A screening tool for therapeutic monoclonal antibodies: Identifying the most stable protein and its best formulation based on thioflavin T binding

The lack of a fast selection method to identify the most stable protein is one of the major challenges for developing successful therapeutic protein formulations more rapidly. The swift and accurate detection of small amounts of aggregates is another problem since aggregates may trigger an immunological response and the aggregation decreases the biological activity of the antibody. Here we present an alternative method for initial screening of the aggregation propensity of proteins, using monoclonal antibodies (mAb) as an example and thioflavin T (ThT) binding. The major advantage of ThT binding is the short duration of testing compared with size-exclusion chromatography (SEC) measurements that can take 6 months or more even under accelerated conditions. The tendency to aggregate of each therapeutic human mAb probed with the ThT assay, together with SEC, is employed to formulate the ranking of mAb aggregation. ThT binding can determine the propensity of proteins to aggregate in a few days, illustrating that ThT binding would be a valuable screening tool.     

分子筛层析法分析重组乙肝表面抗原聚集物的形成

分子筛层析作为分析蛋白质颗粒聚集物的一种有力工具,被用于研究重组乙肝表面抗原聚集物的形成。已去除聚集物的表面抗原放置在不同的理化条件下或经过不同的纯化方法处理后,应用HPLC分析其聚集物的形成。为研究发酵过程中是否形成表面抗原聚集物,酵母细胞破碎后立即用Sepharose 4 FF层析柱分离为不同的组分,并分别进行HPLC分析。结果发现,在纯化过程和酵母发酵阶段都有表面抗原聚集物的产生。     

Aggregation of recombinant hepatitis B surface antigen in Pichia pastoris

The combination of immunoaffinity and size-exclusion chromatography (SEC) is a powerful tool to analyze multiprotein particle assembly. This approach was used to investigate the source of aggregation of recombinant hepatitis B surface antigen (HBsAg) detected in purified material. As HBsAg aggregation does not originate in the stresses, such as the concentration of HBsAg solutions, temperature and chaotropic agents, it is less probable that the HBsAg aggregate is produced during the process. To test whether aggregation takes place in vivo, crude yeast extract containing the expressed HBsAg was fractioned on a Sephacryl S-400 column just after cell disruption, and each fraction immunopurified individually. As a result, the HBsAg aggregate was isolated from a fraction corresponding to the elution of large particle aggregates only, not native HBsAg particles. It was biologically active, which demonstrates aggregate formation by specific assembly of partially or wholly folded HBsAg intermediates.     

Structural and kinetic properties of Bacillus subtilis S-adenosylmethionine synthetase expressed in Escherichia coli

S-adenosylmethionine (SAM) synthetase (EC 2.5.1.6) catalyzes the synthesis of S-adenosylmethionine using l-methionine and ATP as substrates. SAM synthetase gene (metE) from Bacillus subtilis was cloned and over-expressed, for the first time, in the heterologus host Escherichia coli as an active enzyme. Size-exclusion chromatography (SEC) revealed a molecular weight of ~180 kDa, suggesting that the enzyme is a homotetramer stabilized by non-covalent interactions. SAM synthetase exhibited optimal activity at pH 8.0 and 45 degrees C with the requirement of divalent cation Mg(2+), and stimulated by the monovalent cation K(+). The enzyme followed sequential mechanism with a V(max) of 0.362 micromol/min/mg, and a K(m) of 920 microM and 260 microM for ATP and l-methionine, respectively. The urea-induced unfolding equilibrium of the recombinant enzyme revealed a multistate process, comprising partially unfolded tetramer, structural dimer, structural monomer and completely unfolded monomer, as evidenced by intrinsic and extrinsic fluorescence, circular dichroism (CD) and SEC. Absence of trimer in the SEC implicates that the enzyme is a dimer of dimer. Concordance between results of SEC and enzyme activity in the presence of urea amply establishes that tetramer alone with intersubunit active site(s) exhibits enzyme activity.     

Peptides for disrupting and degrading amyloids

Amyloid proteins can aggregate into insoluble fibrils and form amyloid deposits in the human brain, which is the hallmark of many neurodegenerative diseases. Promising strategies toward pathological amyloid proteins and deposition include investigating inhibitors that can disrupt amyloid aggregation or induce misfolding protein degradation. In this review, recent progress of peptide-based inhibitors, including amyloid sequence–derived inhibitors, designed peptides, and peptide mimics, is highlighted. Based on the increased understanding of peptide design and precise amyloid structures, these peptides exhibit advanced inhibitory activities against fibrous aggregation as well as enhanced druggability.     

Quantitation of soluble aggregates in recombinant monoclonal antibody cell culture by pH-gradient protein A chromatography

Monoclonal antibodies (mAbs) produced from mammalian cell culture may contain significant amounts of dimers and higher order aggregates. Quantitation of soluble aggregates in the cell culture is time-consuming and labor-intensive, usually involving a purification step to remove the impurities that interfere with the subsequent size exclusion chromatography (SEC) analysis. We have developed a novel pH-gradient protein A chromatography for rapid, non-size based separation of the aggregates in mAb cell culture samples. Our results demonstrate that this method has excellent correlation with SEC and can be applied to both human immunoglobulin gamma 1 (IgG1) and IgG2 antibodies. This approach can be useful in the quantitation of soluble aggregates in crude cell culture samples.     

Studies on the intermolecular distribution of industrial pectins by means of preparative size exclusion chromatography

Three industrial high methoxyl pectins have been fractionated by size exclusion chromatography (SEC) on a preparative scale and the chemical composition, viscosity and light scattering behaviour of the fractions have been investigated. Chemical analysis revealed that the composition varies greatly from one SEC fraction to another. In all three pectin samples, the fractions of low molecular size contain most of the free neutral polysaccharides as well as some free pectin ‘hairy regions’. In addition, the lemon pectin samples contain some pectin molecules of large size which are rich in neutral sugars. Phenolic and proteinaceous compounds coelute with neutral sugar-rich fractions. However, in the apple pectin, phenolics and proteins occur predominantly in the fractions of low molecular size. Lemon pectin molecules, especially that of the lemon A sample, are prone to aggregation in the presence of calcium cations. The aggregate fraction can be disrupted by shear forces, heating or the presence of a chelating agent. The formation of such calcium-pectinate aggregates seems to be due to the presence of some molecules with low degrees of methoxylation. Light scattering measurements also suggest that even very narrow SEC fractions remain highly heterogeneous on the basis of their molecular weight, thus indicating large differences in molecular conformation.     

Comparison of selected analytical techniques for protein sizing, quantitation and molecular weight determination

Protein analysis techniques are developing fast due to the growing number of proteins obtained by recombinant DNA techniques. In the present paper we compare selected techniques, which are used for protein sizing, quantitation and molecular weight determination: sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE), lab-on-a-chip or microfluidics technology (LoaC), size exclusion chromatography (SEC) and mass spectrometry (MS). We compare advantages and limitations of each technique in respect to different application areas, analysis time, protein sizing and quantitation performance.     

A case study and use of sedimentation equilibrium analytical ultracentrifugation as a tool for biopharmaceutical development

Analytical ultracentrifugation (AUC) has re-emerged as a powerful technique for protein characterisation. We report the pivotal role sedimentation equilibrium AUC has played in the development of macrophage inflammatory protein-1α (MIP-1α) as a protein therapeutic. MIP-1α has potential clinical applications in cancer but its clinical use is limited, since it associates to form large insoluble aggregates in physiological buffers. Using AUC as a screening technique, we have produced a biologically active variant of MIP-1α, BB-10010, which has a reduced tendency to aggregate in physiological buffers. The aggregation of protein based pharmaceuticals is routinely monitored by size exclusion chromatography (SEC). Comparison of the data acquired by SEC and AUC, demonstrates that owing to the complexity of BB-10010, AUC analysis is required in addition to SEC to provide a rigorous characterisation of molecular association. This work has been extended to include the use of AUC as an analytical tool to monitor the quality of BB-10010 during formulation and stability studies. Accepted: 6 October 1996     

Inhibition of aggregation by media selection,sample loading and elution in size exclusion chromatographic refolding of denatured bovine carbonic anhydrase B

The effects of gel media, sample application and elution flowrate on the activity recovery and aggregation in refolding of bovine carbonic anhydrase B (CAB) by size exclusion chromatography (SEC) were investigated. Variation in aggregation was demonstrated visibly by the comparison of refolding profiles under different operating conditions. Some principles with regard to practical application were proposed. Meanwhile, the analysis of relationship between peak resolution and activity recovery provided evidences for the mechanism of size exclusion chromatography protein refolding.     

Mutational analysis of the propensity for amyloid formation by a globular protein

Acylphosphatase can be converted in vitro, by addition of trifluoroethanol (TFE), into amyloid fibrils of the type observed in a range of human diseases. The propensity to form fibrils has been investigated for a series of mutants of acylphosphatase by monitoring the range of TFE concentrations that result in aggregation. We have found that the tendency to aggregate correlates inversely with the conformational stability of the native state of the protein in the different mutants. In accord with this, the most strongly destabilized acylphosphatase variant forms amyloid fibrils in aqueous solution in the absence of TFE. These results show that the aggregation process that leads to amyloid deposition takes place from an ensemble of denatured conformations under conditions in which non-covalent interactions are still favoured. These results support the hypothesis that the stability of the native state of globular proteins is a major factor preventing the in vivo conversion of natural proteins into amyloid fibrils under non-pathological conditions. They also suggest that stabilizing the native states of amyloidogenic proteins could aid prevention of amyloidotic diseases.     

Aggregation analysis of the microtubule binding domain in tau protein by spectroscopic methods

The microtubule-associated protein tau is a highly soluble protein that shows hardly any tendency to assemble under physiological conditions. In the brains of Alzheimer's disease (AD) patients, however, tau dissociates from the axonal microtubule and abnormally aggregates to form paired helical filaments (PHFs). One of the priorities in Alzheimer research is to clarify the mechanism of PHF formation. In recent years, several factors regulating tau assembly have come to light, yet some important questions remain to be answered. In this work, the His-tagged gene constructs of the four-repeat microtubule binding domain (4RMBD) in tau protein and its three mutants, 4RMBD S305N, N279K, and P301L, were expressed in E. coli and purified. Gel filtration chromatography and dynamic light scattering measurement yielded a Stokes radius of 3.1 nm, indicating that the His-tagged 4RMBD normally exists in buffer solution in a dimer state, which is formed by non-covalent intermolecular interactions. This non-covalent dimer can further polymerize to form filaments in the presence of polyanions such as heparin. The kinetics of the in vitro aggregation was monitored by thioflavine S dye fluorescence and CD measurements. The aggregation of 4RMBD was suggested to be a nucleation-dependent process, where the non-covalent dimer acts as an effective structural unit. The aggregation rate was strongly affected by the point mutation. Among the 4RMBD mutants, the rate of S305N was exceptionally fast, whereas N279K was the slowest, even slower than the wild-type. The aggregations were optimal in a weakly reducing environment for all the mutants and the wild type. However, the aggregations were affected differently by buffer pH, depending on the 4RMBD mutation.     

Amylopectin aggregation as a function of starch phosphate content studied by size exclusion chromatography and on-line refractive index and light scattering

Starches with a natural 65-fold span in covalently bound phosphate content were prepared from five different crops including sorghum, cassava, three potato varieties and an exotic ginger plant, Curcuma zedoaria, with extreme starch phosphate content. These starches were subjected to size exclusion chromatography with refractive index detection (SEC/RI). A simple and rapid method for starch solubilisation was used. The conditions during solubilisation (2 M NaOH) and separation (10 mM NaOH, 50°C) were such as enabling >94% recovery of the starch without detectable degradation. The aggregation properties of the starch was investigated using on line refractive index/multi angle laser light scattering (RI/MALLS) detection. Three major regions in the SEC profile were identified, consisting of large amylopectin aggregates, amylopectin particles with radius of gyration (R_g) of approx 200 nm (400 nm blocklets) and amylose. A procedure for correction of light scattering signals spread over the SEC profile as a result of aggregate tailing was developed. The significance of the relative amounts of these three molecular species on standard starch pasting parameters, as measured by a Rapid Visco Analyzer (RVA), was investigated. Starches with a high amount of amylopectin aggregates showed high peak viscosities. Moreover, very high amounts of starch bound phosphate or amylose appears to suppress the content of large aggregates resulting in low viscosity.     

On the use of size exclusion chromatography for the resolution of mixed amyloid aggregate distributions: I. equilibrium partition models

In this study, we investigated the theoretical potential of size exclusion chromatography (SEC) for resolving mixtures of protein aggregates (of various sizes and shapes) produced in the generation of amyloid fibrils. We present our findings in the form of an equilibrium partition model. We first review the general characteristics of SEC and discuss the physicochemical features affecting solute transport and partition. We then develop new methods for estimating the transport and partition coefficients of protein aggregates on the basis of their molecular dimensions and the SEC column properties. We detail how these calculated properties can be used to estimate the likely resolving power of an SEC column. Model predictions were found to be in general agreement with experimental data gained from the measurement of the elution profile of sheared amyloid fibrils prepared from bovine insulin and passed through a Superose 6 precision SEC column. Our formalism should provide a basic appreciation of the competing factors at work and allow an informed choice to be made for optimal selection of SEC column medium to separate a desired size range of aggregate.     

Characterization and analysis of thermal denaturation of antibodies by size exclusion high-performance liquid chromatography with quadruple detection

Size exclusion chromatography (SEC) coupled with online light scattering, viscometry, refractometry, and UV-visible spectroscopy provides a very powerful tool for studying protein size, shape, and aggregation. This technique can be used to determine the molecular weight of the component peaks independent of the retention times in the SEC column and simultaneously measure the hydrodynamic radius and polydispersity of the protein. We applied this technology by coupling an Agilent Chemstation high-performance liquid chromatography system with a diode array UV-visible detector and a Viscotek 300 EZ Pro triple detector (combination of a light scattering detector, refractometer, and differential pressure viscometer) to characterize and compare the molecular properties of a number of monoclonal antibodies. Our studies reveal that different monoclonal immunoglobulin Gs (IgGs) and chimeric IgGs show slightly different retention times and therefore different molecular weights in gel filtration analysis. However, when they are analyzed by light scattering, refractometry, and viscometry, different IgGs have comparable molecular weight, molecular homogeneity (polydispersity), and size. Gel filtration coupled with UV or refractive index detection suggests that antibodies purified and formulated for preclinical and clinical development are more than 95% monomer with little or no detectable soluble aggregates. Light scattering measurements showed the presence of trace amounts of soluble aggregate in all the IgG preparations. The different IgG molecules showed different susceptibility to heat and pH. One of the murine antibodies was considerably less stable than the others at 55 degrees C. The application of this powerful technology for the characterization of monoclonal antibodies of therapeutic potential is discussed.     

Study by PMR spectroscopy and gel chromatography of the unfolding of substituted kerateines in 8 M urea.

Various S-substituted derivatives of the reduced low sulphur and high proteins from wool have been prepared in which the substituted group is hydrogen, carboxymethyl, carboxethyl, methyl, carbamidomethyl, cyanoethyl and aminoethyl. The proton magnetic resonance (PMR) spectra and gel filtration chromatography of these proteins have been examined in 8 M urea solution as a function of pH in order to determine conditions under which the proteins occur as random coils in solution with no evidence for the occurrence of non-covalent interactions. The PMR method described in an earlier paper (1) provides an easier and much more sensitive method for the observation of non-covalent interactions in random coil proteins than does the measurement of elution volumes in gel chromatography. The results obtained by both methods are consistent and show that the widest range of pH for which unfolding occurs in 8 M urea is obtained with the S-carboxymethyl, S-carboxyethyl, S-methyl and S-carbamidomethyl derivatives.     

Expression, purification and preliminary biochemical and structural characterization of the leucine rich repeat namesake domain of leucine rich repeat kinase 2

Mutations in leucine-rich repeat kinase 2 (LRRK2) are the most common cause of familial Parkinson's disease. Much research effort has been directed towards the catalytic core region of LRRK2 composed of GTPase (ROC, Ras of complex proteins) and kinase domains and a connecting COR (C-terminus of ROC) domain. In contrast, the precise functions of the protein-protein interaction domains, such as the leucine-rich repeat (LRR) domain, are not known. In the present study, we modeled the LRRK2 LRR domain (LRR(LRRK2)) using a template assembly approach, revealing the presence of 14 LRRs. Next, we focused on the expression and purification of LRR(LRRK2) in Escherichia coli. Buffer optimization revealed that the protein requires the presence of a zwitterionic detergent, namely Empigen BB, during solubilization and the subsequent purification and characterization steps. This indicates that the detergent captures the hydrophobic surface patches of LRR(LRRK2) thereby suppressing its aggregation. Circular dichroism (CD) spectroscopy measured 18% α-helices and 21% β-sheets, consistent with predictions from the homology model. Size exclusion chromatography (SEC) and dynamic light scattering measurements showed the presence of a single species, with a Stokes radius corresponding to the model dimensions of a protein monomer. Furthermore, no obvious LRR(LRRK2) multimerization was detected via cross-linking studies. Finally, the LRR(LRRK2) clinical mutations did not influence LRR(LRRK2) secondary, tertiary or quaternary structure as determined via SEC and CD spectroscopy. We therefore conclude that these mutations are likely to affect putative LRR(LRRK2) inter- and intramolecular interactions.     

Counteracting effects of thiocyanate and sucrose on chymotrypsinogen secondary structure and aggregation during freezing, drying, and rehydration.

Studies of numerous proteins with infrared spectroscopy have documented that unfolding is a general response of unprotected proteins to freeze-drying. Some proteins that are unfolded in the dried solid aggregate during rehydration, whereas others refold. It has been proposed for the latter case that aggregation is avoided because refolding kinetically outcompetes intermolecular interactions. In contrast, with proteins that normally aggregate after rehydration, minimizing unfolding during freeze-drying with stabilizer has been shown to be needed to favor the recovery of native protein molecules after rehydration. The purpose of the current study was to examine first the opposite situation, in which a denaturant is used to foster additional unfolding in the protein population during freeze-drying. If the protein is not intrinsically resistant to aggregation under the study conditions (e.g., because of intermolecular charge repulsion) and the denaturant does not disrupt intermolecular interactions during rehydration, this treatment should favor aggregation upon rehydration. With infrared spectroscopy we found that at concentrations of the denaturant Na thiocyanate (NaSCN) that only slightly perturbed chymotrypsinogen secondary structure in solution before freeze-drying, there was a large increase in protein unfolding in the dried solid and in protein aggregation measured after rehydration. Bands assigned to intermolecular beta sheet were present in the spectra of samples dried with NaSCN, indicating that aggregation could also arise in the dried solid. By examining the protein structure in the frozen state, we determined that in the absence of NaSCN the protein remains native. NaSCN caused structural perturbations during freezing, without the formation of intermolecular beta sheet, that were intermediate to structural changes noted after freeze-drying. In contrast, samples treated in the presence of NaSCN and sucrose had native-like spectra in the frozen and dried states, and much reduced aggregation after rehydration. These results indicate that during freezing and drying the sugar can counteract and mostly reverse the structural perturbations induced by NaSCN before and during these treatments.     

Quantitative evaluation of the contribution of weak lysine-binding sites present within apolipoprotein(a) kringle IV types 6-8 to lipoprotein(a) assembly

During lipoprotein(a) (Lp(a)) assembly, non-covalent interactions between apolipoprotein(a) (apo(a)) and low density lipoprotein precede specific disulfide bond formation. Studies have shown that the non-covalent step involves an interaction between the weak lysine-binding sites (WLBS) present within each of apo(a) kringle IV types 6, 7, and 8 (KIV(6-8)), and two lysine residues (Lys(680) and Lys(690)) within the NH(2) terminus of the apolipoprotein B-100 (apoB) component of low density lipoprotein. In the present study, we introduced single point mutations (E56G) into each of the WLBS present in apo(a) KIV(6-8) and expressed these mutations in the context of a 17-kringle (17K) recombinant apo(a) variant. Single mutations that disrupt the WLBS in KIV(6), KIV(7), and KIV(8), as well as mutants that disrupt the WLBS in both KIV(6) and KIV(7), or both KIV(7) and KIV(8), were assessed for their ability to form non-covalent and covalent Lp(a) complexes. Our results demonstrate that both apo(a) KIV(7) and KIV(8), but not KIV(6), are required for maximally efficient non-covalent and covalent Lp(a) assembly. Single mutations in the WLBS of KIV(7) or KIV(8) resulted in a 3-fold decrease in the affinity of 17K recombinant apo(a) for apoB, and a 20% reduction in the rate of covalent Lp(a) formation. Tandem mutations in the WLBS in both KIV(7) and KIV(8) resulted in a 13-fold reduction in the binding affinity between apo(a) and apoB, and a 75% reduction in the rate of the covalent step of Lp(a) formation. We also showed that KIV(7) and KIV(8) specifically bind with high affinity to apoB-derived peptides containing Lys(690) or Lys(680), respectively. Taken together, our data demonstrate that specific interactions between apo(a) KIV(7) and KIV(8) and Lys(680) and Lys(690) in apoB mediate a high affinity non-covalent interaction between apo(a) and low density lipoprotein, which dictates the efficiency of covalent Lp(a) formation.