Monoclonal Antibodies Follow Distinct Aggregation Pathways During Production-Relevant Acidic Incubation and Neutralization.

PURPOSE: Aggregation aspects of therapeutic monoclonal antibodies (mAbs) are of common concern to the pharmaceutical industry. Low pH treatment is applied during affinity purification and to inactivate endogenous retroviruses, directing interest to the mechanisms of acid-induced antibody aggregation. METHODS: We characterized the oligomerization kinetics at pH 3.3, as well as the reversibility upon neutralization, of three model mAbs with identical variable regions, representative of IgG1, IgG2 and IgG4 respectively. We applied size-exclusion high performance liquid chromatography and orthogonal analytical methods, including small-angle X-ray scattering and dynamic light scattering and supplemented the experimental data with crystal structure-based spatial aggregation propensity (SAP) calculations. RESULTS: We revealed distinct solution behaviors between the three mAb models: At acidic pH IgG1 retained monomeric, whereas IgG2 and IgG4 exhibited two-phase oligomerization processes. After neutralization, IgG2 oligomers partially reverted to the monomeric state, while on the contrary, IgG4 oligomers tended to aggregate. Subclass-specific aggregation-prone motifs on the Fc fragments were identified, which may lead to two distinct pathways of reversible and irreversible aggregation, respectively. CONCLUSIONS: We conclude that subtle variations in mAb sequence greatly affect responses towards low-pH incubation and subsequent neutralization, and demonstrate how orthogonal biophysical methods distinguish between reversible and irreversible mAb aggregation pathways at early stages of acidic treatment.

In-depth analysis of subclass-specific conformational preferences of IgG antibodies.

IgG subclass-specific differences in biological function and in vitro stability are often referred to variations in the conformational flexibility, while this flexibility has rarely been characterized. Here, small-angle X-ray scattering data from IgG1, IgG2 and IgG4 antibodies, which were designed with identical variable regions, were thoroughly analysed by the ensemble optimization method. The extended analysis of the optimized ensembles through shape clustering reveals distinct subclass-specific conformational preferences, which provide new insights for understanding the variations in physical/chemical stability and biological function of therapeutic antibodies. Importantly, the way that specific differences in the linker region correlate with the solution structure of intact antibodies is revealed, thereby visualizing future potential for the rational design of antibodies with designated physicochemical properties and tailored effector functions. In addition, this advanced computational approach is applicable to other flexible multi-domain systems and extends the potential for investigating flexibility in solutions of macromolecules by small-angle X-ray scattering.

Small-angle x-ray scattering screening complements conventional biophysical analysis: comparative structural and biophysical analysis of monoclonal antibodies IgG1, IgG2, and IgG4.

A crucial step in the development of therapeutic monoclonal antibodies is the selection of robust pharmaceutical candidates and screening of efficacious protein formulations to increase the resistance toward physicochemical degradation and aggregation during processing and storage. Here, we introduce small-angle X-ray scattering (SAXS) to characterize antibody solution behavior, which strongly complements conventional biophysical analysis. First, we apply a variety of conventional biophysical techniques for the evaluation of structural, conformational, and colloidal stability and report a systematic comparison between designed humanized IgG1, IgG2, and IgG4 with identical variable regions. Then, the high information content of SAXS data enables sensitive detection of structural differences between three IgG subclasses at neutral pH and rapid formation of dimers of IgG2 and IgG4 at low pH. We reveal subclass-specific variation in intermolecular repulsion already at low and medium protein concentrations, which explains the observed improved stability of IgG1 with respect to aggregation. We show how excipients dramatically influence such repulsive effects, hence demonstrating the potential application of extensive SAXS screening in antibody selection, eventual engineering, and formulation development.

High level expression, purification and activation of human dipeptidyl peptidase I from mammalian cells.

Dipeptidyl peptidase I (DPPI) plays a crucial role in maturation of many regulatory peptides and has been suggested as a pharmaceutical target in several inflammatory diseases. It is also a useful processing enzyme for the generation of authentic protein products by catalyzing the removal of N-terminal fusion peptides. We used a robust transient transfection system in human embryonic kidney 293 cells to exploit expression and activation of DPPI from chicken, rat and man for the development of an industrial production process. The expression of human and rat DPPI was significantly higher in the human HEK293 cell line than that obtained with avian DPPI. A CHO K1SV stable cell line was selected as the optimal stable host system for production of human DPPI yielding expression levels higher than 1.5 g/L. The secreted pro-DPPI underwent auto-maturation during defined buffer conditions during the purification steps. Active human DPPI was purified with a three-step purification strategy employing: Butyl Sepharose 4 Fast Flow, Sephadex G-25 Medium and Q Sepharose Fast Flow chromatography. The final yield of active enzyme was approximately 1 g/L cell culture. The enzyme exhibited exopeptidase activity against both a dipeptide-p-nitroanilide substrate and N-terminally extended MEAE-hGH (Met-Glu-Ala-Glu-human growth hormone). In conclusion, an efficient production process for recombinant human DPPI has been developed including a highly efficient and stable CHO cell system and an efficient purification procedure, which is simple and easy to scale for industrial purposes. The present data facilitates not only industrial applications of DPPI as a processing enzyme, but also provides active enzyme useful in the identification of small molecule inhibitors.

Aggregation of a multidomain protein: a coagulation mechanism governs aggregation of a model IgG1 antibody under weak thermal stress.

Using an IgG1 antibody as a model system, we have studied the mechanisms by which multidomain proteins aggregate at physiological pH when incubated at temperatures just below their lowest thermal transition. In this temperature interval, only minor changes to the protein conformation are observed. Light scattering consistently showed two coupled phases: an initial fast phase followed by several hours of exponential growth of the scattered intensity. This is the exact opposite of the lag-time behavior typically observed in protein fibrillation. Dynamic light scattering showed the rapid formation of an aggregate species with a hydrodynamic radius of about 25 nm, which then increased in size throughout the experiment. Theoretical analysis of our light scattering data showed that the aggregate number density goes through a maximum in time providing compelling evidence for a coagulation mechanism in which aggregates fuse together. Both the analysis as well as size-exclusion chromatography of incubated samples showed the actual increase in aggregate mass to be linear and reach saturation long before all molecules had been converted to aggregates. The CH2 domain is the only domain partly unfolded in the temperature interval studied, suggesting a pivotal role of this least stable domain in the aggregation process. Our results show that for multidomain proteins at temperatures below their thermal denaturation, transient unfolding of a single domain can prime the molecule for aggregation, and that the formation of large aggregates is driven by coagulation.

Thermodynamic characterization of the binding of tetrahydropterins to phenylalanine hydroxylase.

Phenylalanine hydroxylase (PAH) is the key enzyme in the catabolism of L-Phe. The natural cofactor of PAH, 6R-tetrahydrobiopterin (BH4), negatively regulates the enzyme activity in addition to being an essential cosubstrate for catalysis. The analogue 6-methyltetrahydropterin (6M-PH4) is effective in catalysis but does not regulate PAH. Here, the thermodynamics of binding of BH4 and 6M-PH4 to human PAH have been studied by isothermal titration calorimetry. At neutral pH and 25 degrees C, BH4 binds to PAH with higher affinity (Kd = 0.75 +/- 0.18 microM) than 6M-PH4 (Kd = 16.5 +/- 2.7 microM). While BH4 binding is a strongly exothermic process (DeltaH = -11.8 +/- 0.4 kcal/mol) accompanied by an entropic penalty (-TDeltaS = 3.4 +/- 0.4 kcal/mol), 6M-PH4 binding is both enthalpically (DeltaH = -3.3 +/- 0.3 kcal/mol) and entropically (-TDeltaS = -3.2 kcal/mol) driven. No significant changes in binding affinity were observed in the 5-35 degrees C temperature range for both pterins at neutral pH, but the enthalpic contribution increased with temperature rendering a heat capacity change (DeltaCp) of -357 +/- 26 cal/mol/K for BH4 and -63 +/- 12 cal/mol/K for 6M-PH4. Protons do not seem to be taken up or released upon pterin binding. Structure-based energetics calculations applied on the molecular dynamics simulated structures of the complexes suggest that in the case of BH4 binding, the conformational rearrangement of the N-terminal tail of PAH contribute with favorable enthalpic and unfavorable entropic contributions to the intrinsic thermodynamic parameters of binding. The entropic penalty is most probably associated to the reduction of conformational flexibility at the protein level and disappears for the L-Phe activated enzyme. The calculated energetic parameters aid to elucidate the molecular mechanism for cofactor recognition and the regulation of PAH by the dihydroxypropyl side chain of BH4.

Mechanisms underlying responsiveness to tetrahydrobiopterin in mild phenylketonuria mutations.

A subtype of phenylalanine hydroxylase (PAH) deficiency that responds to cofactor (tetrahydrobiopterin, BH4) supplementation has been associated with phenylketonuria (PKU) mutations. The underlying molecular mechanism of this responsiveness is as yet unknown and requires a detailed in vitro expression analysis of the associated mutations. With this aim, we optimized the analysis of the kinetic and cofactor binding properties in recombinant human PAH and in seven mild PKU mutations, i.e., c.194T>C (p.I65T), c.204A>T (p.R68S), c.731C>T (p.P244L), c.782G>A (p.R261Q), c.926C>T (p.A309V), c.1162G>A (p.V388M), and c.1162G>A (p.Y414C) expressed in E. coli. For p.I65T, p.R68S, and p.R261Q, we could in addition study the equilibrium binding of BH4 to the tetrameric forms by isothermal titration calorimetry (ITC). All the mutations resulted in catalytic defects, and p.I65T, p.R68S, p.P244L, and most probably p.A309V, showed reduced binding affinity for BH4. The possible stabilizing effect of the cofactor was explored using a cell-free in vitro synthesis assay combined with pulse-chase methodology. BH4 prevents the degradation of the proteins of folding variants p.A309V, p.V388M, and p.Y414C, acting as a chemical chaperone. In addition, for wild-type PAH and all mild PKU mutants analyzed in this study, BH4 increases the PAH activity of the synthesized protein and protects from the rapid inactivation observed in vitro. Catalase and superoxide dismutase partially mimic this protection. All together, our results indicate that the response to BH4 substitution therapy by PKU mutations may have a multifactorial basis. Both effects of BH4 on PAH, i.e., the chemical chaperone effect preventing protein misfolding and the protection from inactivation, may be relevant mechanisms of the responsive phenotype.

Structural and stability effects of phosphorylation: Localized structural changes in phenylalanine hydroxylase.

Phosphorylation of phenylalanine hydroxylase (PAH) at Ser16 by cAMP-dependent protein kinase increases the basal activity of the enzyme and its resistance to tryptic proteolysis. The modeled structures of the full-length phosphorylated and unphosphorylated enzyme were subjected to molecular dynamics simulations, and we analyzed the energy of charge-charge interactions for individual ionizable residues in the final structures. These calculations showed that the conformational changes induced by incorporation of phosphate were localized and limited mostly to the region around the phosphoserine (Arg13-Asp17) and a region around the active site in the catalytic domain that includes residues involved in the binding of the iron and the substrate L-Phe (Arg270 and His285). The absence of a generalized conformational change was confirmed by differential scanning calorimetry, thermal-dependent circular dichroism, fluorescence spectroscopy, and limited chymotryptic proteolysis of the phosphorylated and unphosphorylated PAH. Our results explain the effect of phosphorylation of PAH on both the resistance to proteolysis specifically by trypsin-like enzymes and on the increase in catalytic efficiency.

Activation of phenylalanine hydroxylase: effect of substitutions at Arg68 and Cys237.

Phenylalanine hydroxylase (PAH) is a multidomain tetrameric enzyme that displays positive cooperative substrate binding. This cooperative response is believed to be of physiological significance as a mechanism that controls L-Phe homeostasis in blood. The substrate induces an activating conformational change in the enzyme affecting the secondary, tertiary, and quaternary structures. Chemical modification and substitution with a negatively charged residue of Cys237 in human PAH (hPAH) also result in activation of the enzyme. As seen in the modeled structure of full-length hPAH, Cys237 is located in the catalytic domain close to residues in the oligomerization and regulatory domains of an adjacent subunit in the dimer, notably to Arg68. This residue is located in a prominent loop (68-75), which also has contacts with the dimerization motif from the same subunit. To investigate further the involvement of Cys237 and Arg68 in the activation of the enzyme, we have prepared mutants of hPAH at these positions, with substitutions of different charge and size. The mutations C237D, R68A, and C237A cause an increase of the basal activity and affinity for L-Phe, while the mutation C237R results in reduced affinity for the substrate and elimination of the positive cooperativity. The conformational changes induced by the mutations were studied by far-UV circular dichroism, fluorescence spectroscopy, and molecular dynamics simulations. All together, our results indicate that the activating mutations induce a series of conformational changes including both the displacement of the inhibitory N-terminal sequence (residues 19-33) that covers the active site and the domain movements around the hinge region Arg111-Thr117, in addition to the rearrangement of the loop 68-75. The same conformational changes appear to be involved in the activation of PAH induced by L-Phe.

Phosphorylation and mutations of Ser(16) in human phenylalanine hydroxylase. Kinetic and structural effects.

Phosphorylation of phenylalanine hydroxylase (PAH) at Ser(16) by cyclic AMP-dependent protein kinase is a post-translational modification that increases its basal activity and facilitates its activation by the substrate l-Phe. So far there is no structural information on the flexible N-terminal tail (residues 1-18), including the phosphorylation site. To get further insight into the molecular basis for the effects of phosphorylation on the catalytic efficiency and enzyme stability, molecular modeling was performed using the crystal structure of the recombinant rat enzyme. The most probable conformation and orientation of the N-terminal tail thus obtained indicates that phosphorylation of Ser(16) induces a local conformational change as a result of an electrostatic interaction between the phosphate group and Arg(13) as well as a repulsion by Glu(280) in the loop at the entrance of the active site crevice structure. The modeled reorientation of the N-terminal tail residues (Met(1)-Leu(15)) on phosphorylation is in agreement with the observed conformational change and increased accessibility of the substrate to the active site, as indicated by circular dichroism spectroscopy and the enzyme kinetic data for the full-length phosphorylated and nonphosphorylated human PAH. To further validate the model we have prepared and characterized mutants substituting Ser(16) with a negatively charged residue and found that S16E largely mimics the effects of phosphorylation of human PAH. Both the phosphorylated enzyme and the mutants with acidic side chains instead of Ser(16) revealed an increased resistance toward limited tryptic proteolysis and, as indicated by circular dichroism spectroscopy, an increased content of alpha-helical structure. In agreement with the modeled structure, the formation of an Arg(13) to Ser(16) phosphate salt bridge and the conformational change of the N-terminal tail also explain the higher stability toward limited tryptic proteolysis of the phosphorylated enzyme. The results obtained with the mutant R13A and E381A further support the model proposed for the molecular mechanism for the activation of the enzyme by phosphorylation.

L-phenylalanine binding and domain organization in human phenylalanine hydroxylase: a differential scanning calorimetry study.

Human phenylalanine hydroxylase (hPAH) is a tetrameric enzyme that catalyzes the hydroxylation of L-phenylalanine (L-Phe) to L-tyrosine; a dysfunction of this enzyme causes phenylketonuria. Each subunit in hPAH contains an N-terminal regulatory domain (Ser2-Ser110), a catalytic domain (Asp112-Arg410), and an oligomerization domain (Ser411-Lys452) including dimerization and tetramerization motifs. Two partially overlapping transitions are seen in differential scanning calorimetry (DSC) thermograms for wild-type hPAH in 0.1 M Na-Hepes buffer, 0.1 M NaCl, pH 7.0. Although these transitions are irreversible, studies on their scan-rate dependence support that the equilibrium thermodynamics analysis is permissible in this case. Comparison with the DSC thermograms for truncated forms of the enzyme, studies on the protein and L-Phe concentration effects on the transitions, and structure-energetic calculations based on a modeled structure support that the thermal denaturation of hPAH occurs in three stages: (i) unfolding of the four regulatory domains, which is responsible for the low-temperature calorimetric transition; (ii) unfolding of two (out of the four) catalytic domains, which is responsible for the high-temperature transition; and (iii) irreversible protein denaturation, which is likely responsible for the observed exothermic distortion in the high-temperature side of the high-temperature transition. Stages 1 and 2 do not appear to be two-state processes. We present an approach to the analysis of ligand effects on DSC transition temperatures, which is based on the general binding polynomial formalism and is not restricted to two-state transitions. Application of this approach to the L-Phe effect on the DSC thermograms for hPAH suggests that (i) there are no binding sites for L-Phe in the regulatory domains; therefore, contrary to the common belief, the activation of PAH by L-Phe seems to be the result of its homotropic cooperative binding to the active sites. (ii) The regulatory domain appears to be involved in cooperativity through its interactions with the catalytic and oligomerization domains; thus, upon regulatory domain unfolding, the cooperativity in the binding of L-Phe to the catalytic domains seems to be lost and the value of the L-Phe concentration corresponding to half-saturation is increased. Overall, our results contribute to the understanding of the conformational stability and the substrate-induced cooperative activation of this important enzyme.

The binding of tyrosine hydroxylase to negatively charged lipid bilayers involves the N-terminal region of the enzyme.

Tyrosine hydroxylase (TH) is the rate-limiting enzyme in the synthesis of catecholamines. We have studied the association of recombinant human TH with model membranes by using either liposomes or silica gel beads coated with single phospholipid bilayers (TRANSIL). The use of TRANSIL beads has allowed the determination of apparent dissociation constants (Kd) for the binding of the enzyme to negatively charged bilayers (Kd=230-380 microM, at pH 6.0-7.0). Binding to the bilayers is accompanied by a decrease in enzyme activity. Proteolysed forms of the enzyme show decreased binding affinity and two putative amphipathic N-terminal alpha-helices are proposed to be involved in membrane binding. As seen by circular dichroism, binding to the bilayer does not seem to induce significant changes on the secondary structure content of the enzyme, but alpha-helical structures appear to be stabilized against thermal denaturation in the membrane-bound state. Thus, amphitropism, a mechanism that regulates the function of peripheral proteins by weak binding to membrane lipids, may add to the factors that regulate both the activity and the stability of TH.