Opalescence is a phenomenon that has been observed in several commercially available monoclonal antibodies, both in liquid and reconstituted lyophilized formulations. In this article, we demonstrated that an increase in the ionic strength of a monoclonal antibody formulation (MAb1) led to opalescence and higher viscosity. When the ionic strength was reduced, no opalescence in the MAb1 formulation was observed. The removal of polysorbate-80 (PS-80) from the formulation resulted in an increase in opalescence in NaCl-containing formulations, whereas it had no effect on formulations lacking NaCl. Differential scanning calorimetry with MAb1 formulations containing increasing amounts of NaCl indicated that formulations with higher ionic strength present a lower apparent melting temperature. Opalescent MAb1 formulations placed on stability remained unchanged after four weeks at 4 °C, whereas at 45 °C, an increase in dimers was observed. Using multi-angle light scattering, the MAb1 formulation was found to have a negative second virial coefficient.
It is well known that ionic strength can affect the behavior of proteins in solution. In most cases, at low and high salt concentrations either salting-in or salting-out occurs, respectively.5 Salting-in is observed when electrostatic interactions between the salt ions and charged residues of the protein are favorable.5 Salting-out occurs when the salt ions are excluded from the protein, which is mainly caused by unfavorable interactions between the salts and hydrophobic regions of the protein.5 There are examples, however, in which proteins are soluble at high salt concentrations.5–7
The ionic strength can also mediate protein–protein interactions. One example is with the protein b-lactoglobulin, which is predominantly monomeric at pH 3 in the absence of salt, but is dimeric in the presence of salt.8 Additionally, the type of salt may also affect the stability of the protein. Bovine serum albumin is stabilized against thermal unfolding with NaSCN and NaClO4, both kosmotropic salts, yet destabilized by chaotropic salts at high ionic strength.9,10 In other examples, aggregation was decreased in the presence of NaCl for both recombinant factor VIII SQ and recombinant keratinocyte growth factor, whereas aggregation increased in the presence of NaCl with recombinant human granulocyte colony stimulating factor.7,11
In the following article, we sought to determine whether ionic strength and excipients mediated the opalescence of an IgG1 formulation (MAb1). It was demonstrated that MAb1 formulations become opalescent as the ionic strength of the formulation is increased. Conversely, MAb1 formulations without salt lack opalescence. The second virial coefficient of MAb1 was negative. Stability studies indicated that opalescent MAb1 formulations have increased amount of irreversible dimers at elevated temperatures.
MATERIALS AND METHODS
Materials and Reagents
MAb1 is a fully human IgG1 monoclonal antibody. MAb1 was purified from Chinese hamster ovary (CHO) cells by Bioprocess Research and Development, Merck Research Laboratories (Whitehouse Station, NJ). The MAb1 formulation contains 24 mg/mL IgG1 in a formulation containing a buffer, NaCl, and polysorbate-80 (PS-80), pH 6. Polysorbate-80 was from Croda Incorporated (Mill Hall, PA). NaCl, KCl, MgCl2, KSCN, Na3PO4, CsCl, Na2SO4, hexamethylenetetramine, and hydrazine sulfate were from Sigma (St. Louis, MO). The filter used was a 0.22 μm Millex GV from Millipore (Billerica, MA).
Opalescence of the samples was assessed according to the European Pharmacopoeia (EP) 5.0 (2.2.1). Opalescence reference suspensions were made using hexamethylenetetramine and hydrazine sulfate. Samples were evaluated at a volume of 11 mL in 20 mL glass vials by comparing them to the reference suspensions in diffused daylight against a black background.
The opalescence of MAb1 was measured by subtracting the optical density (OD) at 350 nm from the OD at 550 nm using an HP spectrophotometer as previously described.12
Size Exclusion High Performance Liquid Chromatography
Size exclusion high performance liquid chromatography (SEC–HPLC) was performed using a TSKgel 3000SWXL column from Tosoh Corporation (Tokyo, Japan) with a mobile phase consisting of 25 mM phosphate, 0.3 M NaCl, pH 7.0. The flow rate was 0.5 mL/min. The temperature of the column was maintained at 25 °C. The samples were detected at 230 nm.
Dynamic Light Scattering (DLS)
The particle sizes of samples tested were measured by dynamic light scattering (DLS) using a Zetasizer Nano System from Malvern Instruments (Malvern, UK). A total of 10 measurements for each sample were taken and the results were reported as the Zave, the average hydrodynamic size.
Viscosities were measured using a LVDV-III Ultra Programmable Rheometer by Brookfield Engineering (Middleboro, MA). Sample volumes of 0.5 mL were tested for each measurement.
Determination of the Osmotic Second Virial Coefficient Values
The osmotic second virial coefficient was obtained using a Dawn Heleos multi-angle light scattering (MALS) instrument from Wyatt Technology Corporation (Santa Barbara, CA). The formulations containing buffer and 150 mM NaCl (no PS-80) were diluted and injected into the instrument as follows: 1:2.5 (8.20 mg/mL), 1:5 (4.10 mg/mL), 1:10 (2.05 mg/mL), and 1:20 (1.03 mg/mL). The Rayleigh equation was used by the computer software to generate data on the Zimm plot. The software also calculated the molecular weight and particle size of the sample.
in which C is the particle concentration (g cm-3 ), Rθ is the the Rayleigh ratio—the ratio of scattered light to incident light of the sample (cm-1), M is the sample molecular weight (g mol-1 ), A2 is the 2nd virial coefficient (cm3 mol g-2), P(θ) is the angular dependence of the sample scattering intensity, and K is the optical constant, which is calculated using Equation 1.1:
in which NA is Avogadro's constant (mol-1), λo is the Laser wavelength (cm), no is the solvent refractive index, and dn/dc is the the differential refractive index increment (cm3 g-1).
For particles that are smaller than the incident light (laser) used to measure the particle size, Equation 1 can be reduced to the linear form shown in the following:13
Increase in Ionic Strength Correlates to an Increase in Opalescence
Effect of Excipients on the Opalescence of MAb1
Opalescence and Impact of Different Salts
Impact of Ionic Strength on Viscosity
Differential Scanning Calorimetry of MAb1 with Increasing Amounts of NaCl
Osmotic Second Virial Coefficient of the MAb1 Formulation
The osmotic second virial coefficient is commonly indicated by the term A2 or B22. When the second virial coefficient is measured using light scattering, it reflects protein–protein interactions as well as contributions from protein–cosolute interactions and protein nonideality.16,17 A positive B22 value correlates to net repulsive forces between solute molecules, and negative B22 values indicate net attractive forces between solute molecules.16,18,19 Several methods can be used to measure the B22 value of a formulation, one of which is the use of multi-angle light scattering with dilutions of the formulation. This method was used to obtain second virial coefficient values for MAb1.
A Zimm plot of MAb1 in the formulation containing a buffer and 150 mM NaCl is depicted in Figure 10. PS-80 was removed from the formulation to prevent potential interference from micelles. It was determined that this formulation had a negative second virial coefficient (–4.3 x 10-5 mol mL/g2). The molecular weight calculated for MAb1 was 1.46 x 105 g/mol (146 kDa), which is close to the molecular weight of MAb1, which is 148 kDa.
The purpose of this study was to investigate whether the ionic strength and excipients in the MAb1 formulation were associated with the opalescence observed. We identified that the ionic strength of the MAb1 formulation played a major role in mediating opalescence.
The opalescence of MAb1 was demonstrated to increase with ionic strength. In the MAb1 formulation containing NaCl, opalescence was pronounced (Reference III opalescence standard) whereas in its absence, the solution was clear (Reference I opalescence standard). It was also observed that the apparent hydrodynamic radius of the MAb1 formulation was larger at higher concentrations (20 mg/mL) and lower at more dilute concentrations (1 mg/mL). The reason for the larger particle size at higher concentrations of MAb1 is not known, and will require further investigation.
We explored whether salts other than NaCl had an impact on opalescence. Salts were selected based on their position in the Hofmeister series, and included KCl, MgCl2, KSCN, Na3PO4, CsCl, and Na2SO4.14,15 Opalescence was observed in the presence of all salts tested, and no specific ion effects were observed.
As the concentration of NaCl was increased, the electrostatic shielding becomes saturated and it is possible that interactions between the hydrophobic regions of MAb1 become dominant. An example of electrostatic shielding and enhancement of hydrophobic effects in the presence of NaCl was demonstrated in studies using lysozyme.5,20
There are other examples of the effect of ionic strength on proteins. It was demonstrated that when recombinant factor VIII SQ was placed in a solution containing 100 mM NaCl at pH 7, the solution turned opalescent within the first hour.7 However, after the first hour, the protein precipitated out of solution.7 At higher concentrations of NaCl, the precipitate could be dissolved, and activity was observed to recover.7
The interactions between MAb1 molecules described in these studies were not strong enough to precipitate the MAb. It is not known, however, if the increase in opalescence correlates to molecular self-association of MAb1 molecules. Sukumar and colleagues have attributed opalescence of an IgG1 formulation to Rayleigh scatter and indicated that opalescence is not caused by noncovalent association.4
Effects of Excipients
PS-80 has been widely used in liquid parenteral formulations to reduce aggregation caused by surface interactions, to prevent denaturation at the air–liquid interface, and to reduce agitation and temperature-induced aggregation.12,21 In this study, it was demonstrated that PS-80 was able to reduce the opalescence of the MAb1 formulation containing 150 mM NaCl. It is possible that PS-80 partially disrupted the hydrophobic–hydrophobic interactions that are dominant following the initial shielding of electrostatic charges.
Effects of Viscosity
We also explored the effect concentration and NaCl had on the viscosity of MAb1 solutions. It was determined that the viscosity increased as the protein concentration increased. Similar results were obtained by Liu and colleagues for an IgG at a concentration range of 30 to 125 mg/mL in a formulation containing 16 mM histidine, 266 mM sucrose, and 0.03% PS-20, pH 6.22 The viscosity of MAb1 solutions also increased with NaCl concentration. The increase in viscosity of MAb1 solutions with increasing NaCl, however, was in contrast to the results obtained with an IgG by Liu and colleagues.22 In their studies, increasing the molarity of NaCl resulted in a decrease in viscosity.22 The differences in the viscosity increase or decrease in the presence of NaCl when comparing MAb1 to the IgG studied by Liu and colleagues may be caused by sequence-specific variability in the complementary determining regions (CDRs) in the heavy and light chains of the molecules.
Differential Scanning Calorimetry
The thermal unfolding of MAb1 presents two main transitions. Based on the amplitude of these transitions, the first transition corresponds to the unfolding of the CH2 domain in the Fc fragment and the second transition corresponds to the unfolding of the Fab fragment and CH3 domain in the Fc fragment of the IgG molecule.25 The apparent melting temperature for both transitions is slightly decreased as the concentration of the salt is increased. In the absence of salt, the second transition presents a shoulder that may reflect a reduced overlap between CH3 and Fab unfolding. This observation suggests that the Fab fragment is more sensitive to the presence of salt than the CH3 domain.
Second Virial Coefficient
In the presence of NaCl, MAb1 had a negative B22. It is not known if the negative second virial coefficient of MAb1 was associated with the opalescence observed or was caused by the properties of the MAb. It could be hypothesized that net attractive interactions are associated with opalescence, however additional studies will be required to test the idea. It would also be interesting to evaluate MAb1 formulations lacking NaCl to determine if the second virial coefficient changes.
Studies with other proteins have shown that the second virial coefficient decreases as the concentration of NaCl increases.26 For example, it was demonstrated that the second virial coefficient of the peptide enfuviritide decreases with an increase in salt.26 In other studies, including one with an IgG, it was demonstrated that the second virial coefficient decreases as the concentration of NaCl increases.22,27
Stability of Opalescent IgG Formulations
It was determined that the ionic strength of the MAb1 formulation does not affect the stability at 4 °C after four weeks. However, at elevated temperatures for four weeks in the presence of NaCl, irreversible dimer formation occurs, as demonstrated by SEC–HPLC. Interestingly, there was no significant change in high-order aggregate formation as the amount of NaCl was increased (data not shown).
The stability of an opalescent IgG1 formulation has been previously described by Sukumar and colleagues. In those studies, it was determined that the hydrodynamic size of formulations ranging from 0.5 to 50 mg/mL at 25 °C remained unchanged up to 300 minutes.4
An example in which NaCl induced dimerization of an IgG occurred was with the monoclonal antibody rhuMAb (VEGF).28 In the presence of 1M NaCl, a decrease in the kD (the dissociation constant) for rhuMAb (VEGF) was observed, which resulted in the formation of dimers.28 In the same study, it was demonstrated that another salt, CaCl2, had the same effect as NaCl, in that the increased ionic strength resulted in dimerization of rhuMAb (VEGF).28 These results parallel the study described here, in which higher levels of NaCl result in dimer formation. In separate studies with an IgG, it was demonstrated that the percentages of IgG aggregates increased with increasing ionic strength.29
Overall, the studies reported here demonstrate that the ionic strength of a formulation as well as the excipients, play an important role in the opalescent appearance of an IgG1 monoclonal antibody. These factors may be relevant to the appearance and stability of other IgG formulations under stressed conditions.
Craig McKelvey and Yang Wang are acknowledged for helpful discussions.
Ning Wang is a research biochemist, Binghua Hu is a research biochemist, Roxana Ionescu is a research fellow, Henryk Mach is a senior investigator, when this article was written, Joyce Sweeney was a senior investigator, Christopher Hamm is a research biochemist, Marc J. Kirchmeier is an associate director, and Brian K. Meyer is a research fellow, all in Bioprocess Analytical and Formulation Sciences, Bioprocess Research and Development, at Merck Research Laboratories, Merck & Co., West Point, PA, 215.652.3992,
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