A comprehensive strategy for the identification of biologics by liquid-chromatography-mass spectrometry for release testing in a regulated environment.

Identification (ID) testing is a regulatory requirement for biopharmaceutical manufacturing, requiring robust, GMP-qualified assays that can distinguish the therapeutic from any other in the facility. Liquid Chromatography-Mass Spectrometry (LC-MS) is a powerful analytical tool used to identify and characterize biologics. While routinely leveraged for characterization, LC-MS is relatively rare in Quality Control (QC) settings due to its perceived complexity and scarcity of MS-trained personnel. However, employing LC-MS for identification of drug products has many advantages versus conventional ID techniques, including but not limited to its high specificity, rapid turn-around time, and ease of method execution. In this work, we outline the development and implementation of a comprehensive LC-MS based ID strategy for biologics release testing. Two main workflows (WFs) were developed: i) WF1, a subunit-based assay measuring the molecular weight of the light chain (LC) and heavy chain (HC) of an antibody upon reduction, and ii) WF2, intact mass measurement of the biologic upon N-deglycosylation by PNGase F. The proposed strategy is shown to be applicable for over 40 diverse model biologics including monoclonal antibodies (mAbs), biobetters such as antibody prodrugs/afucosylated mAbs, fusion proteins, multi-specific antibodies, Fabs, and large peptides, all with excellent mass accuracy (error typically < 20 ppm) and precision. It requires a single-step sample preparation and a single click to run and process the data upon method setup. This strategy has been successfully implemented for release testing in GMP labs. Challenges and considerations for the establishment of QC-friendly methods are discussed. It is also shown that these methods can be applied to the ID of more analytically complex biotherapeutics, such as fixed-dose combination (FDC) and drug products co-formulated with trace-level additives.

Dual-detection approach for a charge variant analysis of monoclonal antibody combination products using imaged capillary isoelectric focusing.

The clinical benefits of treatments with a combination of two or more therapeutic monoclonal antibodies (mAbs) have emerged in recent years. Imaged capillary isoelectric focusing is a frequently used technology in the biopharmaceutical industry for charge variant analysis of protein therapeutics. However, with the wide concentration ranges of combination products, one component may fall within the linear detection range, whereas the other does not. Here, we report a novel methodology to explore charge variants of mAb mixtures using multiple detection techniques simultaneously. We use ultraviolet absorbance to monitor the charge variants of the high-concentration component and native fluorescence (FL) to monitor the variants of the low-concentration one. Charge variants of mixtures that span 40-fold in ratio differences can be accurately quantified with this approach. In contrast to the conventional methods, it is not necessary to prepare and analyze two samples at different concentrations and combine the results for combination product testing. Additionally, the use of FL detection enables the charge variant analysis of highly potent/low abundant mAbs in a mixture. This methodology is more quality-control friendly and efficient for the charge variant analysis of combination products with wide ratios.

Interactions between Soluble Species of ß-Amyloid and α-Synuclein Promote Oligomerization while Inhibiting Fibrillization.

Aggregations of ß-amyloid (Aß) and α-synuclein (αS) into oligomeric and fibrillar assemblies are the pathological hallmarks of Alzheimer's and Parkinson's diseases, respectively. Although Aß and αS affect different regions of the brain and are separated at the cellular level, there is evidence of their eventual interaction in the pathology of both disorders. Characterization of interactions of Aß and αS at various stages of their aggregation pathways could reveal mechanisms and therapeutic targets for the prevention and cure of these neurodegenerative diseases. In this study, we comprehensively examined the interactions and their molecular manifestations using an array of characterization tools. We show for the first time that αS monomers and oligomers, but not αS fibrils, inhibit Aß fibrillization while promoting oligomerization of Aß monomers and stabilizing preformed Aß oligomers via coassembly, as judged by Thioflavin T fluorescence, transmission electron microscopy, and SDS- and native-PAGE with fluorescently labeled peptides/proteins. In contrast, soluble Aß species, such as monomers and oligomers, aggregate into fibrils, when incubated alone under the otherwise same condition. Our study provides evidence that the interactions with αS soluble species, responsible for the effects, are mediated primarily by the C-terminus of Aß, when judged by competitive immunoassays using antibodies recognizing various fragments of Aß. We also show that the C-terminus of Aß is a primary site for its interaction with αS fibrils. Collectively, these data demonstrate aggregation state-specific interactions between αS and Aß and offer insight into a molecular basis of synergistic biological effects between the two polypeptides.

Amyloid Aggregation of Bacillus circulans Xylanase under Native Conditions and its Modulation by ß-Amyloid-Derived Peptide Fragments.

The aggregation of intrinsically disordered proteins into fibrils is implicated in many neurodegenerative diseases. Amyloid aggregation is a generic property of proteins as evidenced by globular proteins that often form amyloid aggregates under partially denaturing conditions. Recently, multiple lines of evidence have suggested that the amyloid aggregation of globular proteins can also occur under native conditions. Unfortunately, amyloid aggregation under native conditions has been demonstrated in only a handful of cases. Engineering a globular protein's amyloid aggregation might benefit from its fusion to an amyloid-derived fragment with reduced aggregation propensity. Unfortunately, the impacts of such fragments on the amyloid aggregation under native conditions have yet to be examined. In this study, we show that a globular protein, Bacillus circulans xylanase (BCX), can aggregate to form amyloid fibrils under native conditions. When BCX was mixed with or fused to the non-self-aggregating fragments, KLVFWAK and ELVFWAE-which were derived from ß-amyloid (Aß)-they modulated the BCX amyloid aggregation to differing extents. This study also provides insight into a correlation between the kinetic stability and amyloid aggregation of BCX, and supports a view that Aß-derived fragments can be useful for the modulating amyloid aggregation of some, though not all, proteins.

Hetero-assembly of a dual ß-amyloid variant peptide system.

Self-assembly of amyloid polypeptides (1) imparts biological effects depending on the size in over 20 amyloid diseases and (2) produces useful yet relatively untapped biomaterials. Unfortunately, our understanding of amyloid polypeptides, as related to biomedical implications and biomaterial applications, is limited by their self-assembling nature. In this study, we report the creation of a dual peptide system, where a pair of ß-amyloid (Aß) variants are not self-assembled but hetero-assembled in the presence of their assembly partners. We provide evidence that the resulting hetero-assemblies share molecular, structural and morphological similarities with typical amyloid self-assemblies formed by a single polypeptide (e.g., Aß). We anticipate that our dual peptide system may readily be adapted for precise control of amyloid assembly, for the study of size-dependent neurotoxicity and precise fabrication of amyloid biomaterials.

Metabolic engineering of sugarcane to accumulate energy-dense triacylglycerols in vegetative biomass.

Elevating the lipid content in vegetative tissues has emerged as a new strategy for increasing energy density and biofuel yield of crops. Storage lipids in contrast to structural and signaling lipids are mainly composed of glycerol esters of fatty acids, also known as triacylglycerol (TAG). TAGs are one of the most energy-rich and abundant forms of reduced carbon available in nature. Therefore, altering the carbon-partitioning balance in favour of TAG in vegetative tissues of sugarcane, one of the highest yielding biomass crops, is expected to drastically increase energy yields. Here we report metabolic engineering to elevate TAG accumulation in vegetative tissues of sugarcane. Constitutive co-expression of WRINKLED1 (WRI1), diacylglycerol acyltransferase1-2 (DGAT1-2) and oleosin1 (OLE1) and simultaneous cosuppression of ADP-glucose pyrophosphorylase (AGPase) and a subunit of the peroxisomal ABC transporter1 (PXA1) in transgenic sugarcane elevated TAG accumulation in leaves or stems by 95- or 43-fold to 1.9% or 0.9% of dry weight (DW), respectively, while expression or suppression of one to three of the target genes increased TAG levels by 1.5- to 9.5-fold. Accumulation of TAG in vegetative progeny plants was consistent with the results from primary transgenics and contributed to a total fatty acid content of up to 4.7% or 1.7% of DW in mature leaves or stems, respectively. Lipid droplets were visible within mesophyll cells of transgenic leaves by confocal fluorescence microscopy. These results provide the basis for optimizations of TAG accumulation in sugarcane and other high yielding biomass grasses and will open new prospects for biofuel applications.

Engineering of a peptide probe for ß-amyloid aggregates.

Aggregation of ß-amyloid (Aß) is central to the pathogenesis of Alzheimer's disease (AD). Aß aggregation produces amyloid assemblies, such as oligomers and fibrils. In contrast to non-toxic Aß monomers, Aß oligomers and fibrils can act directly as major toxic agents and indirectly as pools of the toxic entities, respectively. Thus, the detection of Aß aggregates is of diagnostic interest and should benefit enhanced molecular understanding of AD. Among many molecular platforms, peptide-based ligands hold promise as Aß probes due to their relative simplicity, ease of optimization and facile conjugation to other molecular contexts. In this regard, Aß hydrophobic segments (critical in Aß self-assembly) or variants thereof can serve as lead molecules for Aß probe development. Unfortunately, the resulting peptides are either highly self-aggregation-prone or their probe potential has not been thoroughly examined. In the present study, we characterized a novel peptide ligand, KLVFWAK, which was created by simple point mutations of an Aß hydrophobic segment ((16)KLVFFAE(22)). We found that KLVFWAK displayed low self-aggregation propensity and was preferentially bound to Aß oligomers and fibrils relative to Aß monomers. Interestingly, binding of KLVFWAK to Aß aggregates occurred at a non-homologous Aß segment (e.g., Aß C-terminal domain) rather than the homologous (16)KLVFFAE(22). We also show that detection of Aß aggregates during incubation of fresh Aß was possible with KLVFWAK, further supporting KLVFWAK's high probe potential for Aß aggregates. In short, this study presents creation of a non-self-aggregating peptide ligand for Aß aggregates through simple point mutation of an Aß-derived segment.

Survey of the total fatty acid and triacylglycerol composition and content of 30 duckweed species and cloning of a Δ6-desaturase responsible for the production of γ-linolenic and stearidonic acids in Lemna gibba.

BACKGROUND: Duckweeds, i.e., members of the Lemnoideae family, are amongst the smallest aquatic flowering plants. Their high growth rate, aquatic habit and suitability for bio-remediation make them strong candidates for biomass production. Duckweeds have been studied for their potential as feedstocks for bioethanol production; however, less is known about their ability to accumulate reduced carbon as fatty acids (FA) and oil. RESULTS: Total FA profiles of thirty duckweed species were analysed to assess the natural diversity within the Lemnoideae. Total FA content varied between 4.6% and 14.2% of dry weight whereas triacylglycerol (TAG) levels varied between 0.02% and 0.15% of dry weight. Three FA, 16:0 (palmitic), 18:2Δ9,12 (Linoleic acid, or LN) and 18:3Δ9,12,15 (α-linolenic acid, or ALA) comprise more than 80% of total duckweed FA. Seven Lemna and two Wolffiela species also accumulate polyunsaturated FA containing Δ6-double bonds, i.e., GLA and SDA. Relative to total FA, TAG is enriched in saturated FA and deficient in polyunsaturated FA, and only five Lemna species accumulate Δ6-FA in their TAG. A putative Δ6-desaturase designated LgDes, with homology to a family of front-end Δ6-FA and Δ8-spingolipid desaturases, was identified in the assembled DNA sequence of Lemna gibba. Expression of a synthetic LgDes gene in Nicotiana benthamiana resulted in the accumulation of GLA and SDA, confirming it specifies a Δ6-desaturase. CONCLUSIONS: Total accumulation of FA varies three-fold across the 30 species of Lemnoideae surveyed. Nine species contain GLA and SDA which are synthesized by a Δ6 front-end desaturase, but FA composition is otherwise similar. TAG accumulates up to 0.15% of total dry weight, comparable to levels found in the leaves of terrestrial plants. Polyunsaturated FA is underrepresented in TAG, and the Δ6-FA GLA and SDA are found in the TAG of only five of the nine Lemna species that produce them. When present, GLA is enriched and SDA diminished relative to their abundance in the total FA pool.