“Rheometry of Protein Solutions”
Student Name: Lohitaksh Sharma Student ID: 10074449
Supervisor Name: Dr. Robin Curtis
Abstract
Introduction
Commercial production of antibodies started in the early 1980s with Orthoclone OKT3 in 1986 which was used to prevent kidney transplant rejections. The first chimeric antibodies were approved in 1990s and since then the sales of monoclonal antibodies has increases dramatically amounting to nearly $75 billion in 2013 which is nearly half of the total sales of biopharmaceutical products [1].
With production of monoclonal antibodies come many challenges mainly of which are aggregation, high protein concentration, high viscosity, phase separation and opalescence. Successful Formulation depends on understanding the physio-chemical and biological characteristics of the protein.
Figure 1. The many conformational choices for a polypeptide chain [2].
A newly synthesized protein can fold to the native state through many intermediate steps. Along these steps it may go towards aggregation or degradation. An amyloid fibril is an example of an organized but misfolded structure. The protein may also transform into an oligomer which has relatively few repeating units or may form a fibrous structure which has a native protein structure.
Aggregation may occur during formulation in which the protein may be exposed to unsuitable physical conditions which affect its stability and lead to precipitation [3]. A partially unfolded protein which may a result of the steps used in formulation is more susceptible to aggregation due to exposure of the hydrophobic region which are otherwise not present in denatured state or are hidden in natural state [4][5]. Irreversible or reversible aggregation may occur depending on the stable hydrophobic association or slow dissociation kinetics which may impact the drug potency and immunogenicity affecting the protein response [6, 7, 8, 9].
Storing a protein for a long period can lead to aggregation even with a small amount of aggregate present. To prevent aggregation during storage one can add an excipient or design a predictive screening tool to test the formulation for chances of aggregation.
Administering these monoclonal antibodies is another challenge that has many factors to be taken into account. High protein concentration can lead to high viscosity making it hard to deliver in the case of a subcutaneous injection. High viscosity of a protein can also affect the time and force required to administer the drug to a patient. One of the many solutions can be to introduce inorganic salts [10, 11], addition of lysine or arginine [12].
Opalescence and high viscosity can also pose problems for high concentration formulation of antibodies. A protein solution with high turbidity can be confused with a solution having opalescence which is sometimes simply accountable to Rayleigh scattering [13]. Phase separation is a process in which a homogenous protein solution separates into two phases with different protein concentrations. Non-specific interactions like self-association can cause crystallization, liquid-liquid phase separation, condensation, aggregation impacting the solubility and activity of the protein [14].
For therapeutic proteins to be successful commercially they need to maintain their efficacy and properties over a long period of time of extended storage and be administered directly as compared to lyophilized formulation which require reconstitution before they are administered to the patient. To overcome these problems studies need to be done on specific antibodies/therapeutic proteins overcoming problems like high protein concentrations, phase separation and long term storage which can otherwise lead to aggregation and high viscosity making the protein solution ineffectual.
Background
Antibodies
Antibodies (also known as immunoglobulins) are Y-shaped proteins constituting of 4 polypeptide chains and are produced by B cells (better known as B lymphocytes) in the immune system to counteract against foreign proteins or antigens [15]. A term ‘magic bullet’ (by Paul Ehrlich) was coined to a compound administered with a toxin to selectively target the infected protein and destroy the antigen. This further led to an effective treatment of syphilis by Paul Ehrlich and Élie Metchnikoff in 1910 for which they received the Nobel Prize in Medicine. Synthetically produced antibodies use mouse or human B cells and modify them to act against a specific antigen. Produced synthetically they are called monoclonal antibodies that are bonding site specific and bind to one target or polyclonal antibodies that can bind to multiple sites on an antigen.
A normal structure for an antibody which is also a glycoprotein consists of two small light chains and two large heavy chains. The heavy chains can be classified in five different types based on the crystallisable fragments (Fc) attached to the antigen binding fragments and further grouped into five isotypes. Each heavy chain has a variable and constant region. The constant region is same for each antibody from the same isotype while the variable region differs in antibodies than are made from different B cells. Isotypes are prefixed with ‘Ig’ that stands for immunoglobulin differing in structure, biological properties and ability to deal with a specific antigen. Different suffixes are assigned to Ig: α (alpha), γ (gamma), δ (delta), ε (epsilon), μ (mu) [2] giving IgA, IgD, IgE, IgG and IgM respectively. Heavy chains also have a hinge region for added flexibility and is specific to α, γ and δ isotypes. The heavy chain consists of approximately 110 amino acids. Light chains are classifies into two types: λ (lambda) and κ (kappa) [16] and have one constant and one variable domain. A light chain normally has 211 to 217 amino acids [16].
The Y portion of the antibody can recognize foreign objects or antigens and bind to them therefore called Fab (Fragment, antigen-binding) region. This is made up of one constant and one variable domain of each light and heavy chains of the antibody [17]. The variable domain of Fv is the region where the antigens bind to the protein.
Protein-Protein Interactions
Protein-Protein Interactions play a key feature in determining the function of a target protein. PPI can be specified using non-ideal parameters i.e. second virial coefficient (B_22) and interaction parameter (k_D) for dilute solutions and can be used to discern the character of intermolecular interactions [18]. The second virial coefficient (B_22) has been linked to many factors such as precipitation, liquid-liquid separation, crystallization and aggregation [19] and is primarily measured using static light scattering but recently advanced to the use of size exclusion chromatography [20] and dynamic light scattering [21]. The interaction parameter (k_D) for dilute solutions is determined by using dynamic light scattering from the plot of mutual diffusion coefficient (D_m) against protein concentration.
Studies done on one IgG1 subclass and two IgG4 subclass antibodies [22] measured pI, interactions and viscosity. A positive value of k_D or B22 denotes repulsion between molecules while a negative value represents attraction between molecules. The term k_D represents interparticle interactions that can be defined as:
k_D=2MB_22-k_f-2v Eq (1)
where B22 is the second virial coefficient, M is the molecular weight, k_fis the first order coefficient in the expansion of frictional coefficient and v is the protein particle specific volume.
Figure 2. (A-C) shows high concentration viscosity measurements with respect to changing pH (3.0-10.0) using DLS. (M-O) shows the interaction parameter with respect to changing pH (3.0-10.0) which is derived from Eq (1). Circles and full lines denote 10 mM ionic strength measurements, Squares and dotted lines show 50 mH ionic strength measurements and Triangles plus Dash-dotted lines show 150 mM ionic strength measurements [22].
For mAb-1, mAb-2 and mAb-3 increasing pH at constant ionic strength shows increasing attractive forces between molecules with mAb-1 and mAb-2 retaining their pH-viscosity profile towards high pH, increasing ionic strength at low pH for mAb-1 creates more attractive forces as the value of k_D changes to a negative value while there is no change observed for mAb-2. Increasing ionic strength for mAb-3 drastically lowers the value of k_D at for pH ranging from 4.0 to 9.0. Further this study shows that for mAb-3 PPI at high ionic strength are neutralized as they reach a value of -5.34 mL/g [23] which is a defining value for attractive or repulsive dominant forces between molecules. Also the k_D profile for mAb-3 becomes flat at high ionic strength values affirming the presence of electrostatic forces and presence of oppositely charged particles resulting in high viscosity. The general trend for these observations is that at a more negative value of k_D and a lower net-charge leads to a higher viscosity.
Rheology
Subcutaneous injection are preferred over intravenous due to ease of self-administration and simple dosing but they have a limitation on the amount of dosage given to a patient (~1 ml) which creates a necessity to increase the concentration of the dose. High concentrated solutions (>100 mg/ml) can lead to irreversible aggregation or clustering and cannot be further used as therapeutics [24]. At high concentrations, viscoelasticity [25] and high shear viscosity [24] play an important role in determining if the protein is suitable for therapeutic use. To find a solution to this problem, models like Krieger-Dougherty and Derjaguin–Landau–Verwey–Overbeek (DLVO) [26] for colloidal particles have to been used and are comparable to the exponential increase in viscosity with concentration for a protein but fail at high protein concentrations.
Solution Viscosity depends on many parameters of which some are the pH, temperature, Ionic strength and presence of inorganic salts. Studies also suggest that high viscosity of a solution can be linked to charge-charge interactions [27] and can be considered the main reason for increases viscosity. Studies done by introducing apolar hydrophobic inorganic salts to a protein solution show a decrease in the viscosity [28] by disrupting interactions in between protein molecules. To confirm this another study was done involving sugars lacking apolarity and charge which led to increase in viscosity [29].
A study on IgG2 class monoclonal antibody (mAb) measuring viscosity at different pH values (4.0 -7.0) and temperatures (20 °C-65 °C) was done and the resultant viscosity was measured using Dynamic light scattering [30]. For a temperature of 20 °C and a high protein concentration of 150mg/ml the viscosity is highly dependent on pH, increasing slightly from 5.9-7.9 cP for a pH range 4.0-5.5 but more notably from 7.9-23.9 cP for a pH range 5.5-7.0. Viscosity at a specific pH for changing temperature showed and increasing trend for pH values of 4 while for pH 4.5 it decreased to a minimum of 3.8 cP and then increasing again. The trend similar to pH 4.5 carried on for pH values ranging from 4.5 onwards till 7.0 with each pH being 0.5 apart from each other. The study also showed that at high temperature above 40 °C protein starts to aggregate hence rendering the measurement of viscosity at higher temperatures impractical.
Figure 3. Dynamic viscosity of the protein solution as given above with respect to pH and temperature [30].
The diffusion coefficient d_c can be found out by using dynamic light scattering and η, viscosity can be calculated using viscometry but instead of these approximate methods we can use GSE to calculate and relate viscosity to the diffusion coefficient and osmotic compressibility. Generalized Stokes-Einstein equations (GSE) proposed by Kholodenko and Douglas [31] relates d_c to η and to the square root of osmotic compressibility (isothermal) as:
(d_(c ) (ϕ)η(ϕ))/(d_( 0) η_0 ) √(S(q→0,ϕ) )≈1 Eq (2)
where S denotes the osmotic compressibility, q is the wavenumber and φ the particle volume fraction. This approximate validity of GSE equation is useful in determining η from a DLS experiment. The plausibility of this equation can be verified by using data sets obtained ford_c, η and S(q→0) at low and high concentration of BSA (Bovine Serum Albumin). Experiments done with no salt added and salt added at 150 mM concentration were performed which confirm the correctness of the equation proposed by Kholodenko and Douglas [32]. A plot of the LHS of equation 4 against concentration of particles (c_p) shows that for no added salt the equation in not valid for non-zero concentrations while if salt was added the equation remains valid up to 90 mg/mL.
This study confirms that under low salt concentrations the KD-GSE equation is not valid but can be applied for high salt concentrations. For high values of φ that is above 0.3 the equation loses its validity confirming non applicability to macro molecules.
Optical Microrheology is both scattering and imaging based, and has been a good resource for rheological characterisation for a big range of complex fluids [33] [34]. This method involves the use of tracer probe particles to measure the relation between deformation and stress in materials analogous to mechanical rheometry. Optical microrheology employs the Brownian movement of the tracer particles and hence applies low stress on the fluid which is essential for many proteins which exhibit notable strain sensitivity. Commonly methods utilise Diffusion Wave Spectroscopy which involves using a high concentration of tracer particles which in turn can associate with the protein molecules. To prevent this a study has been done on using Dynamic Light Scattering with lower concentration of tracer particles and change in the tracer surface chemistry [35]. Melamine resin and polystyrene tracer particles were used for this study. Mean squared diameter data was used to validate GSE and melamine resin based tracer particle was preferred with a size lesser than 615 nm to cause no aggregation. This led to studies with the same tracer particle size and viscosity was found using GSE that leads to a confirmation that both particle size and surface chemistry of the tracer particle impact process used. If optimised the process can be used efficiently with less time to get quick measurements with small sample volumes.
Methodology
BSA solution preparation
BSA samples were obtained from Sigma-Aldrich and do not need to be purified. A buffer solution was prepared before each experiment and fixed at specified ionic concentrations using Henderson-Hasselbalch equation. Each buffer solution is filtered through a vacuum filter (Stericup, Thermo Scientific Ltd., U.K.). The protein solution was concentrated using Ultracentrifuge Filter (Merck-Millipore Ltd., Ireland) and further placed onto a dialysis cassette. The dialysis cassette is placed in a dialysis buffer and stirred for a fixed time. The filtered sample is used for further sample dilution experiments.
Static Light Scattering
To determine the second virial coefficient, B_22 static light scattering is used. An equations is given for a system with protein, salt and water relating light scattering [36]:
K_c/R ̅_θ =1/RT (∂Π/∂c)=1/(M_w S(q→0))=1/M_w +B_22 c Eq (3)
where K_c is the light scattering constant, R ̅_θ is excess measured scattering ratio of the protein solution above the solvent, M_w is the molecular weight of the protein, c is the protein mass concentration, Π is osmotic pressure of the protein solution above the salt solution, R is the gas constant. In SLS, R ̅_θ is measured for a protein solutions with varying concentration. To find the value of B_22 as plot of K_c⁄R ̅_θ vs c is made where the slope is the second virial coefficient and inverse of the y intercept is〖 M〗_w. The SLS experiments are carried out using a Wyatt miniDAWN TREOS detector paired with Calypso, an automated syringe delivery system with three pumps.
Dynamic Light Scattering
For a Dynamic Light Scattering experiment we measure the intensity autocorrelation function C(q,t). This is given as a function of intermediate scattering function given as:
C(q,t)=A+B〖g(q,t)〗^2 Eq (4)
where A is instrument dependent optical constant and B is the background term determined for each sample in the limit of delay times.
The density correlation function for a monodispersed particle system can be given as an exponential decay:
g(q,t)=exp(-Dq^2 t) Eq (5)
where q is the scattering vector magnitude given as, q=4πn_0 sin〖(θ/2)/λ〗 , θ is the scattering angle and D is the diffusion coefficient.
The diffusion coefficient can be calculated as a function of concentration, C and interparticle interactions, k_D :
D=D_0 (1+k_D C) Eq (6)
where D_S is the self-diffusion coefficient. The infinite dilution coefficient, D_0 can also be used to calculate the hydrodynamic radius, r_H from the Stokes-Einstein equation with an assumption that the molecules are spherical.
D_0=(k_B T)/(6πηr_H ) Eq (7)
where T is the Temperature in Kelvin, k_B is the Boltzmann constant and η is the solvent viscosity. The diffusion coefficients are measured using a DynaPro PlateReader (Wyatt, Santa Barbara, CA).
DLS tracer particle experiments
Tracer particles are added to the BSA solutions and are allowed to disperse for about 12 hours before they are used for experiments. The dispersion of tracer particles is checked with stability of the count rate with time before the solution is used for measurement. A fixed tracer particle concentration of was added to a given sample size. Dynamic Light Scattering (DLS) is then used to measure the output of light intensity from the tracer particles and protein. This is used to determine the final concentration of tracer particles at which the scattering intensity of the particles is more than the protein because at higher concentration of protein, the protein adsorbs on to the tracer particles making it aggregated and leading to a higher viscosity. Probes are inserted until the particle size distribution covers ~90 % of the total area. The solution viscosity can also be given as a virial expansion of a polynomial:
η=η_0 (1+k_1+k_2 c^2+k_3 c^3+⋯) Eq (8)
where η and η_0 are solution and solvent viscosity respectively, c is concentration in g/mL and the k1, k2 and k3 are respective virial coefficients. A study on an IgG1 antibody was performed to correlate the relation between intrinsic viscosity and molecular size [37]. The reduced viscosity, η_red can be related to the concentration by Huggins relation:
η_red=η_sp/c=[η]+k_H [η]^2 c Eq (9)
where k_H is the Huggins coefficient and η_sp is the specific viscosity which can be defined as:
η_sp=((η-η_0 ))/η_0 Eq (10)
The expression [η] can be obtained by estimating η_sp⁄c to zero and is given as:
[η]_H=lim┬(c→o)〖η_sp/c〗=lim┬(c→o)〖(η_r-1)/c〗 Eq (11)
where η_r is relative viscosity equal to η⁄η_0 . Within the hydrodynamic region Equation 2 can be divided into two parts where B_22 contributes to the thermodynamic regime, k_T and k_f+〖2v〗_2 to the hydrodynamic/drag region,〖 k〗_H [38].
k_D=k_T-k_H Eq (12)
Protein-Protein Interactions can be linked to these theological measurements through the use of viscosity-concentration relations and then can be further used to find the Huggins coefficient,〖 k〗_H.
DLS tracer particle experiments with surfactant
The tracer particles most likely adsorb the protein at higher concentrations, to minimize this we first test the tracer particles at low and high salt concentrations with added surfactant testing scattering of the particles with DLS.
BSA solutions of concentration range 0-250 g/L are then taken and the viscosity is measured with and without added NaCl at pH 5 and 7 with surfactant. These measurements are also taken without added surfactant for the same pH range. All of these measurements include tracer particles to measure light scattering using DLS. The same measurements are also carried out without tracer particles and used to determine interparticle interaction,〖 k〗_D using Equation 1, 6 and the diffusion coefficient, D_S using Stokes-Einstein equation which is also given as Equation 7.
Planning
Impact
We rely mostly on intravenous drugs to deliver antibodies for counteracting against antigens present in our body. High amounts of drugs can be delivered through the blood stream but is limited by the viscosity of the protein. Intravenous drugs are rarely self-administrable and require a person to reach out to a doctor. Subcutaneous drugs often called SubCut are drugs injected in the skin layer which is directly below the dermis and epidermis. They are highly preferred and effective for administration of vaccines and medications but are limited by the amount and concentration of the protein. Here we study the effect of pH, temperature and ionic strength with added salts or excipients on the viscosity of protein ranging from base 0 to 250 mg/L concentration.
Success of this project will lead to advancements in the area of drug delivery for high concentration of proteins. There are many societal benefits to this study one of which is self-administration. People who are unable/do not want to go to a doctor for vaccination may buy these drugs under prescription and may self-administer them. This can lead to an increase in the amount of vaccination performed due to ease of availability if the industry manages to keep up with the demand. Increased production leads to increased supply also impacting the economy.
Personal benefits include ability to further research in the Rheological behavior of other proteins and other parameters influencing the viscosity.
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