A demonstration of analytical similarity comparing a proposed biosimilar pegfilgrastim and reference pegfilgrastim.

BACKGROUND: Recombinant human granulocyte-colony stimulating factor (G-CSF, filgrastim) is used primarily to reduce incidence and duration of severe neutropenia and its associated complications in cancer patients that have received a chemotherapy regimen. The pegylated form of filgrastim, "pegfilgrastim", is a long-acting form that requires only a once-per-cycle administration for the management of chemotherapy-induced neutropenia. Apobiologix, a division of ApoPharma USA, Inc., and Intas Pharmaceuticals Limited have co-developed a proposed pegfilgrastim biosimilar to US-licensed pegfilgrastim. METHODS: The analytical similarity of Apobiologix pegfilgrastim and US-licensed pegfilgrastim with respect to their physicochemical profile was established using a wide range of rigorous orthogonal analytical techniques. Biological function was compared using receptor binding analyses, in vitro proliferation assays, and in vivo hematopoietic progenitor mobilization. RESULTS: Apobiologix pegfilgrastim and the US-licensed pegfilgrastim reference product were found to be highly similar analytically with respect to molecular mass, primary, secondary and tertiary protein structures, purity, charge, and hydrophobicity. No differences in receptor binding affinity were observed, and all samples demonstrated similar in vitro and in vivo bioactivity. CONCLUSION: These studies provide robust evidence supporting the structural and functional similarity between Apobiologix pegfilgrastim and the US-licensed reference pegfilgrastim, and hence their biosimilarity.

Aggregation of human plasma and of human blood induced in vitro by filgrastim originator product; effect of PEGylation.

We herein report that filgrastim product Neupogen® and the filgrastim formulation buffer induced aggregate formation when mixed in vitro with human plasma, and formation of large membranous erythrocyte aggregates when mixed with human blood, similar to the aggregation induced by pegfilgrastim and by pegfilgrastim buffer [T. Arvinte, E. Poirier, N. Ersayin, G. Darpin, A. Cudd, J. Dowd, S. Brokx, Aggregation of human plasma and of human blood induced in vitro by pegfilgrastim originator formulation buffer and pegfilgrastim products, Eur. J. Pharmaceut. Biopharmaceut. (2023), doi: 10.1016/j.ejpb.2023.10.019]. The data identify the filgrastim buffer (which is practically the same in filgrastim and pegfilgrastim products) as the main driver of human plasma and blood aggregation. Kinetic experiments showed differences in the extent of plasma aggregation induced by a filgrastim product manufactured in EU and one manufactured in USA. Human donor variability in the plasma aggregation induced by filgrastim was observed. To study the effect of PEGylation of the filgrastim protein on plasma aggregation we compared filgrastim (Neupogen®) with pegfilgrastim (Neulasta®) solutions at the same protein concentration. These data show that PEGylation has a beneficial effect in inhibiting to an extent plasma aggregation. Interestingly, 20 kDa polyethylene glycol in the filgrastim buffer induced more plasma aggregation compared to the buffer, similar to the aggregation induced by pegfilgrastim. For intravenous infusion filgrastim solutions (300 µg/ml, vials only) may be diluted in 5 % dextrose from a concentration of 300 µg/ml to 5 µg/ml. Aggregation of human plasma was also induced by filgrastim solutions diluted in 5 % dextrose to 50 µg/ml, 15 µg/ml and 5 µg/ml filgrastim, as well as by the filgrastim buffer similarly diluted in 5 % dextrose (1:6, 1:20 and 1:60 dilution). These data show that filgrastim solutions used for intravenous administration in patients induce human plasma aggregation in vitro. Such aggregation phenomena may be related to known infusion side effects of filgrastim therapy.

Extracellular accumulation of recombinant proteins fused to the carrier protein YebF in Escherichia coli.

Bacterial protein secretion is important in the life cycles of most bacteria, in which it contributes to the formation of pili and flagella and makes available extracellular enzymes to digest polymers for nutritional purposes and toxins to kill host cells in infections of humans, animals and plants. It is generally accepted that nonpathogenic laboratory strains of Escherichia coli, particularly K12 strains, do not secrete proteins into the extracellular medium under routine growth conditions. In this study, we report that commonly used laboratory strains secrete YebF, a small (10.8 kDa in the native form), soluble endogenous protein into the medium, challenging the status quo view that laboratory strains do not secrete proteins to the medium. We further show that 'passenger' proteins linked to the carboxyl end of YebF are efficiently secreted. The function of YebF is unknown, but its use as a carrier for transgenic proteins provides a tool to circumvent toxicity and other contamination issues associated with protein production in E. coli.

Part 2: Physicochemical characterization of bevacizumab in 2 mg/mL antibody solutions as used in human i.v. administration: Comparison of originator with a biosimilar candidate.

The physicochemical properties of Avastin® manufactured in the USA (Originator USA) and in Europe (Originator EU) and ABX-BEV, a bevacizumab biosimilar drug product candidate produced by Apobiologix Inc., were characterized at a clinically relevant concentration of 2 mg/mL following dilution of the 25 mg/mL drug products with 0.9% NaCl. Measurements using 14 orthogonal analytical methods performed within 5 h after dilution showed good similarity of the three antibodies as regards secondary structure, conformation, aggregation properties, subvisible and visible particles. No significant protein aggregation was observed within 5 h at 24 °C in the 2 mg/mL bevacizumab solutions. The same solutions that were measured within 5 h after dilution were analyzed again after 24 h overnight refrigeration at 2-8 °C: all 2 mg/mL bevacizumab solutions showed an increase in the number and sizes of aggregates, especially the particles larger than 10 µm and 25 µm. The data show the very good similarity in the physicochemical properties of ABX-BEV with Originators USA and EU at a clinically relevant concentration.

Part 1: Physicochemical characterization of bevacizumab in undiluted 25 mg/mL drug product solutions: Comparison of originator with a biosimilar candidate.

The biosimilarity assessment of the physicochemical properties of high-concentration biopharmaceuticals is usually performed with measurements on diluted solutions, at concentrations below 1 mg/mL. In this study 13 orthogonal, spectroscopy and particle size determination methods were used to characterize the structure and aggregation of undiluted, 25 mg/mL bevacizumab drug products Avastin® manufactured in the USA and in Europe, and ABX-BEV, a bevacizumab biosimilar candidate produced by Apobiologix Inc. Secondary structure, conformation and the potential occurrence of chemical degradation of the monoclonal antibodies were characterized and compared using infrared spectroscopy, intrinsic fluorescence and ANS fluorescence spectroscopy. Protein aggregation and particulate matter in the monoclonal antibody solutions were compared using UV-Vis absorbance, 90° light-scattering, nanoparticle tracking analysis, Nile red fluorescence microscopy, particle flow imaging, ultrasound resonance technology and a new scanner-based method that visualizes protein aggregates inside unopened vials. A data wheel representation was used to plot in one figure the results from the multiple analytical methods and to highlight differences between samples. The 25 mg/mL Avastin® drug product is stored at 2-8 °C during its 2-year shelf life. After a thermal stress of 4 weeks at 40 °C the ABX-BEV solution was turbid, containing particles of 20-100 µm diameter, accompanied by strong changes in antibody structural properties. Characterization of unstressed samples stored at 2-8 °C showed that the physicochemical properties of bevacizumab in ABX-BEV and the two originator drug products were similar, the observed differences between the originators being in the same range as those between ABX-BEV and the originator. To investigate the similarity of the antibodies under stress conditions, a freeze-thaw study was performed. Although freeze-thawing of bevacizumab products is prohibited by the package insert, after two freeze-thaw cycles (24 °C to -80 °C) small changes in the structural and aggregation properties of bevacizumab were observed, changes that were similar for the originator and ABX-BEV. Our study showed a good similarity of the investigated physicochemical properties of bevacizumab in originator and ABX-BEV products. It also provides an analytical approach, based on orthogonal methods, to compare high-concentration formulations of monoclonal antibodies.

Genome-scale protein expression and structural biology of Plasmodium falciparum and related Apicomplexan organisms.

Parasites from the protozoan phylum Apicomplexa are responsible for diseases, such as malaria, toxoplasmosis and cryptosporidiosis, all of which have significantly higher rates of mortality and morbidity in economically underdeveloped regions of the world. Advances in vaccine development and drug discovery are urgently needed to control these diseases and can be facilitated by production of purified recombinant proteins from Apicomplexan genomes and determination of their 3D structures. To date, both heterologous expression and crystallization of Apicomplexan proteins have seen only limited success. In an effort to explore the effectiveness of producing and crystallizing proteins on a genome-scale using a standardized methodology, over 400 distinct Plasmodium falciparum target genes were chosen representing different cellular classes, along with select orthologues from four other Plasmodium species as well as Cryptosporidium parvum and Toxoplasma gondii. From a total of 1008 genes from the seven genomes, 304 (30.2%) produced purified soluble proteins and 97 (9.6%) crystallized, culminating in 36 crystal structures. These results demonstrate that, contrary to previous findings, a standardized platform using Escherichia coli can be effective for genome-scale production and crystallography of Apicomplexan proteins. Predictably, orthologous proteins from different Apicomplexan genomes behaved differently in expression, purification and crystallization, although the overall success rates of Plasmodium orthologues do not differ significantly. Their differences were effectively exploited to elevate the overall productivity to levels comparable to the most successful ongoing structural genomics projects: 229 of the 468 target genes produced purified soluble protein from one or more organisms, with 80 and 32 of the purified targets, respectively, leading to crystals and ultimately structures from one or more orthologues.

Characterization of 14-3-3 proteins from Cryptosporidium parvum.

UNLABELLED: The parasite Cryptosporidium parvum has three 14-3-3 proteins: Cp14ε, Cp14a and Cp14b, with only Cp14ε similar to human 14-3-3 proteins in sequence, peptide-binding properties and structure. Structurally, Cp14a features the classical 14-3-3 dimer but with a uniquely wide pocket and a disoriented RRY triad potentially incapable of binding phosphopeptides. The Cp14b protein deviates from the norm significantly: (i) In one subunit, the phosphorylated C-terminal tail is bound in the binding groove like a phosphopeptide. This supports our binding study indicating this protein was stabilized by a peptide mimicking its last six residues. (ii) The other subunit has eight helices instead of nine, with αA and αB forming a single helix and occluding the peptide-binding cleft. (iii) The protein forms a degenerate dimer with the two binding grooves divided and facing opposite directions. These features conspire to block and disrupt the bicameral substrate-binding pocket, suggesting a possible tripartite auto-regulation mechanism that has not been observed previously. ENHANCED VERSION: This article can also be viewed as an enhanced version in which the text of the article is integrated with interactive 3D representations and animated transitions. Please note that a web plugin is required to access this enhanced functionality. Instructions for the installation and use of the web plugin are available in Text S1.

Characterization of an Escherichia coli sulfite oxidase homologue reveals the role of a conserved active site cysteine in assembly and function.

We report the biochemical and biophysical characterization of YedYZ, a sulfite oxidase homologue from Escherichia coli. YedY is a soluble catalytic subunit with a twin arginine leader sequence for export to the periplasm by the Tat translocation system. YedY is the only molybdoenzyme so far isolated from E. coli with the Mo-MPT form of the molybdenum cofactor. The electron paramagnetic resonance (EPR) signal of the YedY molybdenum is similar to that of known Mo-MPT containing enzymes, with the exception that only the Mo(IV) --> Mo(V) transition is observed, with a midpoint potential of 132 mV. YedZ is a membrane-intrinsic cytochrome b with six putative transmembrane helices. The single heme b of YedZ has a midpoint potential of -8 mV, determined by EPR spectroscopy of YedZ-enriched membrane preparations. YedY does not associate strongly with YedZ on the cytoplasmic membrane. However, mutation of the YedY active site Cys102 to Ser results in very efficient targeting of YedY to YedZ in the membrane, demonstrating a clear role for YedZ as the membrane anchor for YedY. Together, YedYZ comprise a well-conserved bacterial heme-molybdoenzyme found in a variety of bacteria that can be assigned to the sulfite oxidase class of enzyme.

Localization, annotation, and comparison of the Escherichia coli K-12 proteome under two states of growth.

Here we describe a proteomic analysis of Escherichia coli in which 3,199 protein forms were detected, and of those 2,160 were annotated and assigned to the cytosol, periplasm, inner membrane, and outer membrane by biochemical fractionation followed by two-dimensional gel electrophoresis and tandem mass spectrometry. Represented within this inventory were unique and modified forms corresponding to 575 different ORFs that included 151 proteins whose existence had been predicted from hypothetical ORFs, 76 proteins of completely unknown function, and 222 proteins currently without location assignments in the Swiss-Prot Database. Of the 575 unique proteins identified, 42% were found to exist in multiple forms. Using DIGE, we also examined the relative changes in protein expression when cells were grown in the presence and absence of amino acids. A total of 23 different proteins were identified whose abundance changed significantly between the two conditions. Most of these changes were found to be associated with proteins involved in carbon and amino acid metabolism, transport, and chemotaxis. Detailed information related to all 2,160 protein forms (protein and gene names, accession numbers, subcellular locations, relative abundances, sequence coverage, molecular masses, and isoelectric points) can be obtained upon request in either tabular form or as interactive gel images.

Genome-wide analysis of lipoprotein expression in Escherichia coli MG1655.

To gain insight into the cell envelope of Escherichia coli grown under aerobic and anaerobic conditions, lipoproteins were examined by using functional genomics. The mRNA expression levels of each of these genes under three growth conditions--aerobic, anaerobic, and anaerobic with nitrate--were examined by using both Affymetrix GeneChip E. coli antisense genome arrays and real-time PCR (RT-PCR). Many genes showed significant changes in expression level. The RT-PCR results were in very good agreement with the microarray data. The results of this study represent the first insights into the possible roles of unknown lipoprotein genes and broaden our understanding of the composition of the cell envelope under different environmental conditions. Additionally, these data serve as a test set for the refinement of high-throughput bioinformatic and global gene expression methods.

Structural and biochemical identification of a novel bacterial oxidoreductase.

By using a bioinformatics screen of the Escherichia coli genome for potential molybdenum-containing enzymes, we have identified a novel oxidoreductase conserved in the majority of Gram-negative bacteria. The identified operon encodes for a proposed heterodimer, YedYZ in Escherichia coli, consisting of a soluble catalytic subunit termed YedY, which is likely anchored to the membrane by a heme-containing trans-membrane subunit termed YedZ. YedY is uniquely characterized by the presence of one molybdenum molybdopterin not conjugated by an additional nucleotide, and it represents the only molybdoenzyme isolated from E. coli characterized by the presence of this cofactor form. We have further characterized the catalytic subunit YedY in both the molybdenum- and tungsten-substituted forms by using crystallographic analysis. YedY is very distinct in overall architecture from all known bacterial reductases but does show some similarity with the catalytic domain of the eukaryotic chicken liver sulfite oxidase. However, the strictly conserved residues involved in the metal coordination sphere and in the substrate binding pocket of YedY are strikingly different from that of chicken liver sulfite oxidase, suggesting a catalytic activity more in keeping with a reductase than that of a sulfite oxidase. Preliminary kinetic analysis of YedY with a variety of substrates supports our proposal that YedY and its many orthologues may represent a new type of membrane-associated bacterial reductase.