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• W2003684685 abstract "Since immunoglobulins are conformationally dynamic molecules in solution, we studied the effect of stabilizing and destabilizing excipients on the conformational stability and dynamics of two IgG1 monoclonal antibodies (mAbs; mAb-A and mAb-B) using a variety of biophysical approaches. Even though the two mAbs are of the same IgG1 subtype, the unfolding patterns, aggregation behavior, and pretransition dynamics of these two antibodies were strikingly different in response to external perturbations such as pH, temperature, and presence of excipients. Sucrose and arginine were identified as stabilizers and destabilizers, respectively, on the basis of their influence on conformational stability for both the IgG1 mAbs. The two excipients, however, had distinct effective concentrations and different effects on the conformational stability and pretransition dynamics of the two mAbs as measured by a combination of differential scanning calorimetry, high-resolution ultrasonic spectroscopy, and red-edge excitation shift fluorescence studies. Stabilizing concentrations of sucrose were found to decrease the internal motions of mAb-B, whereas arginine marginally increased its adiabatic compressibility in the pretransition region. Both sucrose and arginine did not influence the pretransition dynamics of mAb-A. The potential reasons for such differences in excipient effects between two IgG1 mAbs are discussed. © 2012 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 101:3062–3077, 2012 Since immunoglobulins are conformationally dynamic molecules in solution, we studied the effect of stabilizing and destabilizing excipients on the conformational stability and dynamics of two IgG1 monoclonal antibodies (mAbs; mAb-A and mAb-B) using a variety of biophysical approaches. Even though the two mAbs are of the same IgG1 subtype, the unfolding patterns, aggregation behavior, and pretransition dynamics of these two antibodies were strikingly different in response to external perturbations such as pH, temperature, and presence of excipients. Sucrose and arginine were identified as stabilizers and destabilizers, respectively, on the basis of their influence on conformational stability for both the IgG1 mAbs. The two excipients, however, had distinct effective concentrations and different effects on the conformational stability and pretransition dynamics of the two mAbs as measured by a combination of differential scanning calorimetry, high-resolution ultrasonic spectroscopy, and red-edge excitation shift fluorescence studies. Stabilizing concentrations of sucrose were found to decrease the internal motions of mAb-B, whereas arginine marginally increased its adiabatic compressibility in the pretransition region. Both sucrose and arginine did not influence the pretransition dynamics of mAb-A. The potential reasons for such differences in excipient effects between two IgG1 mAbs are discussed. © 2012 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 101:3062–3077, 2012 Proteins in solution are inherently conformationally dynamic molecules composed of atoms that are in a state of constant motion at ambient temperatures.1.Karplus M. McCammon J.A. 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Numerous lower resolution techniques such as ultraviolet (UV)-absorption, fluorescence, circular dichroism (CD), and light scattering among others have commonly been employed to characterize higher order structures, hydrodynamic properties, and conformational stability of proteins.10.Kamerzell T.J. Middaugh C.R. The complex inter-relationships between protein flexibility and stability.J Pharm Sci. 2008; 97: 3494-3517Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar,36.Mach H. Volkin D.B. Burke C.J. Middaugh C.R. Ultraviolet absorption spectroscopy.Methods Mol Biol. 1995; 40: 91-114PubMed Google Scholar, 37.Wiethoff C.M. Russell Middaugh C. Light-scattering techniques for characterization of synthetic gene therapy vectors.Methods Mol Med. 2001; 65: 349-376PubMed Google Scholar, 38.Esfandiary R. Hunjan J.S. Lushington G.H. Joshi S.B. Middaugh C.R. 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A fluorescent probe of non-polar binding sites.J Mol Biol. 1965; 13: 482-495Crossref PubMed Scopus (1329) Google Scholar Data from these multiple biophysical techniques can be combined in a vector-based stress/response method known as an empirical phase diagram45.Kueltzo L.A. Ersoy B. Ralston J.P. Middaugh C.R. Derivative absorbance spectroscopy and protein phase diagrams as tools for comprehensive protein characterization: A bGCSF case study.J Pharm Sci. 2003; 92: 1805-1820Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar, 46.Maddux N.R. Joshi S.B. Volkin D.B. Ralston J.P. Middaugh C.R. Multidimensional methods for the formulation of biopharmaceuticals and vaccines.J Pharm Sci. 2011; PubMed Google Scholar, 47.Ramsey J.D. Gill M.L. Kamerzell T.J. Price E.S. Joshi S.B. Bishop S.M. Oliver C.N. Middaugh C.R. Using empirical phase diagrams to understand the role of intramolecular dynamics in immunoglobulin G stability.J Pharm Sci. 2009; 98: 2432-2447Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar (EPD). An EPD displays distinct colored regions, which represent different conformational states of proteins and other macromolecular systems as a function of solution conditions such as pH and temperature. In a recent study,47.Ramsey J.D. Gill M.L. Kamerzell T.J. Price E.S. Joshi S.B. Bishop S.M. Oliver C.N. Middaugh C.R. Using empirical phase diagrams to understand the role of intramolecular dynamics in immunoglobulin G stability.J Pharm Sci. 2009; 98: 2432-2447Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar an EPD was generated for an IgG1 monoclonal antibody (mAb-B) based on techniques sensitive to the dynamic properties of proteins such as high-resolution ultrasonic spectroscopy (HR-US), pressure perturbation calorimetry, red-edge excitation shifts (REES), and time-resolved fluorescence spectroscopy. The results showed a more complex pattern of apparent structural transitions at lower temperatures in the pretransition region (below any detectable unfolding event) compared with an EPD generated from biophysical data using static (time-averaged) measurements such as CD, steady-state fluorescence spectroscopy, and light scattering. The pretransition region is defined as a temperature range over which the change in parameters traditionally used to evaluate a protein's secondary structure, tertiary structure, and conformational stability does not deviate from a continuous change with temperature, as studied by methods such as CD, fluorescence spectroscopy, and differential scanning calorimetry (DSC). A better understanding of any relationship between conformational stability and dynamics, especially in the pretransition region, may be important to our understanding of the development and formulation of biopharmaceutical drugs such as monoclonal antibodies (mAbs). Monoclonal antibodies are an important class of dynamic, Y-shaped proteins that are good models for studying the interrelationships between conformational stability and dynamics. The two Fab domains of immunoglobulins are connected to the Fc domain by a highly flexible proline-rich hinge region, which is believed to affect the structure and dynamics of immunoglobulins.48.Hanson D.C. Yguerabide J. Schumaker V.N. Segmental flexibility of immunoglobulin G antibody molecules in solution: A new interpretation.Biochemistry. 1981; 20: 6842-6852Crossref PubMed Scopus (99) Google Scholar,49.Boehm M.K. Woof J.M. Kerr M.A. Perkins S.J. The Fab and Fc fragments of IgA1 exhibit a different arrangement from that in IgG: A study by X-ray and neutron solution scattering and homology modelling.J Mol Biol. 1999; 286: 1421-1447Crossref PubMed Scopus (193) Google Scholar Various analytical techniques have been used to study the flexibility and dynamics of antibodies.20.Kamerzell T.J. Ramsey J.D. Middaugh C.R. Immunoglobulin dynamics, conformational fluctuations, and nonlinear elasticity and their effects on stability.J Phys Chem B. 2008; 112: 3240-3250Crossref PubMed Scopus (34) Google Scholar,27.Kamerzell T.J. Middaugh C.R. Two-dimensional correlation spectroscopy reveals coupled immunoglobulin regions of differential flexibility that influence stability.Biochemistry. 2007; 46: 9762-9773Crossref PubMed Scopus (41) Google Scholar,47.Ramsey J.D. Gill M.L. Kamerzell T.J. Price E.S. Joshi S.B. Bishop S.M. Oliver C.N. Middaugh C.R. Using empirical phase diagrams to understand the role of intramolecular dynamics in immunoglobulin G stability.J Pharm Sci. 2009; 98: 2432-2447Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar, 48.Hanson D.C. Yguerabide J. Schumaker V.N. Segmental flexibility of immunoglobulin G antibody molecules in solution: A new interpretation.Biochemistry. 1981; 20: 6842-6852Crossref PubMed Scopus (99) Google Scholar, 49.Boehm M.K. Woof J.M. 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The conformational stability of antibody drugs, formulated at both low and high concentrations, is significantly influenced by environmental and formulation factors during manufacturing, long-term storage, and administration.55.Kozlowski S. Swann P. Current and future issues in the manufacturing and development of monoclonal antibodies.Adv Drug Deliv Rev. 2006; 58: 707-722Crossref PubMed Scopus (139) Google Scholar, 56.Shire SJ. Formulation and manufacturability of biologics.Curr Opin Biotechnol. 2009; 20: 708-714Crossref PubMed Scopus (237) Google Scholar, 57.Wang W. Singh S. Zeng D.L. King K. Nema S. Antibody structure, instability, and formulation.J Pharm Sci. 2007; 96: 1-26Abstract Full Text Full Text PDF PubMed Scopus (705) Google Scholar The effect of these factors on protein dynamics, however, has not been examined to any great extent. It is therefore important to not only better understand any relationship between conformational stability and dynamics for different mAbs, but to also examine the effect of various environmental factors (e.g., pH, temperature, excipients, etc.) on their conformational stability and dynamics. In this study, the effect of stabilizing and destabilizing excipients on conformational stability and intramolecular protein dynamics of two different IgG1 mAbs (mAb-A and mAb-B) is compared to further understand the relationships between stability and dynamics. The IgG1 mAbs (mAb-A and mAb-B) were provided by MedImmune (Gaithersburg, Maryland). The stock protein solutions were stored as received at 2°C–8°C. The dialysis of stock protein solutions was carried out overnight (at 4°C) using a 10 kDa molecular weight cutoff dialysis cassette (Pierce, Rockford, Illinois) into 20 mM citrate–phosphate buffer at pH values ranging from 3 to 8 at one unit intervals, unless otherwise noted. The final ionic strength of the buffer was adjusted to 0.1 using NaCl. All of the buffer components and other chemicals were purchased from Sigma (St. Louis, Missouri) and Fisher Scientific (Pittsburgh, Pennsylvania). The protein concentration was measured at room temperature by absorbance measurement at 280 nm using an extinction coefficient 1.45 mL/(mg cm) in an Agilent 8453 UV–visible spectrophotometer (Palo Alto, California), and diluted to the final concentration as indicated in each experiment. For each of these four techniques, the experimental method as applied to mAbs has been described previously.47.Ramsey J.D. Gill M.L. Kamerzell T.J. Price E.S. Joshi S.B. Bishop S.M. Oliver C.N. Middaugh C.R. Using empirical phase diagrams to understand the role of intramolecular dynamics in immunoglobulin G stability.J Pharm Sci. 2009; 98: 2432-2447Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar Ultrasonic measurements20.Kamerzell T.J. Ramsey J.D. Middaugh C.R. Immunoglobulin dynamics, conformational fluctuations, and nonlinear elasticity and their effects on stability.J Phys Chem B. 2008; 112: 3240-3250Crossref PubMed Scopus (34) Google Scholar,47.Ramsey J.D. Gill M.L. Kamerzell T.J. Price E.S. Joshi S.B. Bishop S.M. Oliver C.N. Middaugh C.R. Using empirical phase diagrams to understand the role of intramolecular dynamics in immunoglobulin G stability.J Pharm Sci. 2009; 98: 2432-2447Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar,58.Kamerzell T.J. Unruh J.R. Johnson C.K. Middaugh C.R. Conformational flexibility, hydration and state parameter fluctuations of fibroblast growth factor-10: Effects of ligand binding.Biochemistry. 2006; 45: 15288-15300Crossref PubMed Scopus (23) Google Scholar, 59.Shiio H. Ultrasonic interferometer measurements of the amount of bound water. Saccharides.J Am Chem Soc. 1958; 80: 70-73Crossref Scopus (100) Google Scholar, 60.Tamura Y. Gekko K. Compactness of thermally and chemically denatured ribonuclease A as revealed by volume and compressibility.Biochemistry. 1995; 34: 1878-1884Crossref PubMed Scopus (123) Google Scholar were performed using an HR-US 102 Spectrometer (Ultrasonic Scientific, Dublin, Ireland) with a frequency range of 2–18 MHz and a resolution of 0.2 mm/s for velocity and 0.2% for attenuation measurements. The sample and reference cells contained 1 mL of protein and corresponding buffer solution, respectively. The differential velocity and attenuation were monitored at 12 MHz from 10°C to 85°C and pH 3–8 using 5 mg/mL of mAb-A. The temperature of the cells was controlled by a Phoenix P2 water circulator (Thermo Haake, Newington, New Hampshire). The sample and reference solutions were thoroughly degassed before each measurement. Appropriate amounts of sucrose or arginine were added to both protein and buffer solutions while evaluating excipient effects. Data were analyzed using HRUS v4.50.27.25 software (Ultrasonic Scientific, Dublin, Ireland). The coefficient of adiabatic compressibility (βs) was determined using the following equations20.Kamerzell T.J. Ramsey J.D. Middaugh C.R. Immunoglobulin dynamics, conformational fluctuations, and nonlinear elasticity and their effects on stability.J Phys Chem B. 2008; 112: 3240-3250Crossref PubMed Scopus (34) Google Scholar:βs=−1V(∂V∂P)s=−(1v0)(∂v0∂P)s=(βv0)limc→0((β/β0)−V0c)whereV0=ρ−cρ0;andv0=limc→0(1−V0c)β and β0 are the adiabatic compressibility of the solution and buffer, respectively; ρ and ρ0 are the density of the solution and the corresponding buffer; ν0 is the partial specific volume of the IgG; V0 is apparent volume fraction of the buffer; and c is the protein concentration. The adiabatic compressibility of the sample and buffer are related to the density (ρ) and ultrasonic velocity (μ) by the Laplace equation,61.Chalikian T.V. Sarvazyan A.P. Breslauer K.J. Hydration and partial compressibility of biological compounds.Biophys Chem. 1994; 51: 89-107Crossref PubMed Scopus (241) Google Scholar β = 1/ρμ2. The effect of excipients on the compressibility of mAb-A and mAb-B was studied similarly using solution conditions described later in the text. The density of protein samples (5 mg/mL) and corresponding buffer solutions was measured using a DMA-5000 high-precision densitometer (Anton Paar, Graz, Austria) at a precision of 1 × 10−6 g/cm3 and 0.001°C. The densities of degassed solutions were measured from 5°C to 55°C at 2.5°C intervals. The instrument was calibrated daily with dry air and degassed water before analysis. For the excipient studies, both the protein sample and corresponding buffer solution contained equal predetermined quantities of each excipient. The differential scanning calorimetric studies were performed using a MicroCal VP-Capillary DSC with an autosampler (MicroCal, Northampton, Massachusetts). The pH (pH 3–8 at unit intervals) experiments for mAb-A and mAb-B were performed using 1 mg/mL of protein in 20 mM citrate–phosphate buffer (I = 0.1 adjusted by the appropriate addition of NaCl). The temperature ramp was programmed from 10°C to 90°C at a scanning rate of 60°C/h and a filtering period of 16 s. Protein thermograms were obtained by subtracting the corresponding buffer blank from the sample thermogram. The transition midpoints were obtained by determining the baseline using linear or cubic functions, normalizing it to protein concentration, and fitting the processed thermogram to a non-two-state unfolding model. The endothermic peak maximum of the heat capacity was considered to be the apparent transition midpoint (TM) for the individual peaks that could be deconvoluted from the thermogram. The effect of varying concentrations of excipients was studied similarly at pH 4 and pH 4.5 for mAb-A and mAb-B, respectively. Empirical phase diagrams are constructed to visually represent changes in the structural45.Kueltzo L.A. Ersoy B. Ralston J.P. Middaugh C.R. Derivative absorbance spectroscopy and protein phase diagrams as tools for comprehensive protein characterization: A bGCSF case study.J Pharm Sci. 2003; 92: 1805-1820Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar,47.Ramsey J.D." @default.
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• W2003684685 date "2012-09-01" @default.
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• W2003684685 title "Excipients Differentially Influence the Conformational Stability and Pretransition Dynamics of Two IgG1 Monoclonal Antibodies" @default.
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