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Related Concept Videos

Buffers02:56

Buffers

A solution containing appreciable amounts of a weak conjugate acid-base pair is called a buffer solution, or a buffer. Buffer solutions resist a change in pH when small amounts of a strong acid or a strong base are added. A solution of acetic acid and sodium acetate is an example of a buffer that consists of a weak acid and its salt: CH3COOH (aq) + CH3COONa (aq). An example of a buffer that consists of a weak base and its salt is a solution of ammonia and ammonium chloride: NH3 (aq) + NH4Cl...
Buffer Effectiveness02:19

Buffer Effectiveness

Buffer solutions do not have an unlimited capacity to keep the pH relatively constant . Instead, the ability of a buffer solution to resist changes in pH relies on the presence of appreciable amounts of its conjugate weak acid-base pair. When enough strong acid or base is added to substantially lower the concentration of either member of the buffer pair, the buffering action within the solution is compromised.
The buffer capacity is the amount of acid or base that can be added to a given volume...
Protein Buffers in Blood Plasma and Cells01:20

Protein Buffers in Blood Plasma and Cells

The human body utilizes protein buffer systems to maintain a stable pH. These systems capitalize on the dual role of amino acids, which can act as acids or bases by accepting or releasing hydrogen ions in response to pH changes. Protein buffer systems are particularly significant in the extracellular fluid (ECF) and intracellular fluid (ICF) of active cells, where structural and functional proteins provide substantial buffering capacity.
Certain amino acids can exist in a zwitterion state at a...
Phosphate Buffer01:22

Phosphate Buffer

The phosphate buffer system is a critical biological mechanism for maintaining pH stability in the body. This system operates primarily through two components: sodium dihydrogen phosphate (NaH2PO4), which acts as a weak acid, and sodium hydrogen phosphate (Na2HPO4), which serves as a weak base.
Sodium dihydrogen phosphate does not fully dissociate in neutral or acidic solutions. When a strong base, such as sodium hydroxide (NaOH), is introduced into the solution, sodium dihydrogen phosphate...
Buffers: Buffer Capacity01:09

Buffers: Buffer Capacity

Buffer capacity is the quantitative measure of a buffer to resist the change in pH. As shown in the following equation, the buffer capacity, denoted by 'beta', is expressed as the number of moles of acid or base needed to change the pH of a one-liter buffer solution by 1 unit. Here, Ca and Cb indicate the number of moles of acid and base, respectively. Note that dpH represents the change in pH.
In the graph, pH is plotted as a function of the number of moles of base (Cb) added to a weak acid...
Buffers: Overview01:30

Buffers: Overview

Buffers play a crucial role in stabilizing the pH of a solution by mitigating the effects of small amounts of added acid or base. They consist of a weak acid and its conjugate base or a weak base and its conjugate acid. A solution of acetic acid and sodium acetate is an example of a buffer that consists of a weak acid and its salt: CH3COOH (aq) + CH3COONa (aq). An example of a buffer that consists of a weak base and its salt is a solution of ammonia and ammonium chloride: NH3 (aq) + NH4Cl (aq).

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Determination of the Gas-phase Acidities of Oligopeptides
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Determination of the Gas-phase Acidities of Oligopeptides

Published on: June 24, 2013

Asparagine deamidation dependence on buffer type, pH, and temperature.

Amanda L Pace1, Rita L Wong1, Yonghua Taylor Zhang2

  • 1Late Stage Pharmaceutical Development, Genentech Inc., South San Francisco, California 94080.

Journal of Pharmaceutical Sciences
|April 10, 2013
PubMed
Summary

Monoclonal antibody (mAb) deamidation, a key degradation pathway, can be accurately measured using a novel ion exchange chromatography method. This technique helps determine antibody shelf life by analyzing deamidation rates in Fc and Fab fragments under various conditions.

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Area of Science:

  • Biochemistry
  • Analytical Chemistry
  • Pharmaceutical Science

Background:

  • Asparagine deamidation into aspartate and isoaspartate is a primary chemical degradation route for monoclonal antibodies (mAbs).
  • This degradation impacts the shelf life and efficacy of therapeutic antibodies if not properly formulated or stored.
  • Understanding deamidation kinetics is crucial for predicting and extending antibody stability.

Purpose of the Study:

  • To develop and validate a new method for detecting and quantifying mAb deamidation.
  • To investigate the influence of pH and temperature on deamidation rates of different antibody fragments.
  • To generate kinetic parameters for informed shelf-life prediction of therapeutic antibodies.

Main Methods:

  • Developed a novel ion exchange chromatography method to separate papain-digested mAbs into Fc and Fab fragments.
  • Prepared buffers at room temperature and then incubated at stability testing temperatures (e.g., 5°C, 40°C) without pH readjustment.
  • Analyzed deamidation rates across a range of pH conditions and temperatures to determine kinetic parameters.

Main Results:

  • The ion exchange chromatography method successfully quantified deamidation rates for Fc and Fab fragments.
  • Deamidation rates varied with temperature and pH; faster at 40°C in acidic buffers, but reversed at 5°C due to hydroxide ion concentration.
  • The Fc fragment exhibited a V-shaped pH-rate profile, with distinct deamidation sites at high (PENNY peptide) and low (Asn323) pH, while the Fab fragment showed a linear profile.

Conclusions:

  • The developed chromatographic method provides accurate deamidation rate measurements for mAb fragments.
  • Temperature and pH significantly influence mAb deamidation kinetics, with complex interactions affecting stability.
  • Understanding fragment-specific deamidation mechanisms, like those at Asn323 in Fc, is essential for predicting antibody degradation and optimizing formulation for extended shelf life.