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Lyophilization of Protein Pharmaceuticals
Published in Kenneth E. Avis, Vincent L. Wu, Biotechnology and Biopharmaceutical Manufacturing, Processing, and Preservation, 2020
John F. Carpenter, Byeong S. Chang
Several review papers have been published to describe the physical (e.g., aggregation and precipitation) and chemical (i.e., covalent alterations) degradation pathways of proteins during storage in aqueous solution (Wang and Hanson 1988; Manning et al. 1989; Wang and Pearlman 1993; Cleland et al. 1993; Manning et al. 1995). Very little published information is available about the effect of lyophilization on the rate of individual degradation pathways in protein formulations. However, it is important to understand the major degradation pathways arising during storage in the dried solid and to develop the appropriate analytical methods before attempting to optimize the formulation. This is especially important if the protein undergoes chemical degradation (e.g., deamidation), for which very specific formulation adjustments (e.g., reduced pH) are needed to prevent protein damage. To develop the analytical methods needed and to identify the types of degradation products with which one might have to deal, degradation can be accelerated. The protein is lyophilized in the absence of stabilizers and stored at relatively high temperatures (e.g., > 50°C). The protein is then rehydrated and analyzed for changes, such as aggregation and specific chemical alterations. Since this analysis is performed in solution, the approaches outlined in reviews on solution stability can be used. Detailed accounts can be found in Manning and Ahern (1992) and Manning et al. (1989, 1995).
Preformulation of New Biological Entities
Published in Sandeep Nema, John D. Ludwig, Parenteral Medications, 2019
Riccardo Torosantucci, Vasco Filipe, Jonathan Kingsbury, Atul Saluja, Yatin Gokarn
Deamidation is the chemical removal or conversion of the amide group from the side chains of asparagine or glutamine residues. Deamidation of asparagine residues occurs commonly in therapeutic proteins, whereas that of glutamine residues can occur but is far less common, with reaction rates usually hundredfold slower [84]. The generation of deamidated products is influenced by parameters such as pH, temperature, and ionic strength. Therefore, solvent conditions used in cell culture, downstream processing and formulation should be carefully selected for biotherapeutics with labile asparagine or glutamine residues. Of these factors, alkaline pH contributes most significantly to the rate of deamidation [85]. Following the loss of amide (an irreversible process), the resulting succinimide intermediate may undergo a number of reversible processes including the formation of aspartic acid or isoaspartate. These are prone to reversible racemization through an enol intermediate, which leads to the formation of D-aspartic acid and D-isoaspartate. These may be of immunologic concern due to being non-natural amino acids. It should be noted, however, that racemization occurs at a negligible rate unless catalyzed by solvent components such as phosphate ions [86,87]. Therefore, careful control of solvent conditions may mitigate racemization.
Protein a resin lifetime study: Evaluation of protein a resin performance with a model-based approach in continuous capture
Published in Preparative Biochemistry and Biotechnology, 2018
Ketki Behere, Bumjoon Cha, Seongkyu Yoon
Since the study involved a solid–liquid interaction process, the transport of OH− ions across the resin and into the pores is guided by the boundary layer thermodynamics and diffusion kinetics. Hence, one or combination of the following phenomena can dominate the reaction: Transport of OH− ions within the pores of the resin particle by intraparticle diffusion.Interaction of OH− ions with basic hydrophilic amino acids, such as lysine, arginine, and histidine.Interaction of OH− ions with asparagine and glutamine residues on Protein A resin leading to deamidation.Interaction of OH− ions with the polymeric linker chain that links the Protein A ligand to the resin matrix.
Computational analysis of nonenzymatic deamidation of asparagine residues catalysed by acetic acid
Published in Molecular Physics, 2021
Tomoki Nakayoshi, Kota Wanita, Koichi Kato, Eiji Kurimoto, Akifumi Oda
Post-translational deamidation of asparagine (Asn, l-Asn) residues occurs in peptides and proteins, and proceeds nonenzymatically. Asn-residue deamidation in peptides and proteins converts Asn into aspartic acid (Asp) and/or isoaspartic acid (isoAsp) residues via succinimide (Suc) intermediates [1–3]. Scheme 1 shows the Suc-intermediate-mediated pathway for deamidation of Asn residues. First, the side-chain carbonyl carbon of the Asn residue is nucleophilically attacked by the main-chain amide nitrogen of the C-terminal side adjacent residue, i.e. (n + 1) residue in order to form a five-membered ring l-Suc intermediate. l-Suc intermediates are easily hydrolysed to form l-Asp and l-isoAsp residues. The formation of l-Asp and l-isoAsp residues occurs heterogeneously, and l-Asp and l-isoAsp residues typically form in a ratio of 1:3 [1–3]. Some l-Suc intermediates formed in peptides and proteins undergo stereoinversion to form d-Suc intermediates, and d-Asp and d-isoAsp residues are formed by the hydrolysis of d-Suc intermediates [1–3]. The Suc intermediate is relatively stable, and the accumulation of Suc in some peptides and proteins has been experimentally confirmed [4–7]. The formation rate of Suc intermediates by Asn-residue deamidation is strongly affected by the size of the (n + 1) residue [8–11]. Specifically, when the (n + 1) residue is small, as in the case of glycine, alanine, and serine, Asn-residue deamidation proceeds rapidly. Conversely, when the (n + 1) residue is bulky, as with valine, leucine, and isoleucine, Asn-residue deamidation is extremely limited.
Tunable nonenzymatic degradability of N-substituted polyaspartamide main chain by amine protonation and alkyl spacer length in side chains for enhanced messenger RNA transfection efficiency
Published in Science and Technology of Advanced Materials, 2019
Mitsuru Naito, Yuta Otsu, Rimpei Kamegawa, Kotaro Hayashi, Satoshi Uchida, Hyun Jin Kim, Kanjiro Miyata
Most of the degradable polymers contain ester bonds or other biodegradable linkers, such as the disulfide bond and acetal bond. These chemical bonds often restrict the polymer design and/or the handling method. With regard to biodegradable polycations, Schmitt et al. [15] reported that N-substituted polyaspartamides bearing a 2-aminoethyl (-CH2CH2NH2) moiety in the side chain were nonenzymatically degraded in aqueous milieu, and that their degradation rates increased under basic conditions. We previously developed N-substituted polyaspartamide bearing a two-repeated 2-aminoethyl moiety (-(CH2CH2NH)2-H) in the side chains (PAsp(DET)), which elicited nonenzymatic degradation associated with minimal cytotoxicity, and thus enabled the highly efficient transfection of plasmid DNA (pDNA) [16–18], mRNA [19,20], and oligonucleotide/protein complex [21]. Interestingly, the degradability of PAsp(DET) arose from a cleavage of the amide bond in the main chain, which tends to be a stable bond in the absence of degradation enzymes in physiological conditions. It was postulated that the cleavage of the main chain of PAsp(DET) should be ascribed to the nucleophilic attack of the amide nitrogen in the side chain (CONHside) at the amide carbon in the main chain (CONHmain) (Scheme 1(a)) [17]. An analogous reaction, asparagine (or glutamine) deamidation, occurs in the body through the nucleophilic attack of CONHmain on CONHside accompanied by elimination of ammonia from the carbamoyl group in the side chain, resulting in peptide isomerization (Scheme 1(b)) [22,23]. The main chain cleavage in PAsp(DET) is therefore, an unnatural reaction, as the CONHside activation process remains to be elucidated.