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Characterizing High Affinity Antigen/Antibody Complexes by Kinetic and Equilibrium Based Methods

  • Andrew W. Drake
  • David G. Myszka
  • Scott L. Klakamp
Chapter
Part of the Biotechnology: Pharmaceutical Aspects book series (PHARMASP, volume XI)

Abstract

The development and application of monoclonal antibodies as drugs requires accurate and precise characterization of the antigen/antibody binding constants. Anticipating the required affinity for a therapeutically efficacious monoclonal antibody (mAb) is complex. Generally, the equilibrium dissociation constant for the antigen/mAb complex should be less than 100–1,000 picomolar (pM), depending upon the nature of the target, the desired function of the antibody, and the localized concentration of the antigen in the diseased tissue, among other factors. Measurement of the equilibrium dissociation constant (K D), association rate constant (k a), and dissociation rate constant (k d) for these high affinity antibodies is difficult because of three independent reasons: (1) the time for the antigen/antibody complex to reach equilibrium can be very long, on the order of days (2) usually, the k d for such a tight complex is extremely low, requiring long periods of data collection in order to discern enough information to predict complex stability, and (3) in cases where the k d is easily measurable (greater than 5×10−4s−1), the k a can be very fast, greater than 1×107M−1s−1.

Keywords

Equilibrium Dissociation Constant Association Rate Constant Binding Response Bead Pack Biosensor Surface 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Abbreviations

pM

picomolar

nM

nanomolar

fM

femtomolar

mAb

monoclonal antibody

KD

equilibrium dissociation constant

ka

association rate constant

kd

dissociation rate constant

Fc

Flow cell

RU

resonance units

Da

Dalton

cy5

Indodicarbocyanine

pAb

polyclonal antibody

surfactant P-20

poly(oxyethylene)(20)-sorbitane monolaureate

Notes

Acknowledgments.

The computer algorithm for the simulation of the time to equilibrium for a 1:1 reversible interaction was graciously provided to us by Steve Lackie and Tom Glass of Sapidyne, Inc, Boise, ID.

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Copyright information

© American Association of Pharmaceutical Scientists 2010

Authors and Affiliations

  • Andrew W. Drake
    • 1
  • David G. Myszka
    • 2
  • Scott L. Klakamp
    • 3
  1. 1.Amgen Fremont, Inc.FremontUSA
  2. 2.Center for Biomolecular Interaction AnalysisUniversity of UtahSalt Lake CityUSA
  3. 3.Takeda San FranciscoS. San FranciscoUSA

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