Analytical titration curves of glycosyl hydrolase Cel45 by combined isoelectric focusing — electrophoresis

Attanasio, Francesco; Bruschi, Maurizio; Candiano, Giovanni; Galletto, Roberto; Musante, Luca; Schülein, M.; Rialdi, G.

doi:10.1002/(sici)1522-2683(19990601)20:7<1403::aid-elps1403>3.0.co;2-6

Cited by 7 publications

(3 citation statements)

References 29 publications

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“…The upcoming occurrence of high concentrated liquid formulations of monoclonal antibodies suggested the development of self‐buffering antibody solutions . In the literature, the characterization of proteins in vitro with respect to buffer attributes widely focuses on determination or prediction of amino acid side chain p K a values and isoelectric points . Findings in these studies touch on the subject of buffer capacity indirectly since any monovalent buffer exhibits a maximum molar buffer capacity of β M =0.575 at the p K a .…”

Section: Discussionmentioning

confidence: 99%

Buffer capacity of biologics—from buffer salts to buffering by antibodies

Karow

Bahrenburg

Garidel

2013

Biotechnology Progress

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Controlling pH is essential for a variety of biopharmaceutical process steps. The chemical stability of biologics such as monoclonal antibodies is pH-dependent and slightly acidic conditions are favorable for stability in a number of cases. Since control of pH is widely provided by added buffer salts, the current study summarizes the buffer characteristics of acetate, citrate, histidine, succinate, and phosphate buffers. Experimentally derived values largely coincide with values calculated from a model that had been proposed in 1922 by van Slyke. As high concentrated protein formulations become more and more prevalent for biologics, the self-buffering potential of proteins becomes of relevance. The current study provides information on buffer characteristics for pH ranges down to 4.0 and up to 8.0 and shows that a monoclonal antibody at 50 mg/mL exhibits similar buffer capacity as 6 mM citrate or 14 mM histidine (pH 5.0-6.0). Buffer capacity of antibody solutions scales linearly with protein concentration up to more than 200 mg/mL. At a protein concentration of 220 mg/mL, the buffer capacity resembles the buffer capacity of 30 mM citrate or 50 mM histidine (pH 5.0-6.0). The buffer capacity of monoclonal antibodies is practically identical at the process relevant temperatures 5, 25, and 40°C. Changes in ionic strength of ΔI=0.15, in contrast, can alter the buffer capacity up to 35%. In conclusion, due to efficient self-buffering by antibodies in the pH range of favored chemical stability, conventional buffer excipients could be dispensable for pH stabilization of high concentrated protein solutions.

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Section: Discussionmentioning

confidence: 99%

Buffer capacity of biologics—from buffer salts to buffering by antibodies

Karow

Bahrenburg

Garidel

2013

Biotechnology Progress

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“…Titration was performed according to Bruschi et al (24). Both methods for electrophoresis runs and calculations have been described previously (24,25).…”

Section: Electrophoretic Titration Curvesmentioning

confidence: 99%

Active Focal Segmental Glomerulosclerosis Is Associated with Massive Oxidation of Plasma Albumin

Musante¹,

Candiano²,

Petretto

et al. 2007

Journal of the American Society of Nephrology

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The basic mechanism for idiopathic FSGS still is obscure. Indirect evidence in humans and generation of FSGS by oxidants in experimental models suggest a role of free radicals. In vitro studies demonstrate a main role of plasma albumin as antioxidant, its modification representing a chemical marker of oxidative stress. With the use of complementary liquid chromatography electron spray ionization tandem mass spectrometry (LC-ESI-MS/MS) and biochemical methods, plasma albumin was characterized in 34 patients with FSGS; 18 had received a renal transplant, and 17 had IgM mesangial deposition. Patients with FSGS that was in remission or without recurrence after transplantation had normal plasma albumin, and the same occurred in patients with primary and secondary nephrites and with chronic renal failure. In contrast, patients with active FSGS or with posttransplantation recurrence had oxidized plasma albumin. This finding was based on the characterization of albumin Cys 34 with an mass-to-charge ratio of 511.71 in triple charge that was consistent with the formation of a cysteic acid carrying a sulfonic group (alb-SO 3 ؊ ). The exact mass of albumin was increased accordingly (؉48 Da) for incorporation of three oxygen radicals. Direct titration of the free sulfhydryl group 34 of plasma albumin and electrophoretic titration curves confirmed loss of free sulfhydryl group and formation of a fast-moving isoform in all cases with disease activity. This is the first demonstration of in vivo plasma albumin oxidation that was obtained with an adequate structural approach. Albumin oxidation seems to be specific for FSGS, suggesting some pathogenetic implications. Free radical involvement in FSGS may lead to specific therapeutic interventions.

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“…Titration was performed according to Bruschi et al [17]. Both methodology for electrophoresis runs and calculations have been described in previous papers [18]. Briefly, square gel slabs (145 mm × 145 mm × 1.5 mm) were cast to contain acrylamide (%T 5.00, %C 2.67) and 2.5% w/v carrier ampholytes, pH 3.5 ± 10.…”

Section: Electrophoretic Titration Curvesmentioning

confidence: 99%