Dextrose-mediated aggregation of therapeutic monoclonal antibodies in human plasma: Implication of isoelectric precipitation of complement proteins

Many therapeutic monoclonal antibodies (mAbs) are clinically administered through intravenous infusion after mixing with a diluent, e.g., saline, 5% dextrose. Such a clinical setting increases the likelihood of interactions among mAb molecules, diluent, and plasma components, which may adversely affect product safety and efficacy. Avastin® (bevacizumab) and Herceptin® (trastuzumab), but not Remicade® (infliximab), were shown to undergo rapid aggregation upon dilution into 5% dextrose when mixed with human plasma in vitro; however, the biochemical pathways leading to the aggregation were not clearly defined. Here, we show that dextrose-mediated aggregation of Avastin or Herceptin in plasma involves isoelectric precipitation of complement proteins. Using mass spectrometry, we found that dextrose-induced insoluble aggregates were composed of mAb itself and multiple abundant plasma proteins, namely complement proteins C3, C4, factor H, fibronectin, and apolipoprotein. These plasma proteins, which are characterized by an isoelectronic point of 5.5–6.7, lost solubility at the resulting pH in the mixture with formulated Avastin (pH 6.2) and Herceptin (pH 6.0). Notably, switching formulation buffers for Avastin (pH 6.2) and Remicade (pH 7.2) reversed their aggregation profiles. Avastin formed little, if any, insoluble aggregates in dextrose-plasma upon raising the buffer pH to 7.2 or above. Furthermore, dextrose induced pH-dependent precipitation of plasma proteins, with massive insoluble aggregates being detected at pH 6.5–6.8. These data show that isoelectric precipitation of complement proteins is a prerequisite of dextrose-induced aggregation of mAb in human plasma. This finding highlights the importance of assessing the compatibility of a therapeutic mAb with diluent and human plasma during product development.     

A comparison of the dodecyl sulfate-induced precipitation of the myelin basic protein with other water-soluble proteins

The interactions of sodium dodecyl sulfate with a number of proteins were examined at a variety of pH values ranging from 4.8 to 11.6 The dodecyl sulfate-induced precipitation of some of these proteins was observed within a relatively limited range of total dodecyl sulfate concentration. Most of the basic proteins precipitated at low pH but as the isoelectric point of the protein was approached the amount of protein that precipitated decreased. Bovine myelin basic protein was unique in that it precipitated at all pH values examined both above and below its isoelectric point. Thus, the dodecyl sulfate-induced precipitation of myelin basic protein appears to be different from the dodecyl sulfate-induced precipitation of most proteins. A comparison of protein precipitation at equivalent dodecyl sulfate: protein molar or weight ratios revealed very little difference in the precipitation behavior of the proteins studied. When the bovine myelin basic protein was cleaved at its single tryptophan residue, the N-terminal fragment (1–115) formed insoluble dodecyl sulfate complexes at pH values ranging from 4.8 to 9.2. The C-terminal fragment (116–169) precipitated almost completely at pH 4.8 but to a lesser extent at pH 7.4 and 9.2 Equimolar mixtures of the N- and C-terminal fragments precipitated in the presence of dodecyl sulfate at pH 7.4 and 9.2 to an extent greater than the C-terminal fragment alone but comparable to the N-terminal fragment alone or the whole basic protein. These results suggest: (a) that the mechanism by which dodecyl sulfate induces the precipitation of myelin basic protein may be unique compared to other proteins and (b) that the intact myelin basic protein is not necessary for its precipitation by dodecyl sulfate.     

Intermolecular complexation and phase separation in aqueous solutions of oppositely charged biopolymers

Turbidity measurements performed at 450nm were used to follow the process of complex formation, and phase separation in gelatin-agar aqueous solutions. Acid (Type-A) and alkali (Type-B) processed gelatin (polyampholyte) and agar (anionic polyelectrolyte) solutions, both having concentration of 0.1% (w/v) were mixed in various proportions, and the mixture was titrated (with 0.01 M HCl or NaOH) to initiate associative complexation that led to coacervation. The titration profiles clearly established observable transitions in terms of the solution pH corresponding to the first occurrence of turbidity (pH(C), formation of soluble complexes), and a point of turbidity maximum (pH(phi), formation of insoluble complexes). Decreasing the pH beyond pH(phi) drove the system towards precipitation. The values of pH(C) and pH(phi) characterized the initiation of the formation of intermolecular charge neutralized soluble aggregates, and the subsequent formation of microscopic coacervate droplets. These aggregates were characterized by dynamic light scattering. It was found that Type-A and -B gelatin samples formed soluble intermolecular complexes (and coacervates) with agar molecules through electrostatic and patch-binding interactions, respectively.     

The effect of chondromucoprotein on the solubility and peroxidatic properties of haemoglobin and methaemalbumin in aqueous solution and blood

1. Insoluble complexes, formed by electrostatic interaction between chondromucoprotein and chromoproteins (haemoglobin, methaemalbumin), were studied by measurement of precipitated pigment and by decrease in peroxidatic activity, maximum formation from aqueous solution occurring at pH 4·0–4·6. 2. Chondromucoprotein did not form complexes with plasma haptoglobins and haptohaemoglobins under these conditions, and high concentrations had no significant effect on colorimetric estimates of serum haptoglobin, although the peroxidatic activity of haemoglobinaemic serum was depressed owing to formation of chondromucoprotein–methaemalbumin complex. 3. The complexes formed by interaction between chondromucoprotein and plasma proteins contain two protein-bound biologically active components (plasminogen, haematin), as a result of co-precipitation after interaction between their carriers and chondromucoprotein. The possible presence of other biologically active trace components is discussed. 4. The results are related to complex-formation between other plasma proteins and chondromucoprotein, and possible implications arising from the complex-forming properties of tissue and urine chondromucoprotein are referred to. It is concluded that the inability of chondromucoprotein to form complexes with normal urine proteins is due to a deficiency of fibrinogen, β-lipoproteins and chromoproteins, which, in plasma, form a large proportion of the proteins involved in complex-formation.     

Influence of hetero-association on the precipitation of proteins by poly(ethylene glycol)

The influence of hetero-association on the precipitation of proteins by poly(ethylene glycol) was investigated by comparing the precipitation of binary mixtures to that of the individual proteins. Pronounced enhancement of precipitation was observed for several mixtures, with maximum effect at low ionic strength at a pH between the pI's. Measurements of sedimentation velocity and/or fluorescence polarization of dansyl-labeled components revealed that conditions fostering precipitation of a given mixture also enhanced the formation of soluble hetero-complexes in the absence of poly(ethylene glycol). Conversely, enhanced precipitation was not observed under conditions where complexes were shown to be absent. Poly(ethylene glycol) does not appear to influence such interactions and thus can be used to detect the presence of hetero-complexes in a binary mixture whose precipitation curve is shifted relative to those of its components.     

Compaction of DNA in an anionic micelle environment followed by assembly into phosphatidylcholine liposomes

A difficult problem concerning the interaction of DNA with amphiphiles of opposite charge above their critical micelle concentration is the propensity for aggregation of the condensed DNA complexes. In this study, this problem was addressed by attenuating amphiphile charge density within a cholate micelle environment. The amphiphile consisted of a cationic peptide, acetyl-CWKKKPKK-amide, conjugated to dilaurylphosphatidylethanolamine. In the presence of cholate, multiple equivalents of cationic charge were required to bring about the completion of DNA condensation. At the end point of condensation, stable, soluble DNA–micelle complexes were formed, which by dynamic light scattering exhibited apparent hydrodynamic diameters between 30 and 60 nm. Aggregation, as measured by static light scattering at 90° and by turbidity, was not observed until further additions of peptide–lipid conjugate were made beyond the end point of DNA condensation. Liposome complexes containing the non-aggregated, compacted DNA were formed by adding dioleoylphosphatidylcholine followed by removing the cholate by dialysis. The resulting complexes were distributed within a narrow density range, the DNA was quantitatively assembled into the liposomes, and liposomes without DNA were not detected. Small particles were formed with a mean hydrodynamic diameter of 77 nm. The liposomal DNA showed complete retention of its supercoiled form and no detectable sensitivity to DNase (25 U/10 µg DNA, 1.5 h, 37°C). The use of an anionic, dialyzable amphiphile to attenuate charge interactions between DNA and cationic amphiphiles is a useful technology for the quantitative assembly of compacted DNA into conventional liposomes, with complete protection against nuclease activity.     

Symmetric and asymmetric squarylium dyes as noncovalent protein labels: a study by fluorimetry and capillary electrophoresis

Noncovalent interactions between two squarylium dyes and various model proteins have been explored. NN127 and SQ-3 are symmetric and asymmetric squarylium dyes, respectively, the fluorescence emissions of which have been shown to be enhanced upon complexation with proteins such as bovine serum albumin (BSA), human serum albumin (HSA), beta-lactoglobulin A, and trypsinogen. Although these dyes are poorly soluble in aqueous solution, they can be dissolved first in methanol followed by dilution with aqueous buffer without precipitation, and are then suitable for use as fluorescent labels in protein determination studies. The nature of interactions between these dyes and proteins was studied using a variety of buffer systems, and it was found that electrostatic interactions are involved but not dominant. Dye/protein stoichiometries in the noncovalent complexes were found to be 1:1 for SQ-3, although various possible stoichiometries were found for NN127 depending upon pH and protein. Association constants on the order of 10(5) and 10(7) were found for noncovalent complexes of SQ-3 and NN127, respectively, with HSA, indicating stronger interactions of the symmetric dye with proteins. Finally, HSA complexes with NN127 were determined by capillary electrophoresis with laser-induced fluorescence detection (CE-LIF). In particular, NN127 shows promise as a reagent capable of fluorescently labeling analyte proteins for analysis by CE-LIF without itself being significantly fluorescent under the aqueous solution conditions studied herein.     

New procedure for isolation of amino acids based on selective hydrolysis of trimethylsilyl derivatives

A rapid procedure for the isolation of amino acids from physiological fluids by class separation suitable for gas chromatographic and gas chromatographic—mass spectrometric analysis is described. A physiological fluid such as plasma is adjusted to pH 2 and extracted with diethyl ether to remove organic acids and neutrals. After precipitation of proteins with trichloroacetic acid, the aqueous plasma is dried and derivatized by trimethylsilylation. Organic compounds like sugars and amino acids are rendered soluble in petroleum ether leaving inorganic salts when the soluble layer is transferred. Separation of sugars from amino acids is achieved by taking advantage of the different rates of aqueous hydrolysis of the trimethylsilyl (TMS) derivatives. Mixing the petroleum ether extract with a small volume of water results in two phases. The petroleum ether layer contains TMS-sugar constituents of plasma and the aqueous layer contains free amino acids and amines. This procedure was used to isolate L-dopa, 3-O-methyldopa and tyrosine from human plasma in a quantitation assay using ¹⁵O-labelled amino acids and gas chromatography—mass spectrometry.     

Protein precipitation by caprylic acid: Equilibrium composition data

The precipitation equilibria of hen egg-white lysozyme and bovine serum albumin from aqueous solutions by caprylic acid were studied. A thermodynamic equilibrium was obtained, and the compositions of both the precipitate and the aqueous phases were measured to establish phase diagrams for both systems. The precipitate was not pure protein, but also contained large amounts of water and caprylic acid. At constant initial pH and ionic strength, the composition of both phases depended only on the overall composition of the system. This suggests that protein precipitation by fatty acids should be pictured as liquid-liquid rather than solid-liquid equilibria. The precipitated proteins retained high biological activity. (c) 1996 John Wiley & Sons, Inc.     

Complex Coacervation and Precipitation Between Soluble Pea Proteins and Apple Pectin

Complex formation (leading to either coacervation or precipitation) offers a tool to generate plant-based novel food structures and textures. This study investigated the formation of complexes between soluble pea proteins and apple pectin upon varying the protein-to-pectin ratio (r?=?2:1 to 10:1), pH (3–7), and temperature (25 and 85 °C) with a total biopolymer concentration set to 1% (w/w). The results showed that predominantly soluble biopolymer complexes were formed at pH 5, and at low ratio (r?=?2:1), whereas lowering the pH to more acidic condition, and to higher ratios (r?=?4:1–10:1) induced the formation of more insoluble biopolymer complexes. In general, the mean particle sizes of the biopolymer complexes ranged between approximately 20 and 100 μm. Upon heating to 85 °C, the amount of insoluble biopolymer complexes increased at pH 3–5 at all ratios, except at r?=?2:1. In addition, the complex sizes became somewhat larger at r?=?2:1 to 6:1 upon heat treatment, whereas only trivial size changes were observed at higher ratios (r?=?8:1 to 10:1). Overall, electrostatic and hydrophobic interactions played a major role in the complex formation between the soluble pea proteins and apple pectin. These findings are important for designing solely plant-based food structures.

     

Purification of MAP–kinase protein complexes and identification of candidate components by XL–TAP–MS

The purification of low-abundance protein complexes and detection of in vivo protein–protein interactions in complex biological samples remains a challenging task. Here, we devised crosslinking and tandem affinity purification coupled to mass spectrometry (XL–TAP–MS), a quantitative proteomics approach for analyzing tandem affinity-purified, crosslinked protein complexes from plant tissues. We exemplarily applied XL–TAP–MS to study the MKK2–Mitogen-activated protein kinase (MPK4) signaling module in Arabidopsis thaliana. A tandem affinity tag consisting of an in vivo-biotinylated protein domain flanked by two hexahistidine sequences was adopted to allow for the affinity-based isolation of formaldehyde–crosslinked protein complexes under fully denaturing conditions. Combined with ¹⁵N stable isotopic labeling and tandem MS we captured and identified a total of 107 MKK2–MPK4 module-interacting proteins. Consistent with the role of the MPK signaling module in plant immunity, many of the module-interacting proteins are involved in the biotic and abiotic stress response of Arabidopsis. Validation of binary protein–protein interactions by in planta split-luciferase assays and in vitro kinase assays disclosed several direct phosphorylation targets of MPK4. Together, the XL–TAP–MS approach purifies low abundance protein complexes from biological samples and discovers previously unknown protein–protein interactions.

XL–TAP–MS: a novel technique that allows purification of crosslinked, low abundant protein complexes from plant tissues under denatured conditions and detection of in vivo protein–protein interactions.     

Glycosaminoglycan–Protein Interactions: The First Draft of the Glycosaminoglycan Interactome

The six mammalian glycosaminoglycans (GAGs), chondroitin sulfate, dermatan sulfate, heparin, heparan sulfate, hyaluronan, and keratan sulfate, are linear polysaccharides. Except for hyaluronan, they are sulfated to various extent, and covalently attached to proteins to form proteoglycans. GAGs interact with growth factors, morphogens, chemokines, extracellular matrix proteins and their bioactive fragments, receptors, lipoproteins, and pathogens. These interactions mediate their functions, from embryonic development to extracellular matrix assembly and regulation of cell signaling in various physiological and pathological contexts such as angiogenesis, cancer, neurodegenerative diseases, and infections. We give an overview of GAG–protein interactions (i.e., specificity and chemical features of GAG- and protein-binding sequences), and review the available GAG–protein interaction networks. We also provide the first comprehensive draft of the GAG interactome composed of 832 biomolecules (827 proteins and five GAGs) and 932 protein–GAG interactions. This network is a scaffold, which in the future should integrate structures of GAG–protein complexes, quantitative data of the abundance of GAGs in tissues to build tissue-specific interactomes, and GAG interactions with metal ions such as calcium, which plays a major role in the assembly of the extracellular matrix and its interactions with cells. This contextualized interactome will be useful to identify druggable GAG–protein interactions for therapeutic purpose:     

THE INTERACTION OF PARTICULATE HORSERADISH PEROXIDASE (HRP)-ANTI HRP IMMUNE COMPLEXES WITH MOUSE PERITONEAL MACROPHAGES IN VITRO

The uptake, distribution, and fate of particulate horseradish peroxidase (HRP)-anti HRP aggregates has been studied in homogeneous monolayers of mouse macrophages in vitro. Macrophages rapidly interiorize the immune complexes after binding to the cell surface. The rate of interiorization is maximal for complexes formed in a broad zone of 4-fold antibody excess to equivalence and corresponds to a rate of 10% of the administered load/10⁶ cells per hour. This rate is 4000-fold greater than the uptake of soluble HRP. The binding and endocytosis of HRP-anti HRP by macrophages is mediated by the trypsin insensitive F_c receptor. Cytochemically, intracellular HRP is localized within membrane bound vacuoles. After uptake of HRP, the enzymatic activity is degraded exponentially with a half-life of 14–18 hr until enzyme is no longer detectable. This half-life is twice as long as that previously observed for soluble uncomplexed HRP and is related to the combination of HRP with anti-HRP rather than the absolute amounts of enzyme or antibody ingested. The half-life of HRP-¹²⁵I was 30 hr. Exocytosis of cell associated enzyme or TCA precipitable counts was not detected, nor were persistent surface complexes demonstrable. The extensive capacity of macrophages to interiorize and destroy large amounts of antigen after the formation of antibody illustrates a role of this cell in the efferent limb of the immune response.     

Interactions between glycosaminoglycans and proteins, with particular reference to a new technique for isolating serum glycoproteins

1. Chondroitin sulphate–serum protein complexes (A, B and C), successively precipitated by adding chondroitin sulphate to serum at three arbitrary descending pH values (5·2, 4·3 and 3·1), were dissociated at pH 6·7 and chromatographed on DEAE-Sephadex, when the liberated serum proteins were simultaneously freed of chondroitin sulphate and separated into five fractions. Evidence that serum proteins were precipitated as a result of electrostatic interactions with dissociated carboxylate groups on the glycosaminoglycan is presented. 2. Serum proteins (fraction G), unable to form complexes with chondroitin sulphate, contained 4·4% of sialic acid and accounted for 4 and 26% of the total protein and protein-bound sialic acid in serum respectively. This fraction interacted electrostatically with chondroitin sulphate only when rendered more basic by removal of sialic acid residues with neuraminidase. The heat stability, solubility properties and high carbohydrate content of fraction G classified it as a seromucoid fraction. 3. Fraction G contained several glycoprotein and hexuronic acid-containing fractions, including a hitherto undetected brown-pigmented high-molecular-weight serum component, which migrated in starch gel between the origin and Sα₂-globulin and contained 3·1 and 4·1% of sialic acid and hexuronic acid respectively. 4. Glycosaminoglycan–protein interactions are discussed in relation to protein fractionation. By prior removal of less acidic proteins by these interactions, a new technique is available for isolating serum seromucoids in higher yields and under milder conditions than existing methods.     

Nuclear Import of Cdk/Cyclin Complexes: Identification of Distinct Mechanisms for Import of Cdk2/Cyclin E and Cdc2/Cyclin B1

Reversible phosphorylation of nuclear proteins is required for both DNA replication and entry into mitosis. Consequently, most cyclin-dependent kinase (Cdk)/cyclin complexes are localized to the nucleus when active. Although our understanding of nuclear transport processes has been greatly enhanced by the recent identification of nuclear targeting sequences and soluble nuclear import factors with which they interact, the mechanisms used to target Cdk/cyclin complexes to the nucleus remain obscure; this is in part because these proteins lack obvious nuclear localization sequences. To elucidate the molecular mechanisms responsible for Cdk/cyclin transport, we examined nuclear import of fluorescent Cdk2/cyclin E and Cdc2/cyclin B1 complexes in digitonin-permeabilized mammalian cells and also examined potential physical interactions between these Cdks, cyclins, and soluble import factors. We found that the nuclear import machinery recognizes these Cdk/cyclin complexes through direct interactions with the cyclin component. Surprisingly, cyclins E and B1 are imported into nuclei via distinct mechanisms. Cyclin E behaves like a classical basic nuclear localization sequence–containing protein, binding to the α adaptor subunit of the importin-α/β heterodimer. In contrast, cyclin B1 is imported via a direct interaction with a site in the NH₂ terminus of importin-β that is distinct from that used to bind importin-α.     

Cavities in protein–DNA and protein–RNA interfaces

An analysis of cavities present in protein–DNA and protein–RNA complexes is presented. In terms of the number of cavities and their total volume, the interfaces formed in these complexes are akin to those in transient protein–protein heterocomplexes. With homodimeric proteins protein–DNA interfaces may contain cavities involving both the protein subunits and DNA, and these are more than twice as large as cavities involving a single protein subunit and DNA. A parameter, cavity index, measuring the degree of surface complementarity, indicates that the packing of atoms in protein–protein/DNA/RNA is very similar, but it is about two times less efficient in the permanent interfaces formed between subunits in homodimers. As within the tertiary structure and protein–protein interfaces, protein–DNA interfaces have a higher inclination to be lined by β-sheet residues; from the DNA side, base atoms, in particular those in minor grooves, have a higher tendency to be located in cavities. The larger cavities tend to be less spherical and solvated. A small fraction of water molecules are found to mediate hydrogen-bond interactions with both the components, suggesting their primary role is to fill in the void left due to the local non-complementary nature of the surface patches.     

Interactions between chondroitin sulfate and concanavalin A

Chondroitin sulfate, the major extracellular matrix glycosaminoglycan, formed an insoluble complex with concanavalin A at pH 5.4 or below. Concanavalin A (500 μg/ml) reacted only with a relatively narrow concentration range of chondroitin sulfate (optimally between 5 and 50 μg/ml) at pH 5.4 in 0.05 M buffer. Similar precipitin-like interactions were seen between concanavalin A and hyaluronic acid or heparin. No precipitating complexes formed between concanavalin A and the glycosaminoglycans at these concentrations in physiological salt solutions (approx. 0.15 M) unless the pH was below 4.5. Precipitating self-aggregates of concanavalin A appeared to be promoted by chondroitin sulfate at pH 7.3, but no significant precipitation occurred between the reactants at this pH even at very high concentrations, nor did soluble complexes form as determined by affinity chromatography on Sephadex G-200 or fractionation on Bio-Gel P-200. Thus, binding between the lectin and glycosaminoglycans appeared to depend upon reversible non-specific electrostatic interactions observed only at low pH and low ionic strength. Stable interactions were not seen in experiments using physiologically balanced salts at near neutral pH.     

Beauty Is in the Eye of the Beholder: Proteins Can Recognize Binding Sites of Homologous Proteins in More than One Way

Understanding the mechanisms of protein–protein interaction is a fundamental problem with many practical applications. The fact that different proteins can bind similar partners suggests that convergently evolved binding interfaces are reused in different complexes. A set of protein complexes composed of non-homologous domains interacting with homologous partners at equivalent binding sites was collected in 2006, offering an opportunity to investigate this point. We considered 433 pairs of protein–protein complexes from the ABAC database (AB and AC binary protein complexes sharing a homologous partner A) and analyzed the extent of physico-chemical similarity at the atomic and residue level at the protein–protein interface. Homologous partners of the complexes were superimposed using Multiprot, and similar atoms at the interface were quantified using a five class grouping scheme and a distance cut-off. We found that the number of interfacial atoms with similar properties is systematically lower in the non-homologous proteins than in the homologous ones. We assessed the significance of the similarity by bootstrapping the atomic properties at the interfaces. We found that the similarity of binding sites is very significant between homologous proteins, as expected, but generally insignificant between the non-homologous proteins that bind to homologous partners. Furthermore, evolutionarily conserved residues are not colocalized within the binding sites of non-homologous proteins. We could only identify a limited number of cases of structural mimicry at the interface, suggesting that this property is less generic than previously thought. Our results support the hypothesis that different proteins can interact with similar partners using alternate strategies, but do not support convergent evolution.     

Dextrose-mediated aggregation of therapeutic monoclonal antibodies in human plasma: Implication of isoelectric precipitation of complement proteins

Many therapeutic monoclonal antibodies (mAbs) are clinically administered through intravenous infusion after mixing with a diluent, e.g., saline, 5% dextrose. Such a clinical setting increases the likelihood of interactions among mAb molecules, diluent, and plasma components, which may adversely affect product safety and efficacy. Avastin® (bevacizumab) and Herceptin® (trastuzumab), but not Remicade® (infliximab), were shown to undergo rapid aggregation upon dilution into 5% dextrose when mixed with human plasma in vitro; however, the biochemical pathways leading to the aggregation were not clearly defined. Here, we show that dextrose-mediated aggregation of Avastin or Herceptin in plasma involves isoelectric precipitation of complement proteins. Using mass spectrometry, we found that dextrose-induced insoluble aggregates were composed of mAb itself and multiple abundant plasma proteins, namely complement proteins C3, C4, factor H, fibronectin, and apolipoprotein. These plasma proteins, which are characterized by an isoelectronic point of 5.5–6.7, lost solubility at the resulting pH in the mixture with formulated Avastin (pH 6.2) and Herceptin (pH 6.0). Notably, switching formulation buffers for Avastin (pH 6.2) and Remicade (pH 7.2) reversed their aggregation profiles. Avastin formed little, if any, insoluble aggregates in dextrose-plasma upon raising the buffer pH to 7.2 or above. Furthermore, dextrose induced pH-dependent precipitation of plasma proteins, with massive insoluble aggregates being detected at pH 6.5–6.8. These data show that isoelectric precipitation of complement proteins is a prerequisite of dextrose-induced aggregation of mAb in human plasma. This finding highlights the importance of assessing the compatibility of a therapeutic mAb with diluent and human plasma during product development.     

Interactions between chondroitin sulfate and concanavalin A.

Chondroitin sulfate, the major extracellular matrix glycosaminoglycan, formed an insoluble complex with concanavalin A at pH 5.4 or below. Concanavalin A (500 microgram/ml) reacted only within a relatively narrow concentration range of chondroitin sulfate (optimally between 5 and 50 microgram/ml) at pH 5.4 in 0.05 M buffer. Similar precipitin-like interactions were seen between concanavalin A and hyaluronic acid or heparin. No precipitating complexes formed between concanavalin A and the glycosaminoglycans at these concentrations in physiological salt solutions (approx. 0.15 M) unless the pH was below 4.5. Precipitating self-aggregates of concanavalin A appeared to be promoted by chondroitin sulfate at pH 7.3, but no significant precipitation occurred between the reactants at this pH even at very high concentrations, nor did soluble complexes form as determined by affinity chromatography on Sephadex G-200 or fractionation on Bio-Gel P-200. Thus, binding between the lectin and glycosaminoglycans appeared to depend upon reversible non-specific electrostatic interactions observed only at low Ph and low ionic strength. Stable interactions were not seen in experiments using physiologically balanced salts at near neutral pH.