scholarly journals Modelling of pH-dependence to develop a strategy for stabilising mAbs at acidic steps in production

2019 ◽  
Author(s):  
Max Hebditch ◽  
Ryan Kean ◽  
Jim Warwicker
Keyword(s):  
Experimental Data ◽  
Design Principle ◽  
Ph Dependence ◽  
Low Ph ◽  
Viral Clearance ◽  
Acidic Ph ◽  
Antibody Therapeutics ◽  
Acid Environment ◽  
Pass Through ◽  
Acid Stable

AbstractEngineered proteins are increasingly being required to function or pass through environmental stresses for which the underlying protein has not evolved. A major example in health are antibody therapeutics, where a low pH step is used for purification and viral clearance. In order to develop a computational model for analysis of pH-stability, predictions are compared with experimental data for the relative pH-sensitivities of antibody domains. The model is then applied to proteases that have evolved to be functional in an acid environment, showing a clear signature for low pH-dependence of stability in the neutral to acidic pH region, largely through reduction of saltbridges. Interestingly, an extensively acidic protein surface can maintain contribution to structural stabilisation at acidic pH through replacement of basic sidechains with polar, hydrogen-bonding groups. These observations form a design principle for engineering acid-stable proteins.

scholarly journals Reinvestigation of the reaction of chymotrypsin with N-furylacryloyltryptophan derivatives at acidic pH

1979 ◽  
Vol 181 (3) ◽  
pp. 733-736 ◽  
Author(s):  
A L Fink ◽  
R Feldman ◽  
J Zehnder
Keyword(s):  
Methyl Ester ◽  
Wide Temperature Range ◽  
Methyl Esters ◽  
Ph Dependence ◽  
Gel Filtration ◽  
Low Ph ◽  
Time Dependent ◽  
Acidic Ph ◽  
Spectral Changes ◽  
Tryptophan Methyl Ester

The reaction of alpha-chymotrypsin with N alpha-3-(2-furyl)acryloyl-L-tryptophan methyl ester (FA-Trp-OMe) and amide has been investigated in aqueous and dimethylsulphoxide cryosolvent solutions from pH2 to 7 and over a wide temperature range. Previous reports have suggested that an intermediate preceding the acyl-enzyme can be detected spectrophotometrically in the reaction with methyl esters of FA-Trp and FA-Tyr at low pH [Yu & Viswanatha (1969) Eur. J. Biochem. 11, 347–352), and that this intermediate is an oxazolinone [Coletti-Previero et al. (1970) FEBS Lett. 11, 213–217]. We show that the previous interpretations of the time-dependent spectral changes were incorrect, and that the only detected intermediate is the acyl-enzyme. This may be isolated by gel filtration at pH less than 2.5, 1 degree C, owing to its relative stability. The pH-dependence of the rates of acylation and deacylation from pH 8.5 to 2.0 are consistent with a single ionization of pK congruent to 7.0 in both aqueous and cryosolvent solutions.

scholarly journals Role of Conserved Histidine Residues in the Low-pH Dependence of the Semliki Forest Virus Fusion Protein

2009 ◽  
Vol 83 (9) ◽  
pp. 4670-4677 ◽  
Author(s):  
Zhao-ling Qin ◽  
Yan Zheng ◽  
Margaret Kielian
Keyword(s):  
Fusion Protein ◽  
Membrane Fusion ◽  
Semliki Forest Virus ◽  
Ph Dependence ◽  
Low Ph ◽  
Endocytic Pathway ◽  
Acidic Ph ◽  
Virus Growth ◽  
Histidine Residues

ABSTRACT A wide variety of enveloped viruses infects cells by taking advantage of the low pH in the endocytic pathway to trigger virus-membrane fusion. For alphaviruses such as Semliki Forest virus (SFV), acidic pH initiates a series of conformational changes in the heterodimeric virus envelope proteins E1 and E2. Low pH dissociates the E2/E1 dimer, releasing the membrane fusion protein E1. E1 inserts into the target membrane and refolds to a trimeric hairpin conformation, thus driving the fusion reaction. The means by which E1 senses and responds to low pH is unclear, and protonation of conserved E1 histidine residues has been proposed as a possible mechanism. We tested the role of four conserved histidines by mutagenesis of the wild-type (wt) SFV infectious clone to create virus mutants with E1 H3A, H125A, H331A, and H331A/H333A mutations. The H125A, H331A, and H331A/H333A mutants had growth properties similar to those of wt SFV and showed modest change or no change in the pH dependence of virus-membrane fusion. By contrast, the E1 H3A mutation produced impaired virus growth and a markedly more acidic pH requirement for virus-membrane fusion. The dissociation of the H3A heterodimer and the membrane insertion of the mutant E1 protein were comparable to those of the wt in efficiency and pH dependence. However, the formation of the H3A homotrimer required a much lower pH and showed reduced efficiency. Together, these results and the location of H3 suggest that this residue acts to regulate the low-pH-dependent refolding of E1 during membrane fusion.

scholarly journals Acidic pH Mediates Changes in Antigenic and Oligomeric Conformation of Herpes Simplex Virus gB and Is a Determinant of Cell-Specific Entry

2018 ◽  
Vol 92 (17) ◽  
Author(s):  
Darin J. Weed ◽  
Stephen J. Dollery ◽  
Tri Komala Sari ◽  
Anthony V. Nicola
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Epithelial Cells ◽  
Conformational Changes ◽  
Viral Entry ◽  
Ph Dependence ◽  
Low Ph ◽  
Acidic Ph ◽  
Class Iii ◽  
Simplex Virus

ABSTRACTHerpes simplex virus (HSV) is an important human pathogen with a high worldwide seroprevalence. HSV enters epithelial cells, the primary site of infection, by a low-pH pathway. HSV glycoprotein B (gB) undergoes low pH-induced conformational changes, which are thought to drive membrane fusion. When neutralized back to physiological pH, these changes become reversible. Here, HSV-infected cells were subjected to short pulses of radiolabeling, followed by immunoprecipitation with a panel of gB monoclonal antibodies (MAbs), demonstrating that gB folds and oligomerizes rapidly and cotranslationally in the endoplasmic reticulum. Full-length gB from transfected cells underwent low-pH-triggered changes in oligomeric conformation in the absence of other viral proteins. MAbs to gB neutralized HSV entry into cells regardless of the pH dependence of the entry pathway, suggesting a conservation of gB function in distinct fusion mechanisms. The combination of heat and acidic pH triggered irreversible changes in the antigenic conformation of the gB fusion domain, while changes in the gB oligomer remained reversible. An elevated temperature alone was not sufficient to induce gB conformational change. Together, these results shed light on the conformation and function of the HSV-1 gB oligomer, which serves as part of the core fusion machinery during viral entry.IMPORTANCEHerpes simplex virus (HSV) causes infection of the mouth, skin, eyes, and genitals and establishes lifelong latency in humans. gB is conserved among all herpesviruses. HSV gB undergoes reversible conformational changes following exposure to acidic pH which are thought to mediate fusion and entry into epithelial cells. Here, we identified cotranslational folding and oligomerization of newly synthesized gB. A panel of antibodies to gB blocked both low-pH and pH-neutral entry of HSV, suggesting conserved conformational changes in gB regardless of cell entry route. Changes in HSV gB conformation were not triggered by increased temperature alone, in contrast to results with EBV gB. Acid pH-induced changes in the oligomeric conformation of gB are related but distinct from pH-triggered changes in gB antigenic conformation. These results highlight critical aspects of the class III fusion protein, gB, and inform strategies to block HSV infection at the level of fusion and entry.

scholarly journals fus-1, a pH Shift Mutant of Semliki Forest Virus, Acts by Altering Spike Subunit Interactions via a Mutation in the E2 Subunit

1998 ◽  
Vol 72 (5) ◽  
pp. 4281-4287 ◽  
Author(s):  
Sallie Glomb-Reinmund ◽  
Margaret Kielian
Keyword(s):  
Ammonium Chloride ◽  
Conformational Changes ◽  
Semliki Forest Virus ◽  
Ph Dependence ◽  
Low Ph ◽  
Spike Protein ◽  
Acidic Ph ◽  
Fusion Activity ◽  
Protein Subunit ◽  
Subunit Interactions

ABSTRACT Semliki Forest virus (SFV), an enveloped alphavirus, is a well-characterized paradigm for viruses that infect cells via endocytic uptake and low-pH-triggered fusion. The SFV spike protein is composed of a dimer of E1 and E2 transmembrane subunits, which dissociate upon exposure to low pH, liberating E2 and the fusogenic E1 subunit to undergo independent conformational changes. SFV fusion and infection are blocked by agents such as ammonium chloride, which act by raising the pH in the endosome and inhibiting the low-pH-induced conformational changes in the SFV spike protein. We have previously isolated an SFV mutant, fus-1, that requires more acidic pH to trigger its fusion activity and is therefore more sensitive to inhibition by ammonium chloride. The acid shift in the fusion activity offus-1 was here shown to be due to a more acidic pH threshold for the initial dissociation of the fus-1 spike dimer, thereby resulting in a more acidic pH requirement for the subsequent conformational changes in both fus-1 E1 andfus-1 E2. Sequence analysis demonstrated that thefus-1 phenotype was due to a mutation in the E2 spike subunit, threonine 12 to isoleucine. fus-1 revertants that have regained the parental fusion phenotype and ammonium chloride sensitivity were shown to have also regained E2 threonine 12. Our results identify a region of the SFV E2 spike protein subunit that regulates the pH dependence of E1-catalyzed fusion by controlling the dissociation of the E1/E2 dimer.

scholarly journals A Rationalization of the Acidic pH Dependence for Stromelysin-1 (Matrix Metalloproteinase-3) Catalysis and Inhibition

2000 ◽  
Vol 275 (15) ◽  
pp. 11026-11033 ◽  
Author(s):  
Linda L. Johnson ◽  
Alexander G. Pavlovsky ◽  
Adam R. Johnson ◽  
Jeffrey A. Janowicz ◽  
Chiu-Fai Man ◽  
...  
Keyword(s):  
Matrix Metalloproteinase ◽  
Ph Dependence ◽  
Acidic Ph ◽  
Matrix Metalloproteinase 3

scholarly journals Impact of the acidic environment on gene expression and functional parameters of tumors in vitro and in vivo

2021 ◽  
Vol 40 (1) ◽  
Author(s):  
Mandy Rauschner ◽  
Luisa Lange ◽  
Thea Hüsing ◽  
Sarah Reime ◽  
Alexander Nolze ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Death ◽  
Western Blot ◽  
Tumor Cell ◽  
Low Ph ◽  
Strong Increase ◽  
Acidic Ph ◽  
The Impact

Abstract Background The low extracellular pH (pHe) of tumors resulting from glycolytic metabolism is a stress factor for the cells independent from concomitant hypoxia. The aim of the study was to analyze the impact of acidic pHe on gene expression on mRNA and protein level in two experimental tumor lines in vitro and in vivo and were compared to hypoxic conditions as well as combined acidosis+hypoxia. Methods Gene expression was analyzed in AT1 prostate and Walker-256 mammary carcinoma of the rat by Next Generation Sequencing (NGS), qPCR and Western blot. In addition, the impact of acidosis on tumor cell migration, adhesion, proliferation, cell death and mitochondrial activity was analyzed. Results NGS analyses revealed that 147 genes were uniformly regulated in both cell lines (in vitro) and 79 genes in both experimental tumors after 24 h at low pH. A subset of 25 genes was re-evaluated by qPCR and Western blot. Low pH consistently upregulated Aox1, Gls2, Gstp1, Ikbke, Per3, Pink1, Tlr5, Txnip, Ypel3 or downregulated Acat2, Brip1, Clspn, Dnajc25, Ercc6l, Mmd, Rif1, Zmpste24 whereas hypoxia alone led to a downregulation of most of the genes. Direct incubation at low pH reduced tumor cell adhesion whereas acidic pre-incubation increased the adhesive potential. In both tumor lines acidosis induced a G1-arrest (in vivo) of the cell cycle and a strong increase in necrotic cell death (but not in apoptosis). The mitochondrial O2 consumption increased gradually with decreasing pH. Conclusions These data show that acidic pHe in tumors plays an important role for gene expression independently from hypoxia. In parallel, acidosis modulates functional properties of tumors relevant for their malignant potential and which might be the result of pH-dependent gene expression.

pH dependence of kinetics and steady-state block of cardiac sodium channels by lidocaine

1993 ◽  
Vol 264 (5) ◽  
pp. H1588-H1598 ◽  
Author(s):  
D. J. Wendt ◽  
C. F. Starmer ◽  
A. O. Grant
Keyword(s):  
Steady State ◽  
Sodium Channel ◽  
Local Anesthetic ◽  
Ph Dependence ◽  
Membrane Depolarization ◽  
Low Ph ◽  
Proton Exchange ◽  
Phasic Block ◽  
Cardiac Sodium Channels ◽  
External Ph

The local anesthetic-class antiarrhythmic drugs produce greater depression of conduction in ischemic compared with normal myocardium. The basis for this relatively selective action is uncertain. A model of the pH-dependent interaction of tertiary amine drugs with the sodium channel suggests that the low pH occurring during ischemia slows drug dissociation from the channel by changing the drug's protonation. The importance of the proton exchange reaction and the effect of overall slowing of drug dissociation on steady-state sodium channel blockade is uncertain. We have measured whole cell sodium channel current in rabbit atrial myocytes during control and exposure to lidocaine while external pH was varied between 6.8 and 7.8 at membrane potentials of -140, -120, and -100 mV. Tonic blockade was little influenced by external pH. Decreasing the external pH from 7.8 to 6.8 slowed both the rate of development of phasic block and recovery from the block. Decreasing the membrane potential from -140 to -100 mV increased the degree of phasic block attained in the steady state. Block was further enhanced when low pH was combined with membrane depolarization. Experiments in which deuterium ions were substituted for protons suggest that the kinetics of proton exchange is not rate limiting in the dissociation of drugs from the sodium channel. We conclude that it is the combined effect of low pH and membrane depolarization that may be critical in the enhanced blocking action of local anesthetic-class drugs during ischemia.

Low pH reduces the activity of ceftolozane/tazobactam in human urine, but confirms current breakpoints for urinary tract infections

2019 ◽  
Vol 75 (3) ◽  
pp. 593-599 ◽  
Author(s):  
Alina Karoline Nussbaumer-Pröll ◽  
Sabine Eberl ◽  
Birgit Reiter ◽  
Thomas Stimpfl ◽  
Christoph Dorn ◽  
...  
Keyword(s):  
Urinary Tract ◽  
Urinary Tract Infections ◽  
Human Urine ◽  
Negative Impact ◽  
Low Ph ◽  
Antibiotic Activity ◽  
Clinical Isolates ◽  
Acidic Ph ◽  
Bacterial Strains ◽  
Tract Infections

Abstract Background Acidic pH has been shown to impact the antibiotic activity of non-β-lactams in urine. Objectives To investigate the in vitro activity of ceftolozane/tazobactam compared with meropenem at different pH settings in urine. Methods We determined the MICs for 30 clinical isolates of Escherichia coli, 25 clinical isolates of Klebsiella pneumoniae and 24 clinical isolates of Proteus mirabilis in pooled human urine and standard growth medium at pH 5 and 7. Time–kill curves were produced for one representative clinical isolate of tested bacterial strains in urine at pH 5, 6 and 7 for both antibiotics at concentrations above and below the MIC. HPLC analysis of the stability of ceftolozane/tazobactam and meropenem was performed at different pH values. Results The median MICs of both antibiotics were up to 8-fold higher at pH 5 than at pH 7. Bacterial growth of E. coli was not impacted by pH, while for K. pneumoniae and P. mirabilis low pH slightly reduced growth. Compared with pH 7, pH 5 resulted in a significant decrease in antibiotic activity with a delta of up to 3 log10 bacterial counts after 24 h. Impact of acidic pH was lowest for P. mirabilis; however, this strain metabolically increased the pH during experiments. Stability was not impacted by low pH. Conclusions Acidic pH had a significant negative impact on the activity of ceftolozane/tazobactam and meropenem in urine. Considering concentrations achieved in urine, our results confirm existing breakpoints and do not advocate increasing ceftolozane/tazobactam breakpoints for urinary tract infections.

scholarly journals Effect of water temperature on a simple suspension method for lansoprazole orally disintegrating tablets

2019 ◽  
Vol 15 (1) ◽  
pp. 4-9
Author(s):  
Sumio Chono ◽  
Megumi Matsui ◽  
Katsuki Nakamura
Keyword(s):  
Physical Properties ◽  
Water Temperature ◽  
Gastric Acid ◽  
Warm Water ◽  
Orally Disintegrating Tablets ◽  
Acid Environment ◽  
Enteric Coated ◽  
Pass Through ◽  
Lukewarm Water ◽  
Coated Granules

In this study, we examined the physical properties, including disintegration, passage through a nasogastric administration tube, and acidoresistance, of one branded and five generic formulations of lansoprazole orally disintegrating (OD) tablets containing enteric-coated granules to examine the feasibility of a simple suspension method. The generic tablets immediately disintegrated in warm (55°C) and lukewarm water (35°C) and released the enteric-coated granules, which passed through an administration tube. Moreover, the released enteric-coated granules were stable under a simulated gastric acid environment. However, although the branded tablet disintegrated in warm water, the released enteric-coated granules formed aggregates that did not pass through the administration tube. Meanwhile, the granules released from the branded tablet in lukewarm water did not form aggregates. The present study demonstrated the applicability of a simple suspension method using warm or lukewarm water for generic lansoprazole OD tablets. Additionally, the method is applicable to branded lansoprazole OD tablets using lukewarm water.

scholarly journals PHR2 of Candida albicans encodes a functional homolog of the pH-regulated gene PHR1 with an inverted pattern of pH-dependent expression.

1997 ◽  
Vol 17 (10) ◽  
pp. 5960-5967 ◽  
Author(s):  
F A Mühlschlegel ◽  
W A Fonzi
Keyword(s):  
Candida Albicans ◽  
Mutant Strain ◽  
Low Ph ◽  
Predicted Amino Acid Sequence ◽  
Acidic Ph ◽  
Null Mutant ◽  
Alkaline Ph ◽  
Ph Values ◽  
Functional Homolog ◽  
Morphological Defects

Deletion of PHR1, a pH-regulated gene of Candida albicans, results in pH-conditional defects in growth, morphogenesis, and virulence evident at neutral to alkaline pH but absent at acidic pH. Consequently, we searched for a functional homolog of PHR1 active at low pH. This resulted in the isolation of a second pH-regulated gene, designated PHR2. The expression of PHR2 was inversely related to that of PHR1, being repressed at pH values above 6 and progressively induced at more acidic pH values. The predicted amino acid sequence of the PHR2 protein, Phr2p, was 54% identical to that of Phr1p. A PHR2 null mutant exhibited pH-conditional defects in growth and morphogenesis analogous to those of PHR1 mutants but manifest at acid rather than alkaline pH values. Engineered expression of PHR1 at acid pH in a PHR2 mutant strain and PHR2 at alkaline pH in a PHR1 mutant strain complemented the defects in the opposing mutant. Deletion of both PHR1 and PHR2 resulted in a strain with pH-independent, constitutive growth and morphological defects. These results indicate that PHR1 and PHR2 represent a novel pH-balanced system of functional homologs required for C. albicans to adapt to environments of diverse pH.

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