Phase separation in solutions of monoclonal antibodies and the effect of human serum albumin

Ying Wanga,1, Aleksey Lomakina, Ramil F. Latypovb, and George B. Benedeka,c,d,1

aMaterials Processing Center; cDepartment of Physics; and dCenter for Materials Science and Engineering, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 02139; and bProcess and Product Development, Amgen Inc., Seattle, WA 98119

Contributed by George B. Benedek, July 27, 2011 (sent for review June 23, 2011)

We report the observation of liquid-liquid phase separation in a tant as an underlying metastable phase transition, which substan- solution of human monoclonal antibody, IgG2, and the effects tially affects kinetics of these other condensation processes. of human serum albumin, a major blood protein, on this phase Recently, LLPS of several pharmaceutical antibodies have separation. We find a significant reduction of phase separation been reported (5–9). There are five isotypes of mammalian anti- temperature in the presence of albumin, and a preferential parti- bodies with distinct Fc regions, including IgA, IgD, IgE, IgG, tioning of the albumin into the antibody-rich phase. We provide a and IgM. For each isotype, there are also large numbers of idio- general thermodynamic analysis of the antibody-albumin mixture types with different Fab regions. Due to this great variety of anti- phase diagram and relate its features to the magnitude of the bodies, their condensation may occur at noticeably different effective interprotein interactions. Our analysis suggests that conditions. As a cooperative phenomenon, LLPS is sensitive to additives (HSA in this report), which have moderate attraction with rather small changes in the average interprotein interaction, and antibody molecules, may be used to forestall undesirable proetin thereby can provide a useful tool to evaluate the propensities condensation in antibody solutions. Our findings are relevant to of different antibodies to condense. understanding the stability of pharmaceutical solutions of antibo- High concentrations of both monoclonal and polyclonal anti- dies and the mechanisms of cryoglobulinemia. bodies also occur in the blood of patients with immunopro- liferative disorders associated with a number of diseases, such as: biopharmaceuticals ∣ coexistence curve ∣ critical point ∣ cryoprecipitation ∣ multiple myeloma, hepatitis C, and HIV. In these cases, excessive immunoglobulin endogenous antibodies (mainly IgG, IgM, and their mixtures) precipitate in blood at temperatures lower than 37 °C. This med- – ntibodies are widely used in research and biotechnology, as ical phenomenon is called cryoglobulinemia (10 12). Sometimes, intravascular condensation of antibodies can even occur at body well as in medical and pharmaceutical applications. In some A temperature and have adverse physiological consequences such cases, concentrated solutions of specific antibodies are required. as auto immunogenicity, increase in blood viscosity, and deposi- In particular, monoclonal antibodies (MAb) have become a ma- tion in blood vessels. The cryoglobulinemia is reversible upon jor category of drugs in the treatment of a variety of diseases (1). raising the temperature, and antibodies may maintain their ability In some drug delivery routes e.g., subcutaneous administration, to bind to antigen. These characteristics are consistent with LLPS. formulations with concentrated antibody solutions are required In order to investigate the propensity of antibodies to undergo to achieve therapeutic dosing (2). protein condensation in vivo, both in the case of cryoglobulinemia The physiological functions of antibodies are mostly determined and in the pharmaceutical applications, the solution conditions by antibody-antigen and antibody-receptor specific interactions. of blood serum must be taken into account. Here we report the However, the nonspecific interactions between antibodies (i.e., study of the LLPS of a monoclonal human antibody, which is self-association) in the concentrated antibody solutions can also denoted by IgG2-A as in ref. 5, under solution conditions mimick- affect their functions. Nonspecific attractive interactions can cause ing those in a blood serum. Specifically, we investigated LLPS various forms of condensation, including liquid-liquid phase separa- at physiological pH (pH ¼ 7.4) in the presence of human serum tion, aggregation, and crystallization. Upon such condensation, albumin (HSA), which is the major protein component in blood antibodies lose their solubility, and may lose their biological activity. serum. Particularly, in pharmaceutical industry, these processes impact The solution conditions, such as protein concentration, com- storage stability and safety of protein therapeutics thus impeding position, temperature, buffer properties, etc., under which LLPS drug development (3). For example, immunogenicity of some bio- occurs are represented by a phase diagram. The phase diagram logics has been attributed to formation of protein aggregates (4). may be viewed as a collection of coexistence curves which repre- The mechanisms of protein condensation are complex and depend sent the dependence of phase separation temperature on the on protein concentration, buffer composition, temperature, etc. protein concentration at various conditions. In this work, we have Clearly, the factors which affect protein condensation throughout determined the coexistence curves of a MAb solution in the the shelf-life must be understood and controlled to ensure biother- presence of various concentrations of HSA. Here we show that apeutic effectiveness. the MAb solutions have much lower critical concentration and One important condensation process is liquid-liquid phase much wider coexistence curve as compared to solution of com- separation (LLPS). In LLPS, a homogeneous protein solution pact globular proteins. We ascribe this difference to extended spontaneously separates into two coexisting phases with differ- Y-like shape of MAb molecules. Further, we find that HSA pre- ent protein concentrations. This phenomenon takes place upon ferentially partitions into protein-rich phase and lowers phase changing the temperature or other solution conditions, and is reversible. In contrast to aggregation or crystallization, LLPS, “ ” Author contributions: Y.W., A.L., R.F.L., and G.B.B. designed research; Y.W., A.L., and while highly sensitive to the average net attractive interaction R.F.L. performed research; Y.W., A.L., and R.F.L. contributed new reagents/analytic tools; between proteins, are much less sensitive to the distribution Y.W., A.L., and G.B.B. analyzed data; and Y.W., A.L., R.F.L., and G.B.B. wrote the paper. pattern and the nature of the “local” interactions on the protein The authors declare no conflict of interest. surface. As a result, LLPS exhibits universal features applicable 1To whom correspondence may be addressed. E-mail: [email protected] or [email protected]. to a variety of proteins. LLPS is often superseded by aggregation, This article contains supporting information online at www.pnas.org/lookup/suppl/ gelation, or crystallization. In such cases, LLPS can still be impor- doi:10.1073/pnas.1112241108/-/DCSupplemental.

16606–16611 ∣ PNAS ∣ October 4, 2011 ∣ vol. 108 ∣ no. 40 www.pnas.org/cgi/doi/10.1073/pnas.1112241108 Downloaded by guest on September 30, 2021 0 0 0 c = 54 mg/mL A 1 B -1 c = 98 mg/mL c = 0 mg/mL -1 1 -1 2 (°C) (°C) -2 ph

ph c = 4 mg/mL -2 2 T -2 T (°C) -3

ph c = 8 mg/mL T -3 -4 -3 2

-5 -4 c = 12mg/mL -4 2

-6 Temperature, Temperature, -5 -5 -7 0 5 10 15 20 50 100 150

Temperature, Concentration of HSA, c (mg/mL) Concentration of MAb, c (mg/mL) -6 2 1 T -7 Fig. 2. (A) Decrease of LLPS temperature, ph, vs. the HSA concentration, c c T c c 050100150200250 2, at fixed MAb concentration, 1. Linear fitting of ph vs 2 at each 1 is Concentration of MAb, c (mg/mL) shown by the dashed line. (B) LLPS boundaries at fixed c2 shift to lower 1 temperature as HSA concentration, c2, increases (The data were obtained Fig. 1. Liquid-liquid phase separation of MAb solutions in 0.1 M Tris·HCl by interpolation of the data in SI Appendix; Fig. S1). buffer at pH 7.4. The eye guide for the LLPS boundary is indicated by the dashed line. The crossed square is the critical point determined at the max- Liquid-Liquid Phase Separation of MAb-HSA-Water Ternary Solutions. imum of the phase boundary. In view of the fact that HSA is a major component of blood ser- um, we have measured the effect of HSA on the phase separation separation temperature. Finally, we present the theoretical ana- of MAb in aqueous solutions at physiological pH. We find that, lysis of these phenomena, show that they imply an attractive regardless of MAb concentration, the addition of HSA lowers the interaction between MAb and HSA, and evaluate the magnitude phase separation temperature in direct proportion to the HSA of this interaction. c ð∂T ∕∂c Þ concentration, 2. We list in Table 1 the values of ph 2 c1. We see that this derivative is approximately independent of c1, Results and has the average value: −0.24 0.03 °C · mL∕mg. Therefore, The Coexistence Curve of MAb-Water Binary Solution at Physiological at the typical concentration of HSA in blood of ∼40 mg∕mL (15), pH. We have measured the temperature for phase separation, ∼9 T c HSA reduces the phase separation temperature by °C. Thus, ph, of our MAb as a function of antibody concentration, 1, HSA may have a significant role in preventing condensation of and obtained the coexistence curve shown in Fig. 1. In a binary antibodies in blood at body temperature. solution, the maximum temperature occurs at the critical point. The values in Table 1 were found by measuring the decrease in Thus, in Fig. 1 we observe that the critical temperature, Tc,is phase separation temperature upon adding HSA at fixed MAb −0 6 0 1 c equal to . . °C, and the critical concentration, c,is concentration. T decreases linearly with increase of c2 as shown 90 9 ∕ ph mg mL. For temperatures greater than the critical tem- in Fig. 2A at two representative values of c1. (See Fig. S1 in SI perature, the MAb solution remains in a stable homogeneous Appendix for the entire dataset). In Fig. 2B we plot the coexis- T T c phase for all concentrations. For temperatures below c, the tence curves ( ph, 1) at several values of HSA concentration, coexistence curve specifies the concentrations of the two coexist- c2. We see that the entire coexistence curve shifts downwards ing liquid phases corresponding to that temperature. as c2 increases. Fig. 2 A and B represent two cross sections of Using the value of 0.71 mL∕g for the protein specific volume a phase diagram, which describes the solution conditions required (13), we find that the critical concentration corresponds to a for LLPS of MAb in the presence of HSA. Due to the diversity critical volume fraction of 6.3%. This value is quite small consid- of antibodies, the critical temperatures, Tc, of different antibo- ering that in solutions of spherical particles the critical volume dies may vary widely. Indeed, in the case of cryoglobulinemia, fraction varies from 13% to 23% as the spatial range of the inter- antibodies form condensates even at body temperature. Thus, BIOPHYSICS AND particle interaction varies from infinity to zero (14). The small the phase diagram can become a clinically important representa- value of the critical volume fraction of MAb reflects the extended, tion of the conditions under which pathophysiological protein COMPUTATIONAL BIOLOGY highly nonspherical shape of antibody molecules. This value condensation can occur in blood. implies that the volume of a spherical particle, which matches the In LLPS, the concentrations of MAb as well as the concentra- observed critical concentration, is at least twice as large as the tions of HSA are different in the two coexisting phases. The actual volume of the antibody molecule. actual partitioning of these two proteins depends on the magni- The phenomenon of separation into coexisting liquid phases tude and the sign of the interprotein interactions between pairs signifies attractive interactions between the antibody molecules of MAb-MAb, MAb-HSA and HSA-HSA. These interprotein CHEMISTRY (14). Such attractive interactions can also lead to crystallization interactions are also responsible for the suppression of LLPS temperature of MAb solutions upon the addition of HSA. There- and aggregation of the antibody molecules. All these condensa- fore, it is important to measure quantitatively the actual compo- tion phenomena of pharmaceutical or endogenous antibodies sitions of MAb and HSA in the two coexisting phases. can have serious pathophysiological consequences in vivo such as immunogenicity, increase in blood viscosity, and deposition Partitioning of MAb and HSA in the Coexisting Phases. At any fixed in blood vessels. From this perspective it is important to investi- temperature beneath Tc, the concentration of each protein in gate how the condensation of antibodies can be affected by other each coexisting phase depends upon the initial concentrations components of blood serum. of the two proteins in the starting solution. We measured the concentrations of MAb and HSA in pairs of coexisting phases Table 1. The rate of change of the phase separation temperature, at fixed temperature. In Fig. 3 we present our results for two dif- T c c ph, with HSA concentration, 2, at a fixed MAb concentration 1 ferent temperatures. In Fig. 3, each pair of two data points repre- Measurement 1 2 3 4 5 senting the two coexisting phases are connected by a so-called “tie-line.” Fig. 3 shows that the concentration of HSA in the c1 (mg∕mL) 37 54 98 114 140 ð∂T ∕∂c Þ − − − − − MAb-rich phase is higher than that in the MAb-poor phase, ph 2 c1 0.22 0.27 0.22 0.24 0.26 i.e., HSA preferentially partition into the protein-rich phase. This (°C · mL∕mg) ±0.03 ±0.03 ±0.03 ±0.03 ±0.03 observation implies that the interprotein interaction between

Wang et al. PNAS ∣ October 4, 2011 ∣ vol. 108 ∣ no. 40 ∣ 16607 Downloaded by guest on September 30, 2021 8 Quasielastic Light-Scattering (QLS) Study of the MAb-HSA Mixture So- lution. We have measured the apparent diffusion coefficients, D, A T = −2.2 °C 7 of protein molecules in pure MAb solutions and in MAb-HSA (mg/mL) 2 6 mixtures containing 30% (w∕w) HSA, as a function of total 5 protein concentration (Fig. S2 in SI Appendix). We deduced the apparent diffusion coefficient, D0, of proteins for infinitely dilute 4 pure and mixed solutions. Using these D0s, we calculated that the 3 0 apparent hydrodynamic radius, Rh , of pure MAb solutions is 0 2 equal to 5.9 0.1 nm. Similarly, we have found the Rh for pure HSA monomers to be equal to 4.1 0.2 nm. In the MAb-HSA 1 0 mixture, the apparent Rh is equal to 5.7 0.3 nm. This value 0 indicates that no heterodimerization or other strong interactions Concentration of HSA, c HSA, of Concentration 050100150200250 between MAb and HSA takes place. The apparent diffusion coefficients decrease with the total protein concentration both 20 in pure MAb solutions and in MAb-HSA mixtures. The negative B T = −4.2 °C value of dD∕dc is indicative of attractive interactions. The value 16 0 (mg/mL) of the normalized slope, dðD∕D Þ∕dc, is less negative for the 2 mixture than that for the pure MAb solution, which implies 12 that HSA diminishes the effective interprotein attraction. This observation is in accord with the suppression of LLPS upon the 8 addition of HSA.

4 Discussion In this work, we report the observation of LLPS of an IgG2 mono- 0 clonal antibody at physiological pH, as well as in the presence of human serum albumin. While LLPS in solutions of globular

Concentration of HSA, c HSA, of Concentration 050100150200250 – Concentration of MAb, c (mg/mL) proteins is well documented (16, 18 21), it is often preempted 1 by aggregation or crystallization. Recently, reports have appeared Fig. 3. Partitioning of MAb and HSA upon LLPS at fixed temperature of such LLPS in solutions of antibodies (5–9, 22). Antibodies can (A) T ¼ −2.2 °C; and (B) T ¼ −4.2 °C. The points representing the two coex- be present in blood at relatively high concentrations. Further- isting phases are connected by the solid lines, i.e., the tie lines. Dashed lines more, antibodies are widely and increasingly used in concentrated are eye guides for the binodal curves fitted from both cloud-point measure- solutions as pharmaceutical drugs. In view of these facts, it is ments (open triangles) and partitioning measurements (solid circles). The very important to quantitatively investigate phase separation critical points are represented by the crossed square. phenomena for these proteins. Indeed the loss of homogeneity MAb and HSA is attractive. In part this attraction may be attrib- due to the formation of droplets of condensed phases can have uted to the electrostatic interaction between MAb and HSA. In- adverse effects both physiologically, and in the manufacturing deed, the isoelectric point of MAb is pI ¼ 8.8 and that of HSA is and storage of MAb-based therapeutics. pI ¼ 5.7 (calculated using www.expasy.org). Thus, at the physio- Phase Diagram of MAb Aqueous Solutions. As is the case with other logical pH 7.4, MAb and HSA carry charges of opposite sign. proteins, the MAb phase diagram provides a comprehensive In Fig. 3, we also designate the binodal curve (c1, c2) at con- delineation of the solution conditions under which phase separa- stant temperature by fitting data from both partitioning measure- tion can occur. Theoretical analysis of this diagram can provide ment and T measurement. Using the method of analysis ph insights into the intermolecular interactions responsible for the described previously (16), we also estimated the positions of condensation of the protein. In previous studies of globular the critical points of the MAb-HSA-water ternary solution. In c 105 proteins, the main features of the coexistence curve such as the Fig. 3, we show that 1 at the critical points are equal to critical temperature, critical concentration, and the width of 10 ∕ mg mL at both measured temperatures. The critical concen- the coexistence curve were successfully explained in terms of the tration of MAb in the ternary solution is equal to that found for effective magnitude, range and anisotropy of the interprotein the pure MAb solution, within experimental error. interactions (14, 23). However, these previous theoretical ∼80% In our samples, of MAb molecules have two pyrogluta- studies were predicated on the model of proteins as spherical mate residues at the heavy chain N termini. In the remaining particles (14, 23). We shall see here that such theories may have fraction, only one of the heavy chains has pyroglutamate, whereas limited applicability to the phase behavior of Y-shaped antibody the other chain has the original N-terminal glutamine. We denote molecules. these two species by the symbols pEpEMAb and QpEMAb respec- Indeed, one of the striking features of the coexistence curve of tively. These two species can be differentiated by CEX HPLC (5, pure MAb is the very small value of the critical concentration, cc, ϕ ¼ c v v ¼ 0 71 ∕ 17). We have measured (Table S1 in SI Appendix) molar ratios of or critical volume fraction, c c sp, where sp . mL g x is the specific volume of protein molecules (13). In Table 2, we QpEMAb to pEpEMAb, , in both coexisting phases as well as in the original solutions. In the protein-poor phase, x ¼ 0.283 0.002. compare the critical volume fractions found theoretically for In the protein-rich phase, x ¼ 0.312 0.004. In the original solu- spherical particles in the limit of very short and very long range tions, x ¼ 0.302 0.001. Table S1 shows that upon phase separa- of attraction, as well as the experimentally observed critical tion the relative proportion of MAb to MAb is slightly but volume fractions for various proteins. We believe that the small QpE pEpE critical volume fraction of antibodies is a consequence of its x consistently increased in the protein-rich phase. The value of in extended, Y-like shape. the two coexisting phases does not depend on HSA concentration In Fig. 4, we show several coexistence curves plotted using the within the experimental errors. Observation of the difference in scaled variables T∕Tc and c∕cc. Curve 1 shows the mean-field the partitioning of QpEMAb and pEpEMAb signifies that alteration prediction for a solution of attractive hard spheres with a Carna- of a single amino acid residue could affect the interprotein inter- han-Starling approximation for the entropy. We may quantify the action and thereby the phase behavior of the protein solution. width, w, of each coexistence curve by fitting the curve in the

16608 ∣ www.pnas.org/cgi/doi/10.1073/pnas.1112241108 Wang et al. Downloaded by guest on September 30, 2021 Table 2. Theoretical and experimental values of the critical and the preferential partitioning of HSA into the concentrated volume fraction, ϕc MAb phase. In the following discussion, we will connect these two “ ” ϕ features to the effective energies of MAb-HSA interactions c and to the excluded volume entropies for both of these molecules. Spherical particles with very long range of interactions 0.13* In the limit of a small mole fraction of HSA, x ¼ N2∕N1 ≪ 1, Spherical particles with very short range of interactions 0.27* where N1 and N2 are the numbers of molecules of MAb and Human lens γD crystallin 0.13† ‡ HSA respectively, the HSA-HSA interaction is negligible. Chicken egg white lysozyme 0.16 F ¼ F0þ γ γ γ γ γ § Hence, we may write the Helmholtz free energy as: Bovine lens crystallins (including B, C, D, E ) 0.21 0 Immunoglobulins: (IgG2-A, IgG2¶, IgG1∥) 0.063 N2kT lnðϕ2∕eαÞþN2E12. Here, F ðϕ1;TÞ is the Helmholtz free energy of a pure MAb solution. The remaining terms, linear in The values are taken from refs. 14, 16, 24, and 18. The value listed for N2, represent the entropic and energetic contributions of HSA. (IgG2-A) is taken from Fig. 1 and is consistent with data reported for The entropic term is written as the entropy of an ideal solution other immunogolobulins in refs. 6 and 7. V *The values are taken from ref. 14. of HSA in the volume, eff , accessible to it. Thus, the quantity †ref. 16. αðϕ1;TÞ represents the fraction of the total volume accessible to ‡ α ¼ V ∕V ref. 24. HSA, i.e., eff . The energetic component, per HSA mo- § ref. 18. lecule, due to the MAb-HSA interaction is denoted as E12ðϕ1;TÞ. ¶ ref. 6. In the high temperature approximation, both α and E12 are inde- ∥ ref. 7. pendent of T.

neighborhood of the critical point using a phenomenological The Reduction of the Phase Separation Temperature of MAb Solutions ½ðc − c Þ∕c 2 ¼ wðT − T Þ∕T asymptotic expression: c c c ph c.For in the Presence of HSA. We now examine the factors, which deter- curve 1, the width is w ¼ 6.15. Curve 2 shows the theoretical mine the change of T in a MAb solution, ΔT , upon addition γ ph ph fit (20) of the data for bovine B crystallin taken from ref. 18. of a small mole fraction, x,(x ≪ 1) of HSA. Using the equilibrium Because of the short range and highly anisotropic (aeolotopic) I II condition, μ1 ¼ μ1 , and general thermodynamic relations (27), interactions (14, 23), this coexistence curve has width w ¼ 27 ΔT ϕ we have derived ph at constant volume fraction of MAb, 1 which is much wider than curve 1. The data points shown for (see SI Appendix): other nearly spherical proteins follow coexistence curves similar     to curve 2 (16, 24). Interestingly, the coexistence curve for our I I II II dT ∕dϕ1 kT ðϕ ∕ϕ − ϕ ∕ϕ Þ ∂Π ΔT ¼ ph − ph 2 1 2 1 þ ϕ MAb (curve 3) is even wider than that observed for nearly sphe- ph I II 2 ∂Π∕∂ϕ1 Ω2ð1∕ϕ − 1∕ϕ Þ ∂ϕ2 rical proteins, and is asymmetrical: being much wider on the high 1 1 ϕ1;T concentration side than on the low concentration side. For curve [1] 3, w ¼ 120. Coexistence curves of other reported MAb’s also T share similar broad and asymmetrical shapes (6, 7). We believe Here, ph is the phase separation temperature of the pure MAb that these characteristics of the MAb coexistence curve result solution, Π is the osmotic pressure of the solution, and Ω2 is the from the highly nonspherical Y-like antibody shape (25, 26), volume of one HSA molecule. The slope of the coexistence curve dT ∕dϕ ϕ and possibly from its flexibility. Thus, it appears that currently of pure MAb solution, ph 1, is positive at 1 smaller than the used model free energies, which assume a spherical shape of critical volume fraction ϕc and is negative at ϕ1 larger than ϕc. protein molecules, have limited applicability in the description of The osmotic incompressibility in a pure MAb solution, ∂Π∕∂ϕ1, ΔT the thermodynamic properties of solutions of antibody. is always positive in a stable phase. Because experimentally ph is negative for all values of ϕ1, it follows that the bracketed term Phase Diagrams of MAb-HSA Aqueous Solutions. There are two in Eq. 1 must have opposite signs on the two sides of the coex- important features of the phase diagrams of MAb-HSA-water istence curve. The first term in the brackets describes the effect T of HSA partitioning. The experimentally observed partitioning of ternary solutions: the reduction of ph upon addition of HSA BIOPHYSICS AND I II HSA is small (Fig. 3): ϕ2 ≈ ϕ2 . This observation is actually 1.005 COMPUTATIONAL BIOLOGY Bovine gB crystallin remarkable because the condensed phase has much less free Human gD crystallin V volume, eff , to accommodate the HSA than has the dilute phase. 1.000 Lysozyme I II MAb When ϕ2 ¼ ϕ2 the partitioning term in Eq. 1 becomes equal to −kT ϕ2∕Ω2. The second term in the bracket characterizes 0.995 ph the change of osmotic pressure upon addition of HSA. Using c T / Π ¼ −ð∂F∕∂VÞN1;N2;T , and our expression for the Helmholtz 0.990 CHEMISTRY ph

T ð∂Π∕∂ϕ Þ ¼ kT ∕Ω þ ϕ ð∂ðE − free energy, it follows that 2 ϕ1;T ph 2 1 12 kT αÞ∕∂ϕ Þ∕Ω 0.985 ph ln 1 2. The first term here represents the ideal (van’t Hoff) contribution to the osmotic incompressibility. Under I II 0.980 the conditions of no partitioning (ϕ2 ¼ ϕ2 ), this contribution cancels the first term in the brackets in Eq. 1. This cancellation 0.975 reflects the fact that adding an ideal, noninteracting solute should 00.511.522.53 c /c produce no change in the coexistence curve. The second term: c ϕ ð∂ðE − kT αÞ∕∂ϕ Þ 1 12 ph ln 1 , represents the nonideal contribution Fig. 4. Coexistence curves in the units of scaled phase separation tempera- of HSA to the osmotic incompressibility. This term involves two T ∕T c∕c tures, ph c, and the scaled protein concentrations, c. Curve 1 (short elements: the MAb-HSA interaction energy E12 and the excluded dashed line) shows the theoretical coexistence curve of spherical particles −kT α volume entropy ph ln . Using a Monte Carlo simulation and using mean-field approximation of attraction and Carnahan-Starling expres- a three-sphere model for the Y-shaped MAb molecule (see SI sion for entropy. Curve 2 (long dashed line) shows the theoretical fit for the Appendix), we have evaluated the free volume fraction α as a data of bovine γB crystallin (open squares) taken from (18). Curve 3 (solid line) function of ϕ1. A graph of ∂ð− ln αÞ∕∂ϕ1 vs. ϕ1 is shown in (Fig. S6 shows the eye guide for the coexistence curve of MAb (solid circles). Data SI Appendix points on the coexistence curves of two other globular proteins (16, 24): in ). This excluded volume contribution is positive human γD crystallin (open diamonds) and chicken egg white lysozyme (open and monotonically increases with ϕ1. At the critical point, this ϕ ð∂ðE − kT αÞ∕∂ϕ Þ triangles) are also shown. derivative is equal to 10. Because 1 12 ph ln 1

Wang et al. PNAS ∣ October 4, 2011 ∣ vol. 108 ∣ no. 40 ∣ 16609 Downloaded by guest on September 30, 2021 changes sign at the critical point, it follows that there ∂E12∕∂ϕ1 ¼ In conclusion, we have observed LLPS of a monoclonal anti- −10kT E ϕ ph. The energy 12 is a smooth monotonic function of 1, body, IgG2-A, at physiological pH. The phase diagram of our thus ∂E12∕∂ϕ1 is not expected to vary dramatically. Indeed, in the MAb solution is distinctly different from that of nearly spherical mean-field approximation, E12 ¼ ϵ12ϕ1, this derivative would be globular proteins. Our experiments, together with the data avail- ϵ ¼ −10kT ϕ E – constant, 12 ph, over the entire range of 1, and 12 will able for other antibodies (5 9), show that: the immunoglobulins be −1.4kTc at the critical point. The negative value of this energy have a markedly lower critical concentration and a much broader, is consistent with an attractive MAb-HSA interaction. This signif- asymmetric coexistence curve. We believe that these features icant attraction compensates for the low entropy of HSA in the are associated with the highly nonspherical shape of an IgG protein-rich phase and produces the nearly equal values of HSA molecule, i.e., the Carnahan-Starling form for excluded volume volume fractions in the two phases. entropy of hard spheres is unsuitable for thermodynamic analysis of antibody solutions. We have also examined the effect of HSA, Partitioning of MAb and HSA. As has been seen above, the partition- the major protein component in blood, on the LLPS of MAbs. ing of HSA into the two coexisting phases is closely connected We have found that the phase separation temperature decreases T 1 as HSA concentration increases. This result is remarkable as it with the magnitude and sign of the change in ph (Eq. ). The partitioning of HSA is controlled by its chemical potential: implies that HSA may play a significant role in maintaining the stability of antibodies in blood. By applying a general thermo- μ2 ¼ð∂F∕∂N2ÞN1;V;T . Using the expression for Helmholtz free dynamic analysis, we have attributed the reduction of phase energy, we derived that: μ2 ¼ kT lnðϕ2∕αÞþE12. The partition- I separation temperature of MAb solutions in the presence of HSA ing of HSA between the two phases, i.e., the relation between ϕ2 II I II to the attractive interaction between MAb and HSA. This phe- and ϕ2 , is determined by the equilibrium condition, μ2 ¼ μ2 , which has the form: nomenon shows the role of the protein-additive interaction in tuning the phase separation temperature of the protein solution. I II II I II I Furthermore, the partitioning of HSA (or any other excipient) kT lnðϕ α ∕ϕ α Þ¼E12ðϕ Þ − E12ðϕ Þ: [2] ph 2 2 1 1 also depends on the energy of MAb-additive interaction. In a special case of MAb, a minor antibody isoform, we have This equation connects the ratio of HSA volume fractions in the QpE two phases to the excluded volume entropies and the MAb-HSA given a further analysis of the relative partitioning, and conclude that the MAb- MAb attraction is stronger than the interaction energies. With αðϕ1Þ determined by Monte Carlo QpE pEpE simulation, Eq. 2 provides an alternative way to evaluate E12. pEpEMAb-pEpEMAb attraction. Using the simulation results and the experimental data from five This investigation along with other recent findings (5–9) sug- II I tie-lines (Fig. 3), we have deduced ΔE12 ≡ E12ðϕ1 Þ − E12ðϕ1 Þ gests that LLPS may be a ubiquitous phenomenon in antibody −1 8kT −1 1kT and found that this quantity ranged from . ph to . ph. solutions. This fact is of obvious importance for biotechno- The negative value of ΔE12 implies an attractive interaction logical and pharmaceutical applications and for understanding between MAb and HSA. This attraction is the driving force the origin of cryoglobulinemia which is a condition observed in for the partitioning of HSA. In the mean-field approximation, a number of human diseases. The present work provides a con- II I ceptual experimental and theoretical framework for further stu- E12 ¼ ϵ12ϕ1, then ϵ12 ¼ ΔE12∕ðϕ1 − ϕ1 Þ has a value ranging −14 0kT −13 3kT dies in this emerging field. from . ph to . ph. Considering the approximations involved, this result is quite consistent with the estimation above, Materials and Methods ϵ ¼ −10kT 12 ph, based on the shift of the coexistence curve upon Preparation and Purification of MAb and HSA. MAb, IgG2-A, was produced at

adding HSA. Amgen Inc. The original MAb solution contained QpEMAb and pEpEMAb iso- Furthermore, it is interesting to examine the relative partition- forms corresponding to partial and complete cyclization of the heavy chain N termini (Fig. S3 in SI Appendix). The two isoforms were identified by peptide ing of QpEMAb and pEpEMAb. QpEMAb and pEpEMAb are iden- tical in shape and size and are different at only one amino acid mapping coupled with mass-spectrometry (performed at Amgen Inc.). HSA was purchased from Sigma. The dimers and oligomers of HSA were removed position. For these “similar” proteins, the difference between the x using a preparative chromatographic system (AKTA prime plus, Amersham molar ratios of QpEMAb to pEpEMAb, , in the two coexisting Biosciences) and a size-exclusion column (Superdex 200, GE Healthcare). phases, is solely determined, when x is small, by the difference Solution Preparation. The purified MAb and HSA proteins were dialyzed ex- between the energies of QpEMAb-pEpEMAb like-unlike interac- E haustively into Tris·HCl buffer (0.1 M, pH 7.4). Solutions containing dilute tion, 12, and that of pEpEMAb-pEpEMAb like-like interaction, · E MAb and HSA in Tris HCl buffer were concentrated by ultrafiltration (Amicon, 11, in these two phases. We have previously shown (16) that this 10 kDa) and Centrifugation (Amicon Ultra, 10 kDa). The concentrations of relative partitioning of similar proteins can be described by: MAb and HSA in the mixture solutions were determined using HPLC with kT ð xI − xIIÞ¼ΔE − ΔE ΔE ≡ E ðϕ IIÞ− μ ph ln ln 12 11, where 12 12 1 a CEX column (wide pore CBx 5 , J.T.Baker). The column was equilibrated I II I with 20 mM potassium phosphate buffer and eluted with 0 to 100% E12ðϕ1 Þ and ΔE11 ≡ E11ðϕ1 Þ − E11ðϕ1 Þ. The driving force for 500 mM potassium phosphate over 30 min at pH 6. This column was precali- the relative partitioning of MAb is: ΔE12 − ΔE11. Using the QpE brated with standard MAb and HSA solutions respectively. The concentra- experimental data in Table S1 in SI Appendix, we can deduce tions of standard solutions were measured by an UV spectrometer at −1 −1 ΔE12 − ΔE11 ≈−0.1kT. The negative value of ΔE12 − ΔE11 sug- 280 nm using the extinction coefficient value of 1.48 mg ·mL·cm for MAb and 0.52 mg−1 ·mL·cm−1 for HSA (www.expasy.org). The ratios of gests that the QpEMAb-pEpEMAb attraction is stronger than the ΔE QpEMAb to pEpEMAb in solutions were determined using a CEX HPLC method pEpEMAb-pEpEMAb attraction. The magnitude of 12 is larger described in refs. 5, 17. The ratios of QpEMAb to pEpEMAb were calculated than that of ΔE11 by one tenth of the thermal energy kT. This from the integrated peak areas using 280 nm detection. change in interaction energy is caused by the alteration of a single ΔE T amino acid residue. While the small difference between 12 Measurement of ph. A test tube containing the sample was placed in a ther- and ΔE11 is expected to have a small effect on the phase separa- mostated light-scattering stage, whose temperature was initially set above tion temperature, this difference produces an observable parti- the phase separation temperature so that the solution was transparent. tioning of these similar proteins. In vivo, as well as in biophar- The transmitted intensity of a 4-mW He-Ne laser was recorded by a photo- diode. The temperature of the sample was then step wise lowered by 0.1 K maceutical production we frequently encounter mixtures of T various antibody isoforms. The case of MAb and MAb every 5 min. At a well defined temperature, cloud, the sample became visibly QpE pEpE cloudy. The temperature was then step wise raised by 0.1 K every 5 min. provides an important example of phase separation partitioning The minimum temperature at which the solution became clear again was T T in such mixtures of closely related antibody variants. denoted by clarify. The phase separation temperature ph is estimated as

16610 ∣ www.pnas.org/cgi/doi/10.1073/pnas.1112241108 Wang et al. Downloaded by guest on September 30, 2021 T T T T the average of clarify and cloud. The difference between cloud and clarify is Coherent He-Ne laser (35 mW, 632.8 nm; Coherent Radiation). The measure- hysteresis which reflects the nucleation rate (19). Because hysteresis depends ments were performed at a scattering angle of 90°. The measured correlation on kinetic processes, all the cooling and heating steps were set with a stan- functions were analyzed by the Precision Deconvolve 5.5 software (Precision dard time interval (5 min). Detectors). The correlation functions were used to calculate the apparent diffusion coefficients, D, of proteins in solutions with given total protein Measurement of MAb-HSA Partitioning. The solutions having known c1 and c2 concentration, c, at different HSA weight fraction, w ¼ 0%, 30%, and were quenched to a temperature below T in a thermostated water bath. ph 100%. Dðc ¼ 0Þ were obtained by extrapolating DðcÞ to c ¼ 0. The hydro- After an incubation time of one week, a sharp interface formed between w w R ’ w two liquid phases. The formation of the sharp interface was taken as an in- dynamic radii, h s, of proteins in solutions with fixed were calculated from Dðc ¼ 0Þ dication that equilibrium was reached. The MAb and HSA in both phases w using Stokes-Einstein relation. were separated and their concentrations were measured using precalibrated CEX HPLC at pH 6. ACKNOWLEDGMENTS. We thank Sabine Hogan, John F. Valliere-Douglass (both of Amgen Inc.) and Olutayo Ogun (MIT) for technical support and QLS. All protein samples were filtered through a 0.1 μm Millipore filter and to thank Suresh Vunnum, Jaby Jacob, Alison Wallace, Gerald W. Becker, placed in a test tube. QLS experiments were performed on a light-scattering Linda O. Narhi, Michael J. Treuheit, and David N. Brems (all of Amgen Inc.) apparatus using a PD2000DLSPLUS correlator (Precision Detectors) and a for helpful discussions. We acknowledge the financial support of Amgen Inc.

1. An Z (2009) Therapeutic monoclonal antibodies: from bench to clinic (John Wiley & 15. Omenn GS (2006) Exploring the human plasma proteome (Wiley-VCH, Weinheim) Sons, Hoboken, NJ.). p xxii, 372. 2. Shire SJ, Shahrokh Z, Liu J (2009) Challenges in the development of high protein con- 16. Wang Y, Lomakin A, McManus JJ, Ogun O, Benedek GB (2010) Phase behavior of centration formulations. Current trends in monoclonal antibody development and mixtures of human lens proteins Gamma D and Beta B1. Proc Natl Acad Sci USA manufacturing, eds SJ Shire, W Gombotz, K Bechtold-Peters, and J Andya (Springer, 107:13282–13287. – New York), pp 131 147. 17. Lau H, et al. (2010) Investigation of degradation processes in IgG1 monoclonal anti- 3. Wang W, Roberts CJ (2010) Aggregation of therapeutic proteins (Wiley, Hoboken, NJ). bodies by limited proteolysis coupled with weak cation-exchange HPLC. J Chromatogr 4. Rosenberg AS (2006) Effects of protein aggregates: an immunologic perspective. AAPS B 878:868–876. J 8:E501–507. 18. Broide ML, Berland CR, Pande J, Ogun OO, Benedek GB (1991) Binary-liquid phase 5. Chen S, Lau H, Brodsky Y, Kleemann GR, Latypov RF (2010) The use of native cation- separation of lens protein solutions. Proc Natl Acad Sci USA 88:5660–5664. exchange chromatography to study aggregation and phase separation of monoclonal antibodies. Protein Sci 19:1191–1204. 19. Liu C, et al. (1996) Phase separation in aqueous solutions of lens gamma-crystallins: – 6. Mason BD, Zhang-van Enk J, Zhang L, Remmele RL, Jr, Zhang J (2010) Liquid-liquid special role of gamma s. Proc Natl Acad Sci USA 93:377 382. phase separation of a monoclonal antibody and nonmonotonic influence of Hofme- 20. Thomson JA, Schurtenberger P, Thurston GM, Benedek GB (1987) Binary liquid phase ister anions. Biophys J 99:3792–3800. separation and critical phenomena in a protein/water solution. Proc Natl Acad Sci USA 7. Nishi H, et al. (2010) Phase separation of an IgG1 antibody solution under a low ionic 84:7079–7083. strength condition. Pharm Res 27:1348–1360. 21. Delaye M, Clark JI, Benedek GB (1981) Coexistence curves for the phase separation in 8. Lewus RA, Darcy PA, Lenhoff AM, Sandler SI (2011) Interactions and phase behavior of the calf lens cytoplasm. Biochem Biophys Res Commun 100:908–914. a monoclonal antibody. Biotechnol Progr 27:280–289. 22. Jion AI, Goh LT, Oh SK (2006) Crystallization of IgG1 by mapping its liquid-liquid phase 9. Trilisky E, Gillespie R, Osslund TD, Vunnum S (2011) Crystallization and liquid-liquid separation curves. Biotechnol Bioeng 95:911–918. phase separation of monoclonal antibodies and Fc-fusion proteins: screening results. 23. Lomakin A, Asherie N, Benedek GB (1999) Aeolotopic interactions of globular pro- – Biotechnol Progr, 27 pp:1054 1067. teins. Proc Nat’l Acad Sci USA 96:9465–9468. 10. Charles ED, Dustin LB (2009) Hepatitis C virus-induced cryoglobulinemia. Kidney Int 24. Taratuta VG, Holschbach A, Thurston GM, Blankschtein D, Benedek GB (1990) Liquid- 76:818–824. liquid phase separation of aqueous lysozyme solutions—effects of pH and salt identity. 11. Fabris P, et al. (2003) Prevalence and clinical significance of circulating cryoglobulins in J Phys Chem 94:2140–2144. HIV-positive patients with and without Co-infection with hepatitis C virus. J Med Virol 25. Sandin S, Ofverstedt LG, Wikstrom AC, Wrange O, Skoglund U (2004) Structure and 69:339–343. flexibility of individual immunoglobulin G molecules in solution. Structure 12:409–415. 12. Dimopoulos MA, Alexanian R (1994) Waldenstroms macroglobulinemia. Blood 83:1452–1459. 26. Harris LJ, Larson SB, Skaletsky E, McPherson A (1998) Comparison of the conformations – 13. Schurtenberger P, Chamberlin RA, Thurston GM, Thomson JA, Benedek GB (1989) of two intact monoclonal antibodies with hinges. Immunol Rev 163:35 43. Observation of critical phenomena in a protein-water solution. Phys Rev Lett 27. Landau LD, Lifshitz EM, Pitaevskii LP (1980) Statistical physics (Pergamon Press, Oxford; 63:2064–2067. New York), 3d rev. and enl. Ed. 14. Lomakin A, Asherie N, Benedek GB (1996) Monte Carlo study of phase separation in 28. Silverton EW, Navia MA, Davies DR (1977) Three-dimensional structure of an intact aqueous protein solutions. J Chem Phys 104:1646–1656. human immunoglobulin. Proc Natl Acad Sci USA 74:5140–5144. BIOPHYSICS AND COMPUTATIONAL BIOLOGY CHEMISTRY

Wang et al. PNAS ∣ October 4, 2011 ∣ vol. 108 ∣ no. 40 ∣ 16611 Downloaded by guest on September 30, 2021