ABSTRACT
Subvisible particle formation, which occurs after the sterile filtration step of the fill/finish process, is a challenge that may occur during the development of biotherapeutics with complex molecular structures. Here, we show that a stainless steel pump head from a rotary piston pump produces more protein aggregates, past the limit of the acceptable quality range for subvisible particle counts, in comparison to a ceramic pump head. The quartz crystal microbalance was used to quantify the primary layer, proteins irreversibly adsorbed at the solid-liquid interface, and the secondary diffuse gel-like layer interacting on top of the primary layer. The results showed that the mass of protein irreversibly adsorbed onto stainless steel sensors is greater than on an aluminum oxide surface (ceramic pump mimic). This suggests that the amount of adsorbed protein plays a role in surface-induced protein aggregation at the solid-liquid interface.
Subject(s)
Antibodies, Monoclonal, Humanized/chemistry , Drug Compounding/methods , Immunoglobulin Fc Fragments/chemistry , Immunoglobulin G/chemistry , Stainless Steel/chemistry , Tumor Necrosis Factor-alpha/chemistry , Adsorption , Aluminum Oxide/chemistry , Antibodies, Monoclonal, Humanized/genetics , Ceramics/chemistry , Drug Stability , Immunoglobulin Fc Fragments/genetics , Immunoglobulin G/genetics , Protein Aggregates , Quartz Crystal Microbalance Techniques , Surface Properties , Tumor Necrosis Factor-alpha/geneticsABSTRACT
Changes in formulation may be required during the development of protein therapeutics. Some of the changes may alter the protein higher order structure (HOS). In this note, we show how the change from a trehalose-based formulation to an arginine-based formulation concomitantly impacted the tertiary structure and the thermal stability of a mAb (mAb1). The secondary structure was not disrupted by the formulation change. The destabilization of the tertiary structure did not affect the long-term stability or the bioactivity of mAb1. This indicates that loss of conformational stability was likely compensated by improvements in the colloidal stability of mAb1 in the arginine-based formulation. The formulation-induced changes in HOS were reversible as proven by measurements after dilution in a common buffer (phosphate-buffered saline). For aggregation driven by assembly of aggregates (colloidally limited), small changes in conformational structure and stability as measured by HOS methods may not necessarily be predictive of long-term stability.
Subject(s)
Antibodies, Monoclonal/chemistry , Biological Products/chemistry , Decision Support Techniques , Drug Discovery/methods , Technology, Pharmaceutical/methods , Arginine/chemistry , Buffers , Chemistry, Pharmaceutical , Drug Stability , Hydrogen-Ion Concentration , Protein Aggregates , Protein Conformation , Protein Stability , Structure-Activity Relationship , Temperature , Trehalose/chemistryABSTRACT
Escherichia coli O148 is a nonencapsulated enterotoxigenic (ETEC) Gram negative bacterium that can cause diarrhea, hemorrhagic colitis, and hemolytic uremic syndrome in humans. The surface-exposed O-specific polysaccharide (O-SP) of the lipopolysaccharide of this bacterium is considered both a virulence factor and a protective antigen. It is built up of the linear tetrasaccharide repeating unit [3)-α-L-Rhap-(1â2)-α-D-Glcp-(1â3)-α-D-GlcNAcp-(1â3)-α-L-Rhap-(1â] differing from that of the O-SP of Shigella dysenteriae type 1 (SD) only in that the latter contains a D-Galp residue in place of the glucose moiety of the former. The close similarity of the O-SPs of these bacteria indicated a possible cross-reactivity. To answer this question we synthesized several oligosaccharide fragments of E. coli O148 O-SP, up to a dodecasaccharide, as well as their bovine serum albumin or recombinant diphtheria toxin conjugates. Immunization of mice with these conjugates induced anti-O-SP-specific serum IgG antibody responses. The antisera reacted equally well with the LPSs of both bacteria, indicating cross-reactivity between the SD and E. coli O148 O-SPs that was further supported by Western-blot and dot-blot analyses, as well as by inhibition of binding between the antisera and the O-SPs of both bacteria.
Subject(s)
Cross Reactions/immunology , Escherichia coli/immunology , O Antigens/immunology , Oligosaccharides/chemical synthesis , Oligosaccharides/immunology , Carbohydrate Conformation , Escherichia coli/chemistry , Molecular Sequence Data , O Antigens/chemistry , Oligosaccharides/chemistryABSTRACT
Multivalent glycoconjugates were prepared using generation-4 PAMAM dendrimers, and their interaction with Langmuir monolayers containing GM3 was investigated. Excessive carbohydrate valency adversely affects the carbohydrate-carbohydrate interaction. The GM3 monolayer selectively interacts with lactose-functionalized dendrimers in the presence of calcium ions.
Subject(s)
Carbohydrates/chemistry , Dendrimers/chemistry , G(M3) Ganglioside/chemistry , Glycoconjugates/chemical synthesis , Lactose/chemistry , Glycoconjugates/chemistry , Molecular StructureABSTRACT
A new class of prodigiosenes with stability-enhancing functionalities appended to the C-ring were found to transport chloride anions through liposomal membranes, as well as to induce copper-mediated DNA cleavage.
Subject(s)
Chlorides/chemistry , Copper/chemistry , DNA/chemistry , Prodigiosin/chemistry , Ion TransportSubject(s)
Amides/chemistry , Chlorides/chemistry , Organic Anion Transporters/chemistry , Phthalic Acids/chemistry , Amides/metabolism , Chlorides/metabolism , Crystallography, X-Ray , Hydrogen Bonding , Ion Transport , Lipid Bilayers/chemistry , Lipid Bilayers/metabolism , Liposomes/chemistry , Models, Molecular , Molecular Conformation , Organic Anion Transporters/metabolism , Phthalic Acids/metabolismABSTRACT
The C3-symmetric triamide selectively transports NO3- anions across lipid vesicles: this H+-NO3- co-transporter alters the pH inside of liposomes experiencing a NO3-/Cl- gradient.
Subject(s)
Anions/chemistry , Chlorides/chemistry , Nitrates/chemistry , Crystallography, X-Ray , Hydrogen-Ion Concentration , Liposomes/chemistry , Models, Molecular , Molecular StructureABSTRACT
This paper describes studies of the carbohydrate-carbohydrate interaction (CCI) between micelles of a lactosyl lipid and monolayers of the glycosphingolipid GM(3). The lactose Lac.GM(3) interaction is involved in B16 melanoma cell adhesion and signaling processes, and a thorough understanding of the molecular details of this CCI is important for the design of new anti-adhesive and anti-metastatic agents. In this paper, we examine the influence of variations in divalent cations and subphase ionic strength on the Lac.GM(3) interaction. Our results indicate that, in the absence of divalent cations, the Lac.GM(3) CCI is strengthened at higher sodium chloride concentrations in the subphase. In contrast, when divalent cations are present in solution, the CCI is not as sensitive to ionic strength. These results suggest a role for both cation dependent as well as independent interactions in the Lac.GM(3) CCI.
