8 research outputs found
Quantitative Analysis of Peptide–Matrix Interactions in Lyophilized Solids Using Photolytic Labeling
Peptide–matrix
interactions in lyophilized solids were explored
using photolytic labeling with reversed phase high performance liquid
chromatography (rp-HPLC) and mass spectrometric (MS) analysis. A model
peptide (Ac-QELHKLQ–NHCH<sub>3</sub>) derived from salmon calcitonin
was first labeled with a heterobifunctional cross-linker NHS-diazirine
(succinimidyl 4,4′-azipentanoate; SDA) at Lys5 in solution,
with ∼100% conversion. The SDA labeled peptide was then formulated
with the following excipients at a 1:400 molar ratio and lyophilized:
sucrose, trehalose, mannitol, histidine, arginine, urea, and NaCl.
The lyophilized samples and corresponding solution controls were exposed
to UV at 365 nm to induce photolytic labeling, and the products were
identified by MS and quantified with rp-HPLC or MS. Peptide–excipient
adducts were detected in the lyophilized solids except the NaCl formulation.
With the exception of the histidine formulation, peptide–excipient
adducts were not detected in solution and the fractional conversion
to peptide–water adducts in solution was significantly greater
than in lyophilized solids, as expected. In lyophilized solids, the
fractional conversion to peptide–water adducts was poorly correlated
with bulk moisture content, suggesting that the local water content
near the labeled lysine residue differs from the measured bulk average.
In lyophilized solids, the fractional conversion to peptide–excipient
adducts was assessed using MS extracted ion chromatograms (EIC); subject
to the assumption of equal ionization efficiencies, the fractional
conversion to excipient adducts varied with excipient type. The results
demonstrate that the local environment near the lysine residue of
the peptide in the lyophilized solids can be quantitatively probed
with a photolytic labeling method
Protein G, Protein A and Protein A-Derived Peptides Inhibit the Agitation Induced Aggregation of IgG
Controlling and preventing aggregation is critical to
the development of safe and effective antibody drug products. The
studies presented here test the hypothesis that protein A and protein
G inhibit the agitation-induced aggregation of IgG. The hypothesis
is motivated by the enhanced conformational stability of proteins
upon ligand binding and the specific binding affinity of protein A
and protein G to the Fc region of IgG. The aggregation of mixed human
IgG from pooled human plasma was induced by agitation alone or in
the presence of (i) protein A, (ii) protein G or (iii) a library of
24 peptides derived from the IgG-binding domain of protein A. Aggregation
was assessed by UV spectroscopy, SDS–PAGE, high performance
size-exclusion chromatography (HP-SEC), dynamic light scattering (DLS)
and fluorescence spectroscopy. Additional information on IgG–ligand
interactions was obtained using differential scanning fluorimetry
(DSF) and competitive binding studies. The results demonstrate that
protein A provides near-complete inhibition of agitation-induced aggregation,
while protein G and two peptides from the peptide library show partial
inhibition. The findings indicate that the IgG protein A-binding site
is involved in the agitation-induced aggregation of IgG, and suggest
a dominant role of colloidal interactions
Localized Hydration in Lyophilized Myoglobin by Hydrogen–Deuterium Exchange Mass Spectrometry. 2. Exchange Kinetics
Solid-state hydrogen–deuterium exchange with mass
spectrometric analysis (ssHDX) is a promising method for characterizing
proteins in amorphous solids. Though analysis of HDX kinetics is informative
and well-established in solution, application of these methods to
solid samples is complicated by possible heterogeneities in the solid.
The studies reported here provide a detailed analysis of the kinetics
of hydration and ssHDX for equine myoglobin (Mb) in solid matrices
containing sucrose or mannitol. Water sorption was rapid relative
to ssHDX, indicating that ssHDX kinetics was not limited by bulk water
transport. Deuterium uptake in solids was well-characterized by a
biexponential model; values for regression parameters provided insight
into differences between the two solid matrices. Analysis of the widths
of peptide mass envelopes revealed that, in solution, an apparent
EX2 mechanism prevails, consistent with native conformation of the
protein. In contrast, in mannitol-containing samples, a smaller non-native
subpopulation exchanges by an EX1-like mechanism. Together, the results
indicate that the analysis of ssHDX kinetic data and of the widths
of peptide mass envelopes is useful in screening solid formulations
of protein drugs for the presence of non-native species that cannot
be detected by amide I FTIR
Localized Hydration in Lyophilized Myoglobin by Hydrogen–Deuterium Exchange Mass Spectrometry. 1. Exchange Mapping
The local effects of hydration on myoglobin (Mb) in solid
matrices
containing mannitol or sucrose (1:1 w/w, protein:additive) were mapped
using hydrogen–deuterium exchange with mass spectrometric analysis
(HDX–MS) at 5 °C and compared to solution controls. Solid
powders were exposed to D<sub>2</sub>OÂ(g) at controlled activity (<i>a</i><sub>w</sub>) followed by reconstitution and analysis of
the intact protein and peptides produced by pepsin digestion. HDX
varied with matrix type, <i>a</i><sub>w</sub>, and position
along the protein backbone. HDX was less in sucrose matrices than
in mannitol matrices at all <i>a</i><sub>w</sub> while the
difference in solution was negligible. Differences in HDX in the two
matrices were detectable despite similarities in their bulk water
content. The extent of exchange in solids is proposed as a measure
of the hydration of exchangeable amide groups, as well as protein
conformation and dynamics; pepsin digestion allows these effects to
be mapped with peptide-level resolution
Photolytic Cross-Linking to Probe Protein–Protein and Protein–Matrix Interactions in Lyophilized Powders
Protein
structure and local environment in lyophilized formulations
were probed using high-resolution solid-state photolytic cross-linking
with mass spectrometric analysis (ssPC–MS). In order to characterize
structure and microenvironment, protein–protein, protein–excipient,
and protein–water interactions in lyophilized powders were
identified. Myoglobin (Mb) was derivatized in solution with the heterobifunctional
probe succinimidyl 4,4′-azipentanoate (SDA) and the structural
integrity of the labeled protein (Mb-SDA) confirmed using CD spectroscopy
and liquid chromatography/mass spectrometry (LC–MS). Mb-SDA
was then formulated with and without excipients (raffinose, guanidine
hydrochloride (Gdn HCl)) and lyophilized. The freeze-dried powder
was irradiated with ultraviolet light at 365 nm for 30 min to produce
cross-linked adducts that were analyzed at the intact protein level
and after trypsin digestion. SDA-labeling produced Mb carrying up
to five labels, as detected by LC–MS. Following lyophilization
and irradiation, cross-linked peptide–peptide, peptide–water,
and peptide–raffinose adducts were detected. The exposure of
Mb side chains to the matrix was quantified based on the number of
different peptide–peptide, peptide–water, and peptide–excipient
adducts detected. In the absence of excipients, peptide–peptide
adducts involving the CD, DE, and EF loops and helix H were common.
In the raffinose formulation, peptide–peptide adducts were
more distributed throughout the molecule. The Gdn HCl formulation
showed more protein–protein and protein–water adducts
than the other formulations, consistent with protein unfolding and
increased matrix interactions. The results demonstrate that ssPC–MS
can be used to distinguish excipient effects and characterize the
local protein environment in lyophilized formulations with high resolution
A Cooperative Folding Unit as the Structural Link for Energetic Coupling within a Protein
Previously, we demonstrated
that binding of a ligand to <i>Escherichia coli</i> cofactor-dependent
phosphoglycerate mutase
(dPGM), a homodimeric protein, is energetically coupled with dimerization.
The equilibrium unfolding of dPGM occurs with a stable, monomeric
intermediate. Binding of several nonsubstrate metabolites stabilizes
the dimeric native form over the monomeric intermediate, reducing
the population of the intermediate. Both the active site and the dimer
interface appear to be unfolded in the intermediate. We hypothesized
that a loop containing residues 118–152 was responsible for
the energetic coupling between the dimer interface and the distal
active site and was unfolded in the intermediate. Here, we investigated
the structure of the dPGM intermediate by probing side-chain interactions
and solvent accessibility of the peptide backbone. By comparing the
effect of a mutation on the global stability and the stability of
the intermediate, we determine an equilibrium φ value (φ<sub>eq</sub> value), which provides information about whether side-chain
interactions are retained or lost in the intermediate. Hydrogen/deuterium
exchange coupled with mass spectrometry (HDX-MS) was used to investigate
differences in the solvent accessibility of the peptide backbone in
the intermediate and native forms of dPGM. The results of φ<sub>eq</sub> value analysis and HDX-MS reveal the least stable folding
unit of dPGM, which is unfolded in the intermediate and links the
active site to the dimer interface. The structure of the intermediate
reveals how the cooperative network of residues in dPGM gives rise
to the observed energetic coupling between dimerization and ligand
binding
Probing the Conformation of an IgG1 Monoclonal Antibody in Lyophilized Solids Using Solid-State Hydrogen–Deuterium Exchange with Mass Spectrometric Analysis (ssHDX-MS)
Therapeutic proteins are often formulated
as lyophilized products
to improve their stability and prolong shelf life. The stability of
proteins in the solid-state has been correlated with preservation
of native higher order structure and/or molecular mobility in the
solid matrix, with varying success. In the studies reported here,
we used solid-state hydrogen–deuterium exchange with mass spectrometric
analysis (ssHDX-MS) to study the conformation of an IgG1 monoclonal
antibody (mAb) in lyophilized solids and related the extent of ssHDX
to aggregation during storage in the solid phase. The results demonstrate
that the extent of ssHDX correlated better with aggregation rate during
storage than did solid-state Fourier-transform infrared (ssFTIR) spectroscopic
measurements. Interestingly, adding histidine to sucrose at different
formulation pH conditions decreased aggregation of the mAb, an effect
that did not correlate with structural or conformational changes as
measured by ssFTIR or ssHDX-MS. Moreover, peptide-level ssHDX-MS analysis
in four selected formulations demonstrated global changes across the
structure of the mAb when lyophilized with sucrose, trehalose, or
mannitol, whereas site-specific changes were observed when lyophilized
with histidine as the sole excipient
Solid-State Hydrogen–Deuterium Exchange Mass Spectrometry: Correlation of Deuterium Uptake and Long-Term Stability of Lyophilized Monoclonal Antibody Formulations
Solid
state hydrogen–deuterium exchange with mass spectrometric
analysis (ssHDX-MS) has been used to assess protein conformation and
matrix interactions in lyophilized solids. ssHDX-MS metrics have been
previously correlated to the formation of aggregates of lyophilized
myoglobin on storage. Here, ssHDX-MS was applied to lyophilized monoclonal
antibody (mAb) formulations and correlated to their long-term stability.
After exposing lyophilized samples to D<sub>2</sub>OÂ(g), the amount
of deuterium incorporated at various time points was determined by
mass spectrometry for four different lyophilized mAb formulations.
Hydrogen–deuterium exchange data were then correlated with
mAb aggregation and chemical degradation, which was obtained in stability
studies of >2.5 years. Deuterium uptake on ssHDX-MS of four lyophilized
mAb formulations determined at the initial time point prior to storage
in the dry state was directly and strongly correlated with the extent
of aggregation and chemical degradation during storage. Other measures
of physical and chemical properties of the solids were weakly or poorly
correlated with stability. The data demonstrate, for the first time,
that ssHDX-MS results are highly correlated with the stability of
lyophilized mAb formulations. The findings thus suggest that ssHDX-MS
can be used as an early read-out of differences in long-term stability
between formulations helping to accelerate formulation screening and
selection