Role of Buffers in Protein Formulations

Zbacnik, Teddy J.; Holcomb, Ryan E.; Katayama, Derrick S.; Murphy, Brian M.; Payne, R. W.; Coccaro, Richard C.; Evans, Gabriel J.; Matsuura, James E.; Henry, Charles S.; Manning, Mark C.

doi:10.1016/j.xphs.2016.11.014

Cited by 151 publications

(93 citation statements)

References 329 publications

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“…The pharmaceutical stability profiles encountered during manufacturing and storage are not only dependent on such intrinsic properties (e.g., primary sequence/post-translational modifications, 33 conformational stability, 34 solubility 35 ), but also extrinsic stress factors (e.g., storage temperature, freeze-thaw, agitation). 36,37 Primary Structure, Post-Translational Modifications, and Chemical Stability Profile An additional Met residue identified at N-terminus of each antigen was not unexpected because these antigens were expressed in E. coli and the efficiency of methionine aminopeptidase, enzyme responsible for excision of N-terminal methionine during translation, is about 30%-60% depending on the host. 38 Residue next to the N-terminal Met for each NRRV antigen is Glu with a bulky side chain which could also hinder the efficiency of methionine aminopeptidase by steric effects as noted in the literature.…”

Section: Discussionmentioning

confidence: 99%

Recombinant Subunit Rotavirus Trivalent Vaccine Candidate: Physicochemical Comparisons and Stability Evaluations of Three Protein Antigens

Hickey

Sahni

Toth

et al. 2020

Journal of Pharmaceutical Sciences

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a b s t r a c tAlthough live attenuated Rotavirus (RV) vaccines are available globally to provide protection against enteric RV disease, efficacy is substantially lower in low-to middle-income settings leading to interest in alternative vaccines. One promising candidate is a trivalent nonreplicating RV vaccine, comprising 3 truncated RV VP8 subunit proteins fused to the P2 CD4 þ epitope from tetanus toxin (P2-VP8-P[4/6/8]). A wide variety of analytical techniques were used to compare the physicochemical properties of these 3 recombinant fusion proteins. Various environmental stresses were used to evaluate antigen stability and elucidate degradation pathways. P2-VP8-P[4] and P2-VP8-P[6] displayed similar physical stability profiles as function of pH and temperature while P2-VP8-P[8] was relatively more stable. Forced degradation studies revealed similar chemical stability profiles with Met 1 most susceptible to oxidation, the single Cys residue (at position 173/172) forming intermolecular disulfide bonds (P2-VP8-P[6] was most susceptible), and Asn 7 undergoing the highest levels of deamidation. These results are visualized in a structural model of the nonreplicating RV antigens. The establishment of key structural attributes of each antigen, along with corresponding stability-indicating methods, have been applied to vaccine formulation development efforts (see companion paper), and will be utilized in future analytical comparability assessments.

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Section: Discussionmentioning

confidence: 99%

Recombinant Subunit Rotavirus Trivalent Vaccine Candidate: Physicochemical Comparisons and Stability Evaluations of Three Protein Antigens

Hickey

Sahni

Toth

et al. 2020

Journal of Pharmaceutical Sciences

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“…The most commonly used buffers in pharmaceutical development are acetate, citrate, histidine, phosphate, Tris and glycine [105,109,191]. A complete description of the effects of buffer on peptide aggregation is beyond the scope of this review; however, interested readers are directed towards the recent excellent review by Zbacnik and co-workers [192].…”

Section: Buffering Agentsmentioning

confidence: 99%

Factors affecting the physical stability (aggregation) of peptide therapeutics

Zapadka

Becher

Santos³

et al. 2017

Interface Focus.

264

207

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The number of biological therapeutic agents in the clinic and development pipeline has increased dramatically over the last decade and the number will undoubtedly continue to increase in the coming years. Despite this fact, there are considerable challenges in the development, production and formulation of such biologics particularly with respect to their physical stabilities. There are many cases where self-association to form either amorphous aggregates or highly structured fibrillar species limits their use. Here, we review the numerous factors that influence the physical stability of peptides including both intrinsic and external factors, wherever possible illustrating these with examples that are of therapeutic interest. The effects of sequence, concentration, pH, net charge, excipients, chemical degradation and modification, surfaces and interfaces, and impurities are all discussed. In addition, the effects of physical parameters such as pressure, temperature, agitation and lyophilization are described. We provide an overview of the structures of aggregates formed, as well as our current knowledge of the mechanisms for their formation.

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“…10 Certain parameters are very crucial for higher yield, as well as improving the stability and integrity of protein molecules, including type of buffers and the concentrations of individual components, as well as the pH of the buffer. 11,12 In particular, Tris buffers (pH 6.5-9.5), as well as HEPES or phosphate buffers, are widely used as a result of their ability to maintain the conformational integrity of proteins and high solubility. [13][14][15] Phosphate buffers improve the thermal stability in solutions containing NaCl, 16 delay denaturation of proteins, 17 or stabilize the protein during longterm storage.…”

Section: Introductionmentioning

confidence: 99%

Cytosolic and Transmembrane Protein Extraction Methods of Breast and Ovarian Cancer Cells: A Comparative Study

2018

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Efficient extraction of proteins is a great challenge for numerous downstream proteomic analyses. During the protein extraction procedure, it is critical to maintain the conformational stability, integrity, as well as higher yield of the protein. To do so, 5-different lysis buffers of Tris and HEPES have been used as the primary buffering reagents with variable compositions at different concentrations and pH using human cancer cells. In this study, different protein lysates of human breast cancer cells T47D and MDA-MB-231 and ovarian cancer cell PA-1 were subjected to run SDS-PAGE for separation of proteins based on their molecular size, followed by Coomassie blue, silver staining, and immunoblot assays to compare the extraction yield of total cytoplasmic proteins, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and the integral membrane protein, integrin b-1. Our results revealed that Tris-based lysis buffer with 50 mM concentration, pH 7.5, is relatively the efficient and reliable protein extraction method for a wide range of MW subcellular markers, cytoplasmic GAPDH and transmembrane integrin b-1 proteins. We anticipate that this simple and cost-effective protein extraction protocol might be extremely useful across a broad range of subcellular proteins in different biologic samples.

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Role of Buffers in Protein Formulations

Cited by 151 publications

References 329 publications

Recombinant Subunit Rotavirus Trivalent Vaccine Candidate: Physicochemical Comparisons and Stability Evaluations of Three Protein Antigens

Recombinant Subunit Rotavirus Trivalent Vaccine Candidate: Physicochemical Comparisons and Stability Evaluations of Three Protein Antigens

Factors affecting the physical stability (aggregation) of peptide therapeutics

Cytosolic and Transmembrane Protein Extraction Methods of Breast and Ovarian Cancer Cells: A Comparative Study

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