfolding and transition midpoint (Tm) 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 (Tonset and Tagg) 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.

Structure-Based Design of Small Peptide Ligands to Inhibit Early-Stage Protein Aggregation Nucleation.

We report a structure-based approach to design peptides that can bind to aggregation-prone, partially folded intermediates (PFI) of insulin, thereby inhibiting early stages of aggregation nucleation. We account for the important role of the modular architecture of protein-protein binding interfaces and tertiary structure heterogeneity of the PFIs in the design of peptide inhibitors. The determination of association hotspots revealed that two interface segments are required to capture majority contribution to insulin homodimer binding energy. The selection of peptides that will have a high probability to inhibit insulin self-association was done on the basis of similarity in binding interface coverage of PFI residues in the peptide-PFI complex and the native-PFI dimer. Data on aggregate growth rate and secondary structure for formulations incubated under amyloidogenic conditions show that designed peptides inhibit insulin aggregation in a concentration-dependent manner. The mechanism of aggregation inhibition was probed by determining the enthalpy of peptide-insulin binding and peptide micellization using isothermal titration calorimetry. Finally, the effect of designed peptides on insulin activity was quantified using a spectrophotometric assay for glucose uptake by HepG2 cells.

Assessment of Structural and Functional Comparability of Biosimilar Products: Trastuzumab as a Case Study.

BACKGROUND: Biotherapeutics are protein products generated using recombinant DNA technology and manufactured in prokaryotic or eukaryotic cells. It is often said that "the process is the product" and thereby the effect of the manufacturing process is etched on the final product in the form of its heterogeneity. For any biotherapeutic, the acceptable range of the critical quality attributes is defined based on the expected impact of a specific variation on the product stability, safety, and efficacy. For a biosimilar to receive regulatory approval, the manufacturer must demonstrate analytical and clinical comparability with the originator product. As this is mandatory, every biosimilar manufacturer performs this exercise for each biosimilar product under development. However, few reports of thorough evaluation of the quality of biosimilar products are available in the literature. OBJECTIVE: We examined the structural and functional comparability of biosimilars of trastuzumab, a humanized monoclonal antibody biotherapeutic. The originator product, Herclon (Roche), was compared with four marketed biosimilars: Trasturel from Reliance Life Sciences, Canmab from Biocon, Vivitra from Zydus Ingenia, Hertraz from Mylan. METHODS: Structural comparability was established using mass spectrometry and spectroscopic techniques such as Fourier transform infrared spectroscopy, differential light scattering, circular dichroism, and fluorescence spectroscopy. Stability was compared by performing accelerated thermal stress studies. Functional comparability was established via surface plasmon resonance and biological assays such as antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity. RESULTS: With respect to comparability, one biosimilar exhibited significant differences in multiple attributes, such as lower percentage of monomer content and main charge variant species, lower percentage of aglycosylated glycoform G0, and lower estimated potency values. CONCLUSIONS: Overall, the results indicated general similarity with respect to structure and function, but we found variations with respect to size heterogeneity, charge heterogeneity, and glycosylation pattern in each of the biosimilars.

Selenium treatment differentially affects sulfur metabolism in high and low glucosinolate producing cultivars of broccoli (Brassica oleracea L.).

The effect of selenium (Se) application on the sulfur (S)-rich glucosinolate (GSL)-containing plant, broccoli (Brassica oleracea L. var. italica) was examined with a view to producing germplasm with increased Se and GSL content for human health, and to understanding the influence of Se on the regulation of GSL production. Two cultivars differing in GSL content were compared. Increased Se application resulted in an increase in Se uptake in planta, but no significant change in total S or total GSL content in either cultivar. Also no significant change was observed in the activity of ATP sulfurylase (ATPS, EC 2.7.7.4) or O-acetylserine(thiol) lyase (OASTL, EC 2.5.1.47) with increased Se application. However, in the first investigation of APS kinase (APSK, EC 2.7.1.25) expression in response to Se fertilisation, an increase in transcript abundance of one variant of APS kinase 1 (BoAPSK1A) was observed in both cultivars, and an increase in BoAPSK2 transcript abundance was observed in the low GSL producing cultivar. A mechanism by which increased APSK transcription may provide a means of controlling the content of S-containing compounds, including GSLs, following Se uptake is proposed.

Genotypic variation in sulfur assimilation and metabolism of onion (Allium cepa L.) III. Characterization of sulfite reductase.

Genomic and cDNA sequences corresponding to a ferredoxin-sulfite reductase (SiR) have been cloned from bulb onion (Allium cepa L.) and the expression of the gene and activity of the enzyme characterized with respect to sulfur (S) supply. Cloning, mapping and expression studies revealed that onion has a single functional SiR gene and also expresses an unprocessed pseudogene (φ-SiR). Northern and qPCR analysis revealed differences in expression pattern between the SiR gene and the pseudogene. Western analysis using antibodies raised to a recombinant SiR revealed that the enzyme is present in chloroplasts and phylogenetic analysis has shown that the onion protein groups with lower eudicots. In hydroponically-grown plants, levels of SiR transcripts were significantly higher in the roots of S-sufficient when compared with S-deficient plants of the pungent cultivar 'W202A' but not the less pungent cultivar 'Texas Grano'. In these same treatments, a higher level of enzyme activity was observed in the S-sufficient treatment in leaves of both cultivars before and after bulbing. In a factorial field trial with and without sulfur fertilization, a statistically significant increase in SiR activity was observed in the leaves of the pungent cultivar 'Kojak' in response to added S but not in the less pungent cultivar 'Encore'.