PMID- 22683695
OWN - NLM
STAT- MEDLINE
DA  - 20121204
DCOM- 20130510
IS  - 1873-3441 (Electronic)
IS  - 0939-6411 (Linking)
VI  - 82
IP  - 3
DP  - 2012 Nov
TI  - Biophysical characterisation of thermal-induced precipitates of recombinant
      anthrax protective antigen: evidence for kinetically trapped unfolding domains in
      solid-state.
PG  - 475-84
LID - 10.1016/j.ejpb.2012.05.019 [doi]
LID - S0939-6411(12)00186-5 [pii]
AB  - Insoluble aggregation or precipitation is one of the most common degradation
      pathways observed for biotherapeutics; despite this, the structural mechanisms by
      which this occurs remain poorly understood due to difficulties associated with
      biophysical characterisation of protein particulates. To address this knowledge
      gap, we developed a solid-state circular dichroism (CD) technique, which allows
      in situ measurements of the secondary and tertiary structural changes associated 
      with the formation of visible therapeutic protein aggregates. We demonstrate how 
      solid-state CD, in conjunction with other biophysical and computational methods
      can aid in gaining valuable insights into the mechanisms and pathways of
      thermal-induced precipitation of Bacillus anthracis recombinant protective
      antigen (rPA), the primary immunogen of anthrax subunit vaccine. Using these
      methods, we show the domains d3 and d4 are the most labile of the four
      structurally distinct domains of rPA and play the critical role in nucleating the
      cascade of unfolding and aggregation. During the assembly process, the domains d1
      and d2 become kinetically trapped within the insoluble aggregate and reveal
      previously intractable distinct tertiary structural elements of the rPA native
      structure. These findings reveal a uniquely detailed insight into the role of rPA
      domains on protein stability and provide a mechanistic framework for
      thermal-induced unfolding and precipitation. It also shows that solid-state CD
      provides a novel approach in characterising protein precipitation that may
      facilitate rational improvements to the stability of biopharmaceuticals.
CI  - Copyright (c) 2012 Elsevier B.V. All rights reserved.
AD  - XstalBio Ltd., University Avenue, Glasgow, United Kingdom.
      ashok.ganesan@switch.vib-kuleuven.be
FAU - Ganesan, Ashok
AU  - Ganesan A
FAU - Watkinson, Allan
AU  - Watkinson A
FAU - Moore, Barry D
AU  - Moore BD
LA  - eng
GR  - 1UC1AI67223-01/AI/NIAID NIH HHS/United States
PT  - Journal Article
PT  - Research Support, N.I.H., Extramural
DEP - 20120607
PL  - Netherlands
TA  - Eur J Pharm Biopharm
JT  - European journal of pharmaceutics and biopharmaceutics : official journal of
      Arbeitsgemeinschaft fur Pharmazeutische Verfahrenstechnik e.V
JID - 9109778
RN  - 0 (Antigens, Bacterial)
RN  - 0 (Bacterial Toxins)
RN  - 0 (Recombinant Proteins)
RN  - 0 (anthrax toxin)
SB  - IM
MH  - Antigens, Bacterial/*chemistry
MH  - Bacterial Toxins/*chemistry
MH  - Chemical Precipitation
MH  - Circular Dichroism/*methods
MH  - Hot Temperature
MH  - Protein Folding
MH  - Protein Stability
MH  - Protein Structure, Secondary
MH  - Protein Structure, Tertiary
MH  - Recombinant Proteins/chemistry
EDAT- 2012/06/12 06:00
MHDA- 2013/05/11 06:00
CRDT- 2012/06/12 06:00
PHST- 2012/01/13 [received]
PHST- 2012/04/25 [revised]
PHST- 2012/05/29 [accepted]
PHST- 2012/06/07 [aheadofprint]
AID - S0939-6411(12)00186-5 [pii]
AID - 10.1016/j.ejpb.2012.05.019 [doi]
PST - ppublish
SO  - Eur J Pharm Biopharm. 2012 Nov;82(3):475-84. doi: 10.1016/j.ejpb.2012.05.019.
      Epub 2012 Jun 7.