A comparative study of monoclonal antibodies. 1. phase behavior and protein–protein interactions

Protein phase behavior is involved in numerous aspects of downstream processing, either by design as in crystallization or precipitation processes, or as an undesired effect, such as aggregation. This work explores the phase behavior of eight monoclonal antibodies (mAbs) that exhibit liquid–liquid separation, aggregation, gelation, and crystallization. The phase behavior has been studied systematically as a function of a number of factors, including solution composition and pH, in order to explore the degree of variability among different antibodies. Comparisons of the locations of phase boundaries show consistent trends as a function of solution composition; however, changing the solution pH has different effects on each of the antibodies studied. Furthermore, the types of dense phases formed varied among the antibodies. Protein–protein interactions, as reflected by values of the osmotic second virial coefficient, are used to correlate the phase behavior. The primary findings are that values of the osmotic second virial coefficient are useful for correlating phase boundary locations, though there is appreciable variability among the antibodies in the apparent strengths of the intrinsic protein–protein attraction manifested. However, the osmotic second virial coefficient does not provide a clear basis to predict the type of dense phase likely to result under a given set of solution conditions. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 31:268–276, 2015     

Moving through three‐dimensional phase diagrams of monoclonal antibodies

Protein phase behavior characterization is a multivariate problem due to the high amount of influencing parameters and the diversity of the proteins. Single influences on the protein are not understood and fundamental knowledge remains to be obtained. For this purpose, a systematic screening method was developed to characterize the influence of fluid phase conditions on the phase behavior of proteins in three‐dimensional phase diagrams. This approach was applied to three monoclonal antibodies to investigate influences of pH, protein and salt concentrations, with five different salts being tested. Although differences exist between the antibodies, this extensive study confirmed the general applicability of the Hofmeister series over the broad parameter range analyzed. The influence of the different salts on the aggregation (crystallization and precipitation) probability was described qualitatively using this Hofmeister series, with a differentiation between crystallization and precipitation being impossible, however. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:1103–1113, 2014     

Osmotic second virial cross‐coefficient measurements for binary combination of lysozyme,ovalbumin, and α‐amylase in salt solutions

Interactions measurement is a valuable tool to predict equilibrium phase separation of a desired protein in the presence of unwanted macromolecules. In this study, cross‐interactions were measured as the osmotic second virial cross‐coefficients (B₂₃) for the three binary protein systems involving lysozyme, ovalbumin, and α‐amylase in salt solutions (sodium chloride and ammonium sulfate). They were correlated with solubility for the binary protein mixtures. The cross‐interaction behavior at different salt concentrations was interpreted by either electrostatic or hydrophobic interaction forces. At low salt concentrations, the protein surface charge dominates cross‐interaction behavior as a function of pH. With added ovalbumin, the lysozyme solubility decreased linearly at low salt concentration in sodium chloride and increased at high salt concentration in ammonium sulfate. The B₂₃ value was found to be proportional to the slope of the lysozyme solubility against ovalbumin concentration and the correlation was explained by preferential interaction theory. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:1203–1211, 2013     

Molecular origins of osmotic second virial coefficients of proteins.

The thermodynamic properties of protein solutions are determined by the molecular interactions involving both solvent and solute molecules. A quantitative understanding of the relationship would facilitate more systematic procedures for manipulating the properties in a process environment. In this work the molecular basis for the osmotic second virial coefficient, B22, is studied; osmotic effects are critical in membrane transport, and the value of B22 has also been shown to correlate with protein crystallization behavior. The calculations here account for steric, electrostatic, and short-range interactions, with the structural and functional anisotropy of the protein molecules explicitly accounted for. The orientational dependence of the protein interactions is seen to have a pronounced effect on the calculations; in particular, the relatively few protein-protein configurations in which the apposing surfaces display geometric complementarity contribute disproportionately strongly to B22. The importance of electrostatic interactions is also amplified in these high-complementarity configurations. The significance of molecular recognition in determining B22 can explain the correlation with crystallization behavior, and it suggests that alteration of local molecular geometry can help in manipulating protein solution behavior. The results also have implications for the role of protein interactions in biological self-organization.     

Enhanced crystal packing due to solvent reorganization through reductive methylation of lysine residues in oxidoreductase from <Emphasis Type="Italic">Streptococcus pneumoniae</Emphasis>

Protein crystallization is in part driven by the changes in the entropy of the system, but opinions differ as to whether the solute (protein) or solvent (water) molecules make more of a contribution to the overall entropic change. Methylation of lysine residues in proteins has been used to enhance protein crystallization. We investigated using molecular dynamics simulations with explicit solvent molecules, the behavior of several native proteins and their methylated counterparts chosen from an earlier large-scale study. Methylated lysines are capable of making a variety of interactions including H-bonds with protein residues and solvent. We demonstrate that methylation on the lysine slightly increases its side chain conformational entropy by about 3.5 J mol⁻¹ K⁻¹. Analysis of the radial and spatial distributions of the water molecules around the methylated lysine surface in oxidoreductase from Streptococcus pneumoniae revealed a larger sphere of water molecules with low entropy, as compared with solvent associated with unmethylated lysine. If methylated lysine were to make interactions at the protein–protein interface, the low-entropy water molecules associated with methylated lysines would be released, resulting in a gain of entropy. We show that this gain more than compensates for the loss of protein entropy. Therefore, we propose that lysine methylation favors the formation of crystals through solvent entropic gain.     

Protein-protein interaction on lysozyme crystallization revealed by rotational diffusion analysis

Intermolecular interactions between protein molecules diffusing in various environments underlie many biological processes as well as control protein crystallization, which is a crucial step in x-ray protein structure determinations. Protein interactions were investigated through protein rotational diffusion analysis. First, it was confirmed that tetragonal lysozyme crystals containing fluorescein-tagged lysozyme were successfully formed with the same morphology as that of native protein. Using this nondisruptive fluorescent tracer system, we characterized the effects of sodium chloride and ammonium sulfate concentrations on lysozyme-lysozyme interactions by steady-state and time-resolved fluorescence anisotropy measurements and the introduction of a novel interaction parameter, k_rot. The results suggested that the specific attractive interaction, which was reflected in the retardation of the protein rotational diffusion, was induced depending on the salt type and its concentration. The change in the attractive interactions also correlated with the crystallization/precipitation behavior of lysozyme. Moreover, we discuss the validity of our rotational diffusion analysis through comparison with the osmotic second virial coefficient, B₂₂, previously reported for lysozyme and those estimated from k_rot.     

Theoretical studies of clonal selection: minimal antibody repertoire size and reliability of self-non-self discrimination.

Viewing the immune system as a molecular recognition device designed to identify “foreign shapes”, we estimate the probability that an immune system with N_Ab monospecific antibodies in its repertoire can recognize a random foreign antigen. Furthermore, we estimate the improvement in recognition if antibodies are multispecific rather than monospecific. From our probabilistic model we conclude: (1) clonal selection is feasible, i.e. with a finite number of antibodies an animal can recognize an effectively infinite number of antigens; (2) there should not be great differences in the specificities of antibody molecules among different species; (3) the region of a foreign molecule recognized by an antibody must be severely limited in extent; (4) the probability of recognizing a foreign molecule, P, increases with the antibody repertoire size N_Ab; however, below a certain value of N_Ab the immune system would be very ineffectual, while beyond some high value of N_Ab further increases in N_Ab yield diminishing small increases in P; (5) multispecificity is equivalent to a modest increase (probably less than 10) in the antibody repertoire size N_Ab, but this increase can substantially improve the probability of an immune system recognizing a foreign molecule.Besides recognizing foreign molecules, the immune system must distinguish them from self molecules. Using the mathematical theory of reliability we argue that multisite recognition is a more reliable method of distinguishing between molecules than single site recognition. This may have been an important evolutionary consideration in the selection of weak non-covalent interactions as the basis of antigen-antibody bonds.     

Lipid contribution to the affinity of antigen association with specific antibodies conjugated to liposomes

Immunoliposomes, directed to clinically relevant cell-surface molecules with antibodies, antibody fragments or peptides, are used for site-specific diagnostic evaluation or delivery of therapeutic agents. We have developed intrinsically echogenic liposomes (ELIP) covalently linked to fibrin(ogen)-specific antibodies and Fab fragments for ultrasonic imaging of atherosclerotic plaques. In order to determine the effect of liposomal conjugation on the molecular dynamics of fibrinogen binding, we studied the thermodynamic characteristics of unconjugated and ELIP-conjugated antibody molecules. Utilizing radioimmunoassay and enzyme-linked immunosorbent assay protocols, binding affinities were derived from data obtained at three temperatures. The thermodynamic functions ΔH°, ΔG° and ΔS° were determined from van't Hoff plots and equations of state. The resultant functions indicated that both specific and nonspecific associations of antibody molecules with fibrinogen occurred through a variety of molecular interactions, including hydrophophic, ionic and hydrogen bonding mechanisms. ELIP conjugation of antibodies and Fab fragments introduced a characteristic change in both ΔH° and ΔS° of association, which corresponded to a variable contribution to binding by phospholipid gel-liquid crystal phase transitions. These observations suggest that a reciprocal energy transduction, affecting the strength of antibody-antigen binding, may be a singular characteristic of immunoliposomes, having utility for optimization and further development of the technology.     

The influence of solute structure,temperature, and eluent composition on the chiral separation of 21 aminotetralins on a cellulose tris-3,5-dimethylcarbamate stationary phase in high-performance liquid chromatography

In the present study 21 different chiral aminotetralins were used to investigate the mechanism behind their enantiomeric resolution (R_s) on a commercially available high-performance liquid chromatography (HPLC) cellulose tris-3,5-dimethylcarbamate stationary phase. The differences in the chemical structures of the aminotetralins used were never directly located on the chiral carbon. Their chromatographic behavior was studied for two eluent compositions at six different temperatures. Hydrogen bonding and π? π interactions are two possible solute–chiral stationary phase (CSP) interactions. Differences between the enantiomers in their spatial arrangement of positions involved in solute–CSP interactions were the major forces behind enantiomeric separation. Lowering the temperature increased the R_s for the aminotetralins having π-electrons not directly bonded to that part of the molecule where the hydrogen bonding with the CSP is located. Primary amines and secondary amines, with a sufficiently short N-alkyl substituent, showed a decrease of R_s with lower temperatures, all other aminotetralins yielding an increase of R_s with lower temperatures. © 1992 Wiley-Liss, Inc.     

Reentrant condensation of lysozyme: Implications for studying dynamics of lysozyme in aqueous solutions of lithium chloride

Recent studies have outlined the use of eutectic solutions of lithium chloride in water to study microscopic dynamics of lysozyme in an aqueous solvent that is remarkably similar to pure water in many respects, yet allows experiments over a wide temperature range without solvent crystallization. The eutectic point in a (H₂O)_R(LiCl) system corresponds to R ≈ 7.3, and it is of interest to investigate whether less‐concentrated aqueous solutions of LiCl could be used in low‐temperature studies of a solvated protein. We have investigated a range of concentrations of lysozyme and LiCl in aqueous solutions to identify systems that do not show phase separation and avoid solvent crystallization on cooling down. Compared to the lysozyme concentration in solution, the concentration of LiCl in the aqueous solvent plays the major role in determining systems suitable for low‐temperature studies. We have observed interesting and rich phase behavior reminiscent of reentrant condensation of proteins. © 2013 Wiley Periodicals, Inc. Biopolymers 101: 624–629, 2014.     

Farnesol and geranylgeraniol modulate the structural properties of phosphatidylethanolamine model membranes

The biological activity of farnesol (FN) and geranylgeraniol (GG) and their isoprenyl groups is related to membrane-associated processes. We have studied the interactions of FN and GG with 1,2-dielaidoyl-sn-glycero-3-phosphoethanolamine (DEPE) membranes using DSC and X-ray diffraction. Storage of samples at low temperature for a long time favors a multidomain system formed by a lamellar crystalline (Lc) phase and isoprenoids (ISPs) aggregates. We demonstrate that ISPs alter the thermotropic behavior of DEPE, thereby promoting a H_II growth in a lamellar Lc phase with a reduced degree of hydration. The H_II phase occurs with the same repeat distance (d_HII=5.4 nm) as the Lc phase and upon heating it expands considerably (δd/δT≈0.22 nm/°C). The dimensional stabilization of this H_II phase coincides with the transition temperature of the Lc to L_α phase. Thereafter, the system DEPE/ISP will progress by increasing the nonlamellar-forming propensity and reaching a single H_II phase at high temperature. The cooling scan followed a similar structural path, except that the system went into a stable gel phase L_β with a repeat distance, d_Lβ=6.5 nm, in co-existence with a H_II phase. The formation of ISP microdomains in model PE membranes substantiates the importance of the isoprenyl group in the binding of isoprenylated proteins to membranes and in lipid–lipid interactions through modulation of the membrane structure.     

The thermotropic properties of coenzyme Q10 and its lower homologues

The thermotropic properties of coenzymes Q₁₀, Q₉, Q₈, and Q₇ have been examined by differential scanning calorimetry and wide-angle X-ray diffraction. Typical scanning calorimetry cooling curves of coenzyme Q from the liquid state exhibit a single exothermic phase transition into a crystalline state at a temperature that decreases as the length of the polyisoprenoid side-chain substituent decreases. Upon subsequent heating, the molecules undergo a series of thermal events which precede the main crystalline-to-liquid endothermic phase transition. The temperature of these transitions increases with increasing chain length. The crystallization phase transition temperature depends markedly on the rate at which the sample is cooled and increases with decreasing scan rate; the temperature of the melting endotherm is not markedly affected by the scan rate. Detailed calorimetric studies of coenzyme Q₁₀ indicate that two crystalline states are formed, one at relatively high cooling rates to low temperatures and the other when preparations are cooled slowly from the liquid state to relatively high temperatures. Heating the crystalline phase formed by rapid cooling causes its transformation into the phase observed by cooling slowly. X-ray diffraction analysis confirmed the existence of these two crystal phases in coenzymes Q₉ and Q₁₀ and the transformation from the rapidly crystallized form to the more ordered form associated with slower cooling rates. At body temperature (310 K) under equilibrium conditions coenzyme Q₁₀ exists in an ordered crystalline phase; the implications of the thermotropic behavior of coenzyme Q₁₀ on mitochondrial functionin vitro andin vivo are discussed.     

DNA, rRNA and RNA partition isotherms rapidly labeled in a saline solvent system

Partition isotherms of DNA, rRNA, and rapidly labeled RNA in a salt solvent system are determined. In the salt solvent system PMB (potassium phosphate buffer 1.50 M pH 7.0:2-methoxyethanol:2-butoxyethanol; 3:1:variable volume), equilibrated at the temperature T, the partition isotherms of animal ribosomal RNA and DNA form a series of straight lines according to the basic relation log k = B ? A_T(BuO), where k is the partition coefficient, B and A_T are parameters depending upon the base composition, the molecular weight, the helix content, and conformation of nucleic acids, and (BuO) refers to the 2-butoxyethanol amount in volume percent. This relation is also valid for other nucleic acid compounds (bases, nucleosides, nucleotides, oligoribonucleotides, and transfer RNA). For RNA fractions distributed in different Kirby salt solvent systems, log k is proportional to the relative levels of adenine and guanine expressed as the A/(A+G) ratio. Partition isotherms of high molecular weight RNA from mouse plasmocytoma, labeled during a few hours, are not straight lines. This behavior indicates a heterogeneous RNA population. By plotting the specific activity of this mixture (rRNA and rapidly labeled RNA) determined in the top phase of our salt solvent system as a function of the 2-but-oxyethanol content, we obtained a sigmoidal curve, which characterizes the nature of the RNA population investigated. Formulas are given for the calculation of the specific activities of rRNA and rapidly labeled RNA and for the calculation of their relative ratio.     

Modelling hydrogen bonds in NN-dimethylformamide

N, N-dimethylformamide (DMF) is a ‘universal’ solvent with the simplest amide structure. DMF has different interactions with many polymers and biomolecules. It is therefore necessary to study systematically the interactions in DMF itself first. In this study, both FT-IR and two molecular theoretical methods (MP2 and DFT/B3LYP) were used to study various hydrogen bonding interactions in DMF molecules based on its weak H-bonding donors CH/CH₃ and strong H-bonding acceptor C = O. The possible H-bonding donors and acceptors in DMF molecules were first analysed followed by modelling the effect of different structural environments on vC = O bands in infrared spectra. Finally, H-bonding properties including distance, angles and the energy as well as the probability of H-bonding patterns were obtained. The results showed that there exist five possible different weak types of H-bonding dimers; among them, three dimers consist of a pair of weak H-bonds, whereas two other dimers have two pairs of H-bonds, leading to 14 (including eight different) H-bonds. Two types of dimers were dominant, whereas three others can be omitted.     

Antigen recognition by single-domain antibodies: structural latitudes and constraints

Single-domain antibodies (sdAbs), the autonomous variable domains of heavy chain-only antibodies produced naturally by camelid ungulates and cartilaginous fishes, have evolved to bind antigen using only three complementarity-determining region (CDR) loops rather than the six present in conventional V_H:V_L antibodies. It has been suggested, based on limited evidence, that sdAbs may adopt paratope structures that predispose them to preferential recognition of recessed protein epitopes, but poor or non-recognition of protuberant epitopes and small molecules. Here, we comprehensively surveyed the evidence in support of this hypothesis. We found some support for a global structural difference in the paratope shapes of sdAbs compared with those of conventional antibodies: sdAb paratopes have smaller molecular surface areas and diameters, more commonly have non-canonical CDR1 and CDR2 structures, and have elongated CDR3 length distributions, but have similar amino acid compositions and are no more extended (interatomic distance measured from CDR base to tip) than conventional antibody paratopes. Comparison of X-ray crystal structures of sdAbs and conventional antibodies in complex with cognate antigens showed that sdAbs and conventional antibodies bury similar solvent-exposed surface areas on proteins and form similar types of non-covalent interactions, although these are more concentrated in the compact sdAb paratope. Thus, sdAbs likely have privileged access to distinct antigenic regions on proteins, but only owing to their small molecular size and not to general differences in molecular recognition mechanism. The evidence surrounding the purported inability of sdAbs to bind small molecules was less clear. The available data provide a structural framework for understanding the evolutionary emergence and function of autonomous heavy chain-only antibodies.     

Mobile‐Phase Modulators as Salt Tolerance Enhancers in Phenylboronate Chromatography: Thermodynamic Evaluation of the Mechanisms Underlying the Adsorption of Monoclonal Antibodies

Phenylboronate chromatography has been employed for bioseparation applications though details concerning the mechanisms of interaction between the ligand and macromolecules remain widely unknown. Here, the phenomena underlying the adsorption of an anti‐human interleukin‐8 (anti‐IL8) monoclonal antibody (mAb) onto an m‐aminophenylboronic acid (m‐APBA) ligand in the presence of different mobile‐phase modulators (NaF/MgCl ₂/(NH ₄) ₂SO ₄) and under different pH values (7.5/8.5/9.0) is investigated. Flow microcalorimetry (FMC) is applied to measure instantaneous heat energy transfer, providing insights about the role of specific and nonspecific interactions involved in the adsorptive process. Results show that the adsorption of anti‐IL8 mAb to m‐APBA is enthalpically driven, corroborating the presence of the reversible esterification reaction between boronic acid or boronates and cis‐diol‐containing molecules. Nevertheless, for all mobile‐phase modulators studied, changes in thermogram profiles are observed as well as reductions in the net heat of adsorption when increasing the pH. Overall, FMC and parallel chromatographic experiments data suggest that ligand salt tolerance could be enhanced using mobile‐phase modulators, with all salts studied promoting the specific cis‐diol interactions and reducing nonspecific interactions. The last feature is more noticeable at pH values above ligand's pK _a, mainly due to the ability of NaF and (NH ₄) ₂SO ₄ to diminish electrostatic interactions when compared to the commonly used NaCl.     

Complex formation and characterization of MgCl2/2-(2-ethylhexyloxy)ethanol adduct

Complex formation of MgCl₂ with 2-(2-ethylhexyloxy)ethanol was studied by IR and NMR spectroscopic methods and molecular modeling to determine the binding mode of the alcohol to MgCl₂. According to both experimental and theoretical studies, during the reaction of the alcohol and MgCl₂, two alcohol molecules form an adduct with MgCl₂ through the oxygen atoms of the alcohol and ether groups, giving rise to a chelated structure. Crystallization of the MgCl₂/2-(2-ethylhexyloxy)ethanol complex was attempted by various methods. Toluene was used as solvent in the dissolution of MgCl₂ in alcohol, and heptane was used to adjust the solubility of MgCl₂ during the crystallization. Crystals piled up as thin plates. Single-crystal X-ray structure analysis revealed a chelated structure formed through the oxygen atoms of the ether and alcohol groups. Two water molecules are also bound to the magnesium, as seen in the IR spectrum of the crystals as well.     

Improved assembly of bispecific antibodies from knob and hole half‐antibodies

A process was developed for large‐scale assembly of IgG₁ and IgG₄ bispecific antibodies from knob and hole half‐antibodies. We optimized assembly conditions such as pH, temperature, stabilizers, and reducing agent. We also identified and exploited structural changes unique to knob and hole half‐antibodies with the result of improving assembly outcome, specifically storing half‐antibodies at higher pH will condition them to assemble more rapidly and produce fewer high molecular‐weight species (HMWS). Application of heat to the assemblies resulted in an acceleration of assembly rate, with optimal formation of bispecific achieved at 37°C. IgG₄ half‐antibodies were unusually sensitive to temperature‐dependent formation of HMWS in pre‐assembly conditioning as well as during assembly. We selected l ‐histidine and Polyvinylpyrrolidone (PVP) as stabilizers to prevent HMWS formation in IgG₄, and achieved rapid and high‐efficiency assemblies. Using optimized assembly conditions, we developed and scaled up a method for assembling bispecific antibody with 90% assembly efficiency over 6 h with minimal impact to product quality, generating a pool with bispecific antibody for downstream processing. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:1315–1322, 2015     

Challenges with afucosylation content in antibody‐based drugs: Guidance provided by mathematical modeling

The theory of competitive ligand–receptor binding has been used to analyze the effect of afucosylation‐based antibody heterogeneity on Fc‐FcγRIIIa ligand–receptor binding activity. In vitro activity is found to represent a linear combination of the component antibody activities, weighted by the relative concentrations of the different afucosylated antibody forms. An analysis of ELISA binding activity data has allowed for the dissection of the activity contributions of the different afucosylated antibodies, revealing that the heterogeneous afucosylated antibody exhibits greater activity, on a per mole basis, when compared to the homogeneous afucosylated antibody. The ratio of the afucosylated antibody equilibrium dissociation constants is computed to be K_AF/K_A ≈ 0.6–0.9, where K_AF and K_A denote the dissociation equilibrium constant of the heterogeneous and the homogeneous afucosylated antibodies, respectively. Our analysis also reveals that, in general, activity scales quadratically with the afucosylated glycan content of a sample. Linear activity–afucosylated glycan fraction correlations reported in the literature are shown to represent specific cases of this general scaling and result from oversimplifying the underlying antibody concentration distributions. The implications of our findings for drug development are also discussed. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:775–782, 2015