Quantification and Structure
Elucidation of <i>in Vivo</i> Bevacizumab Modification in
Rabbit Vitreous Humor
after Intravitreal Injection
Kai On Chu
David Ta Li Liu
Kwok Ping Chan
Ya Ping Yang
Gary Hin
Fai Yam
Michael S Rogers
Chi Pui Pang
10.1021/mp3005403.s002
https://acs.figshare.com/articles/journal_contribution/Quantification_and_Structure_Elucidation_of_i_in_Vivo_i_Bevacizumab_Modification_in_Rabbit_Vitreous_Humor_after_Intravitreal_Injection/2464732
Off-label and intravitreal use of bevacizumab, a recombinant
immunoglobulin
against VEGF, has been practiced widely for ophthalmic treatments.
However, longitudinal data of its intravitreal status is unavailable
due to a lack of reliable methods for bevacizumab determination. Thus
its pharmacokinetics and pharmacodynamics are uncertain. We developed
and validated a high performance liquid chromatographic method to
determine bevacizumab in vitreous humor and utilized a novel strategy
to assess <i>in vivo</i> temporal binding changes by affinity
chromatography. Mass spectrometry and tandem mass spectrometry detection
were used for structural evaluation. The coefficient of variation
(CV) for intrabatch imprecision varied from 0.5 to 14.3% and for interbatch
imprecision from 1.9 to 11.6%. The linearity was over 0.9982, lower
limit of quantification 1.95 μg, recoveries over 95%, and accuracy
between 90 and 112% over the range of 1.95–250 μg of
bevacizumab in 100 μL of vitreous humor. Blank vitreous humor
showed no interference peak. It was stable at room temperature for
5 h. Bevacizumab elimination in the vitreous followed first order
kinetics with half-life as 5.7 days and elimination rate as 0.1221
day<sup>–1</sup>. Peptide mapping and tandem mass spectrometry
revealed structural modifications of the <i>in vivo</i> bevacizumab
mainly on the heavy chain in both variable and constant regions 7
days after intravitreal injection. Minor changes were also discovered
on the light chain. Affinity chromatography showed significant affinity
changes in samples 21 days after intravitreal injection. The changes
were consistent with structural modifications as found in endothelial
cell migration assays results. In conclusion, we have established
a robust chromatographic method for determination of bevacizumab and
strategies with affinity chromatography and molecular mass detection
that revealed bevacizumab structural and possible functional changes
in vitreous.
2012-12-03 00:00:00
affinity chromatography
tandem mass spectrometry
VEGF
intravitreal injection
vitreous humor
method
quantification 1.95 μ g
100 μ L
bevacizumab
CV
Rabbit Vitreous Humor
cell migration assays results
samples 21 days
Vivo Bevacizumab Modification
Blank vitreous humor
tandem mass spectrometry detection
regions 7 days