2D NMR Analysis of the Effect of Asparagine Deamidation Versus Methionine Oxidation on the Structure, Stability, Aggregation, and Function of a Therapeutic Protein

Bandi, Swati; Singh, Surinder; Shah, Dinen D.; Upadhyay, Vaibhav; Mallela, Krishna

doi:10.1021/acs.molpharmaceut.9b00719

Cited by 11 publications

(12 citation statements)

References 77 publications

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“…The percentages of correct forms varied between different manufacturers, and relatively stable between different batches, demonstrating the status of disulfide bonds were related to expression procedure. Oxidation of Met residues and deamidation of Asp residues are two of the most common forms of chemical modifications that compromise the efficacy of therapeutic proteins [21]. In intact protein analysis, oxidized IFNα2b was only found in sample 4E (24.2%) and 6P (14.7%), but in peptide mapping, oxidized peptides were found in all seven samples, suggesting that oxidation could happen during sample preparation.…”

Section: Discussionmentioning

confidence: 99%

Analysis of Molecular Heterogeneity in Therapeutic IFNα2b from Different Manufacturers by LC/Q-TOF

Tao

Zhao

et al. 2020

Molecules

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Recombinant human IFNα2b (rhIFNα2b), as an important immune-related protein, has been widely used in clinic for decades. It is also at the forefront of the recent emergence of biosimilar medicines, with numerous products now available worldwide. Although with the same amino acid sequence, recombinant proteins are generally heterogeneous due to post-translational modification and chemical reactions during expression, purification, and long-term storage, which could have significant impact on the final product quality. So therapeutic rhIFNα2b must be closely monitored to ensure consistency, safety, and efficacy. In this study, we compared seven rhIFNα2b preparations from six manufacturers in China and one in America, as well as four batches of rhIFNα2b preparations from the same manufacturer, measuring IFNα2b variants and site-specific modifications using a developed LC/Q-TOF approach. Three main forms of N-terminus, cysteine, methionine, and acetylated cysteine were detected in five rhIFNα2b preparations produced in E. coli (1E~5E) and one in Pseudomonas (6P), but only the native form with N-terminal cysteine was found in rhIFNα2b preparation produced in Saccharomyces cerevisiae (7Y). Two samples with the lowest purity (4E and 6P), showed the highest level of acetylation at N-terminal cysteine and oxidation at methionine. The level of oxidation and deamidation varied not only between samples from different manufacturers but also between different batches of the same manufacturer. Although variable between samples from different manufacturers, the constitution of N-terminus and disulfide bonds was relatively stable between different batches, which may be a potential indicator for batch consistency. These findings provide a valid reference for the stability evaluation of the production process and final products.

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Section: Discussionmentioning

confidence: 99%

Analysis of Molecular Heterogeneity in Therapeutic IFNα2b from Different Manufacturers by LC/Q-TOF

Tao

Zhao

et al. 2020

Molecules

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“…As most proteins are amphiphilic and surface active, they have a tendency towards adsorption at the ALI. Upon adsorption, conformation changes may occur, exposing hydrophobic residues to the interface to avoid contact with water, thus leading to aggregation and unfolding, which are the main factors contributing to protein instability [ 67 , 68 ]. Chemical degradation of proteins (deamidation, oxidation, glycation) may also cause aggregation (either covalent or non-covalent) [ 67 ].…”

Section: Challenges and Considerations In The Development Of Protein mentioning

confidence: 99%

“…These findings revealed that deamidation destabilised IFNA2a and enhanced its tendency to aggregate under stressful conditions, and reduced its function to a greater extent than oxidation. This is the first study that quantitatively compared the effects between deamidation and oxidation of a therapeutic protein [ 68 ]. It would be a good strategy to conduct such studies early on in the development of therapeutic protein candidates in order to identify the chemical modifications that a particular protein would be susceptible to, and to test out various excipients that could resolve specific stability issues.…”

Section: Challenges and Considerations In The Development Of Protein mentioning

confidence: 99%

“…Polysorbates are the most commonly used surfactants, and are already being used to preclude aggregation in formulations of intravenously administered antibodies [ 34 ]. Polysorbate 80 has been reported to lead to stabilisation in various inhaled protein formulations including those for granulocyte-colony stimulating factor (G-CSF), lactate dehydrogenase (LDH), tissue plasminogen activator (t-Pa) and aviscumine (recombinant mistletoe lectin) [ 68 ]. The ability of polysorbates and other surfactants to stabilise a protein and hinder aggregate formation is contingent on the protein-to-surfactant ratio.…”

Section: Approaches To Address the Challenges In Formulating Inhaled mentioning

confidence: 99%

“…Sugars (sucrose, trehalose, raffinose and lactose) and polyols (mannitol) stabilise proteins through the preferential hydration of proteins via steric repulsion of sugar/polyol molecules from the native protein [ 4 , 68 ]. Lactose is often used as a drug carrier in DPIs, however, it may not be suitable for proteins because it is a reducing sugar, and it could interact with amino groups in proteins (Maillard reaction) [ 67 ].…”

Section: Approaches To Address the Challenges In Formulating Inhaled mentioning

confidence: 99%

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Developing inhaled protein therapeutics for lung diseases

Matthews

2020

Mol Biomed

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Biologic therapeutics such as protein/polypeptide drugs are conventionally administered systemically via intravenous injection for the treatment of diseases including lung diseases, although this approach leads to low target site accumulation and the potential risk for systemic side effects. In comparison, topical delivery of protein drugs to the lung via inhalation is deemed to be a more effective approach for lung diseases, as proteins would directly reach the target in the lung while exhibiting poor diffusion into the systemic circulation, leading to higher lung drug retention and efficacy while minimising toxicity to other organs. This review examines the important considerations and challenges in designing an inhaled protein therapeutics for local lung delivery: the choice of inhalation device, structural changes affecting drug deposition in diseased lungs, clearance mechanisms affecting an inhaled protein drug’s lung accumulation, protein stability, and immunogenicity. Possible approaches to overcoming these issues will also be discussed.

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