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Biophysical and Biochemical Characterization of Peptide, Protein, and Bioconjugate Products
Published in Sandeep Nema, John D. Ludwig, Parenteral Medications, 2019
Tapan K. Das, James A. Carroll
Glycoproteins can be either N-linked or O-linked, depending on the type of covalent modification of the glycan to the protein. The type of glycosylation is dependent on both the protein sequence and the expression system used for production. Glycosylation may commonly occur for proteins expressed in mammalian or yeast expression systems, but is not observed for proteins expressed in bacterial systems. N-linked glycosylation occurs at asparagine residues in the consensus sequence of Asn-Xxx-Ser or Asn-Xxx-Thr, where Xxx is any amino acid except proline. While the presence of this motif does not guarantee glycosylation, it makes N-linked glycosylation a predictable attribute. The amino acid sequence can be easily scanned for this sequence motif in order to determine if N-linked glycosylation is a possibility for a given biotherapeutic protein. Analysis of N-linked glycosylation, therefore, begins with an assessment of the site occupancy levels of any possible N-linked glycosylation sites in molecule, referred to as the macroheterogeneity. This can be accomplished using analytical methods which can distinguish size variants, such as electrophoretic or chromatographic separations, or mass spectrometry. For glycoproteins with multiple glycosylation sites, macroheterogeneity can lead to complex mixtures. For example, the therapeutic glycoprotein interferon gamma has two sequence motifs for N-linked glycosylation. Therefore, there are four theoretical forms based on occupancy alone: unoccupied, two different singly occupied forms, and one fully occupied form [48].
Characterization of Biosimilar Biologics
Published in Laszlo Endrenyi, Paul Jules Declerck, Shein-Chung Chow, Biosimilar Drug Product Development, 2017
As referenced earlier, a recombinant form of the glycoprotein erythropoietin required a mammalian cell production platform since glycosylation and the precise glycoform were shown to be essential to in vivo activity (Macdougall et al., 2012). When first produced, in CHO cells, the product was shown to have an enhanced activity in vitro, compared to the natural form isolated from urine, but it was inactive in vivo. This was due to the absence of terminal sialic residues from the three N-linked oligosaccharide moieties; the consequence was exposure of terminal galactose residues, resulting in uptake by the asialo-glycoprotein receptor and rapid clearance through the liver. Process improvements led to a product having ∼30% of sialylated EPO, and methods were developed to harvest/purify this fraction, the remainder being discarded. Since expiry of the patent, many biosimilar EPO products have been generated and approved by regulatory authorities; however, increased incidences of the development of ADA, with consequent pure red cell aplasia (PRCA), have been reported (Macdougall et al., 2012). Prior to patent expiry, the innovator company (Amgen) developed an improved EPO and obtained new patent protection. Improvement was achieved by the introduction of two extra N-linked glycosylation sites, resulting in increased sialylation and enhanced biological activity and in vivo half-life (Elliott et al., 2004; Sinclair, 2013). Interestingly, while EPO cells secreted from CHO cells is heterogeneously glycosylated when expressed as a transmembrane protein, on CHO cells, it is homogeneously glycosylated and fully sialylated; the membrane bound EPO can be released to yield a fully active EPO product (Singh et al., 2015).
Model-guided concurrent data assimilation for calibrating cardiac ion-channel kinetics
Published in IISE Transactions on Healthcare Systems Engineering, 2023
Haedong Kim, Hui Yang, Andrew R. Ednie, Eric S. Bennett
Another aberrant activity of K+ currents was slower inactivation of delayed rectifier currents in MGAT1KO cardiomyocytes. Figure 8 shows the prediction results of the inactivation time constants of IKslow1 and IKslow2. The computer models successfully capture this trend, resulting in the time constant of inactivation in for IKslow1 () being estimated to be significantly slowed in MGAT1KO compared to WT, i.e. 348.9 ms, as shown in Fig. 8(a). IKslow2 is also slowed (see Fig. 8(b)). Inactivation time constants are important because they determine how slowly IKslow1 and IKslow2 inactivate and, in turn, significantly affect the repolarization of the AP. show higher variability than other currents to address most of the decaying portion of IKsum. This is likely due to the fact that while Kv4.2 and Kv2.1 are O-glycosylated, Kv1.5 possess a single occupied N-linked glycosylation. Hence, IKslow1 is more affected by reduced N-glycosylation. A plot is not provided here, but there is no significant effect on IKto for membrane potential greater than 0 mV. It is worth mentioning that the proposed method was able to extrapolate that is greater than 5 s from the 4.5-s-pulse protocol, which shows the generalization power of the in-silico modeling.
Heterologous expression of azurin from Pseudomonas aeruginosa in the yeast Pichia pastoris
Published in Preparative Biochemistry & Biotechnology, 2021
Yagmur Unver, Busra Sensoy Gun, Melek Acar, Seyda Yildiz
Recombinant proteins have mainly N-linked glycosylation with little O-linked glycosylation when they were expressed in P. pastoris.[59] According to Western blot analysis result which was performed with anti-azurin antibody, extracellular recombinant azurin-cMyc-His tag fusion protein had approximately 20 kDa. This value was higher than the expected molecular weight. According to the manufacturer (Invitrogen), myc epitope and polyhistidine tag added 2.5 kDa to recombinant protein size. Therefore, it was thought that the remainder came from the glycosylated portion of recombinant protein. Similarly, staphylokinase (SAK) was expressed in P. pastoris and it was reported that the molecular weight of the recombinant SAK-cMyc-His tag fusion protein was 3.5 kDa higher than the predicted molecular weight because of the glycosylated form of secreted protein.[60] As for the expression of azurin in other expression systems, Yildiz and Askin reported extracellular production of azurin in Lactococcus lactis strain NZ3900 with the Nisin Controlled Gene Expression (NICE) System, and azurin produced by this prokaryotic system had 16.5 kDa molecular weight.[61] Zhang et al., performed in vitro and in vivo azurin secretion using engineered E. coli Nissle 1917 with tumor-targeting capability. They concluded that thanks to azurin expression, B16 melanoma, and orthotopic 4T1 breast tumor growth were inhibited in mice.[62] Also, Chung et al. was reported that a nanoscale biomemory device was fabricated using modified recombinant azurin, expressed with its cysteine residue, in E. coli BL21 (DE3).[63]