Subvisible (2-100 μm) particle analysis during biotherapeutic drug product development: Part 2, experience with the application of subvisible particle analysis.

Measurement and characterization of subvisible particles (including proteinaceous and non-proteinaceous particulate matter) is an important aspect of the pharmaceutical development process for biotherapeutics. Health authorities have increased expectations for subvisible particle data beyond criteria specified in the pharmacopeia and covering a wider size range. In addition, subvisible particle data is being requested for samples exposed to various stress conditions and to support process/product changes.

Subvisible (2-100 μm) Particle Analysis During Biotherapeutic Drug Product Development: Part 1, Considerations and Strategy.

Measurement and characterization of subvisible particles (defined here as those ranging in size from 2 to 100 μm), including proteinaceous and nonproteinaceous particles, is an important part of every stage of protein therapeutic development. The tools used and the ways in which the information generated is applied depends on the particular product development stage, the amount of material, and the time available for the analysis. In order to compare results across laboratories and products, it is important to harmonize nomenclature, experimental protocols, data analysis, and interpretation.

An automated robotic platform for rapid profiling oligosaccharide analysis of monoclonal antibodies directly from cell culture.

Oligosaccharides attached to Asn297 in each of the CH2 domains of monoclonal antibodies play an important role in antibody effector functions by modulating the affinity of interaction with Fc receptors displayed on cells of the innate immune system. Rapid, detailed, and quantitative N-glycan analysis is required at all stages of bioprocess development to ensure the safety and efficacy of the therapeutic. The high sample numbers generated during quality by design (QbD) and process analytical technology (PAT) create a demand for high-performance, high-throughput analytical technologies for comprehensive oligosaccharide analysis.

Elucidation of PEGylation site with a combined approach of in-source fragmentation and CID MS/MS.

Poly(ethylene glycol) (PEG)ylation of peptides and proteins creates significant challenges for detailed structural characterization, such as PEG heterogeneity, site of addition and number of attached PEGylated moieties. Recently, we published a novel LC/MS methodology with a post-column addition of amines to obtain accurate masses of PEGylated peptides and proteins. The accurate masses can be used to assign the structures and number of attached PEGs [15], but the PEGylation site remains unclear in situations where multiple potential attachments are involved.

Linear epitope mapping by native mass spectrometry.

The identification of antigenic epitopes is important for the optimization of monoclonal antibodies (mAbs) intended as therapeutic agents. MS has proven to be a powerful tool for the study of noncovalent molecular interactions such as those involved in antibody-antigen (Ab-Ag) binding. In this work, we described a novel methodology for mapping a linear epitope based on direct mass spectrometric measurement of Ab-Ag complexes.

Analysis of 3-(acetylamino)-6-aminoacridine-derivatized oligosaccharides from recombinant monoclonal antibodies by liquid chromatography-mass spectrometry.

Glycosylation has been established as playing a pivotal role in various aspects of recombinant monoclonal antibodies (MAbs), ranging from pharmacokinetics to enhancement of effector function. Consequently, characterization of these oligosaccharides is of great importance and requires sensitive analytical techniques. Here we present a method for the rapid elucidation of 3-(acetylamino)-6-aminoacridine-labeled N-glycans present on MAbs using liquid chromatography-mass spectrometry.

Characterization of poly(ethylene glycol) and PEGylated products by LC/MS with postcolumn addition of amines.

PEGylation of peptides and proteins presents significant challenges for structural characterization due to the heterogeneity of the poly(ethylene glycol) (PEG), the number of PEG moieties attached, and the site(s) of PEGylation. In this work, a novel and powerful methodology using a postcolumn addition combined with LC/MS was developed and applied to examine high molecular weight (>/=20 kDa) PEG as well as PEGylated peptide and protein products. The PEG and PEGylated compounds were eluted from RP-HPLC, and the HPLC stream was mixed with diethylmethylamine (DEMA) or triethylamine (TEA) through a T mixer coupled to a time-of-flight mass spectrometer.

In vitro stability of insulin lispro in continuous subcutaneous insulin infusion.

Background: The stability of insulin lispro for use in continuous subcutaneous insulin infusion (CSII) therapy was evaluated using a stress test incorporating high temperature and mechanical agitation combined with simulated basal/bolus administration.

Methods: Insulin lispro formulation contained in MiniMed 507c (Medtronic MiniMed, Sylmar, CA), H-TRONplus (Disetronic Medical Systems, St. Paul, MN), and D-TRON CSII (Disetronic Medtronic Systems) devices was subjected to a stress test involving exposure to elevated temperature (37 degrees C) and mechanical agitation (shaking at 100 strokes/min) for 7 days.

Amyloid fibrils of glucagon characterized by high-resolution atomic force microscopy.

Glucagon solutions at pH 2.0 were subjected to mechanical agitation at 37 degrees C in the presence of a hydrophobic surface to explore the details of aggregation and fiber formation. High-resolution intermittent-contact atomic force microscopy performed in solution revealed the presence of aggregates after 0.

The role of formulation in insulin comparability assessments.

The concept of comparability can be applied when changes are made to manufacturing processes for biotechnology products subsequent to pivotal clinical trial studies. For many process changes, comparability can be demonstrated based entirely on relevant in vitro data provided that a detailed knowledge of the process/product exists, suitable analytical methodology is employed, and historical data are available for the assessment. Insulin provides an excellent model system to illustrate many important considerations when dealing with comparability exercises for biotechnology products.

Micronization of insulin from halogenated alcohol solution using supercritical carbon dioxide as an antisolvent.

Insulin was precipitated from solution in 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) using supercritical carbon dioxide (CO2) as an antisolvent. Biosynthetic human insulin crystals were dissolved in HFIP and the solution was sprayed through an ultrasonic nozzle into supercritical CO2. The factors in the 2(3) factorial experimental design with a center point replicate included pressure (83.

Insulin self-association and the relationship to pharmacokinetics and pharmacodynamics.

The treatment of type 1 diabetes requires multiple, daily injections of insulin. While many improvements involving formulation adjustments have been made in an attempt to optimize therapy, clinical experience indicates that the commercially available insulin preparations used for treatment have significant limitations. One principal deficiency relates to poor simulation of the physiological insulin secretion pattern, making achieving normalization of blood glucose concentrations difficult.

Structural studies of a crystalline insulin analog complex with protamine by atomic force microscopy.

Crystallographic studies of insulin-protamine complexes, such as neutral protamine Hagedorn (NPH) insulin, have been hampered by high crystal solvent content, small crystal dimensions, and extensive disorder in the protamine molecules. We report herein in situ tapping mode atomic force microscopy (TMAFM) studies of crystalline neutral protamine Lys(B28)Pro(B29) (NPL), a complex of Lys(B28)Pro(B29) insulin, in which the C-terminal prolyl and lysyl residues of human insulin are inverted, and protamine that is used as an intermediate time-action therapy for treating insulin-dependent diabetes. Tapping mode AFM performed at 6 degrees C on bipyramidally tipped tetragonal rod-shaped NPL crystals revealed large micron-sized islands separated by 44-A tall steps.

Preparation of a microcrystalline suspension formulation of Lys(B28)Pro(B29)-human insulin with ultralente properties.

The monomeric analogue, Lys(B28)Pro(B29)-human insulin (LysPro), has been crystallized using similar conditions employed to prepare extended-acting insulin ultralente formulations. In the presence of zinc ions, sodium acetate and sodium chloride, but without phenolic preservative, LysPro surprisingly forms small rhombohedral crystals with similar morphology to human insulin ultralente crystals with a mean particle size of 20 +/- 1 microm. X-ray powder diffraction studies on the LysPro crystals prior to dilution in ultralente vehicle ([NaCl] = 1.

A novel protein cross-linking reaction in stressed Neutral Protamine Hagedorn formulations of insulin.

The covalent insulin-protamine product molecules formed by heat stress in Neutral Protamine Hagedorn formulations of insulin and the insulin analogue [LysB28,ProB29] were examined by mass spectrometry. The results demonstrated that the covalent cross-link between insulin and protamine was not caused by linkage through the protamine N-terminal amino group, as had been previously thought. Our results indicate that the linkage was formed between the side chain of a protamine arginine and a histidine in the insulin B chain, resulting in a net mass change of -5 Da, compared to the sum of the protamine and insulin molecular masses.

Self-association properties of monomeric insulin analogs under formulation conditions.

Purpose: The purpose of the current study was to investigate the effects of two important excipients, zinc and m-cresol, on the self-association properties of a series of monomeric insulin analogs. In this way, the effects on formulation behavior of individual amino acid substitutions in the C-terminal region of the insulin B-chain could be compared.

Methods: The self-association of ten insulin analogs was monitored by equilibrium and velocity analytical ultracentrifugation under three different conditions: (i) in neutral buffer alone; (ii) in neutral buffer containing zinc ion; and (iii) in neutral buffer containing both zinc ion and phenolic preservative (a typical condition for insulin formulations).

Structural and morphological characterization of ultralente insulin crystals by atomic force microscopy: evidence of hydrophobically driven assembly.

Although x-ray crystal structures exist for many forms of insulin, the hormone involved in glucose metabolism and used in the treatment of diabetes, x-ray structural characterization of therapeutically important long-acting crystalline ultralente insulin forms has been elusive because of small crystal size and poor diffraction characteristics. We describe tapping-mode atomic force microscopy (TMAFM) studies, performed directly in crystallization liquor, of ultralente crystals prepared from bovine, human, and porcine insulins. Lattice images obtained from direct imaging of crystal planes are consistent with R3 space group symmetry for each insulin type, but the morphology of the human and porcine crystals observed by AFM differs substantially from that of the bovine insulin crystals.

Atomic force microscopy of crystalline insulins: the influence of sequence variation on crystallization and interfacial structure.

The self-association of proteins is influenced by amino acid sequence, molecular conformation, and the presence of molecular additives. In the presence of phenolic additives, LysB28ProB29 insulin, in which the C-terminal prolyl and lysyl residues of wild-type human insulin have been inverted, can be crystallized into forms resembling those of wild-type insulins in which the protein exists as zinc-complexed hexamers organized into well-defined layers. We describe herein tapping-mode atomic force microscopy (TMAFM) studies of single crystals of rhombohedral (R3) LysB28ProB29 that reveal the influence of sequence variation on hexamer-hexamer association at the surface of actively growing crystals.

Preparation and characterization of a cocrystalline suspension of [LysB28,ProB29]-human insulin analogue.

Soluble preparations of [LysB28,ProB29]-human insulin analogue (LysPro) exhibit more rapid absorption than human insulin upon subcutaneous injection. Biphasic mixtures of LysPro and intermediate-acting insulin suspensions could provide advantages over current preparations for the treatment of diabetes. To prepare biphasic mixtures of LysPro, a suspension formulation of the analogue is required.

Assembly and dissociation of human insulin and LysB28ProB29-insulin hexamers: a comparison study.

Purpose: Investigations into the kinetic assembly and dissociation of hexameric LysB28ProB29-human insulin (LysPro), a rapid-acting insulin analog produced by the sequence inversion of amino acids at positions B28 and B29, were designed to explain the impact that the sequence inversion has on the formulation and pharmacokinetics of the insulin analog.

Methods: The kinetics of phenolic ligand binding to human insulin and LysPro were studied by stopped-flow spectroscopy. The kinetics of R6 hexamer disruption were studied by extraction of Co(II) with EDTA.

Identification of essential residues for the catalytic function of 85-kDa cytosolic phospholipase A2. Probing the role of histidine, aspartic acid, cysteine, and arginine.

Cytosolic phospholipase A2 (cPLA2) hydrolyzes the sn-2-acyl ester bond of phospholipids and shows a preference for arachidonic acid-containing substrates. We found previously that Ser-228 is essential for enzyme activity and is likely to function as a nucleophile in the catalytic center of the enzyme (Sharp, J. D.

Kinetic analysis of the folding of human growth hormone. Influence of disulfide bonds.

We report the results of a stopped-flow kinetic evaluation of the folding of human growth hormone (hGH). The results are compared with those obtained for a disulfide-modified analog in which the four cysteine residues have been reduced and alkylated to form tetra-S-carbamidomethylated hGH in order to elucidate the role of disulfide bonds in the folding reaction. Multiple detection techniques were applied to monitor both refolding and unfolding processes initiated by guanidine hydrochloride concentration jumps.

Acid stabilization of human growth hormone equilibrium folding intermediates.

Equilibrium denaturation experiments were performed on human growth hormone (hGH) under acidic conditions (pH 1.5-3.0) and different protein concentrations.

Evidence for a self-associating equilibrium intermediate during folding of human growth hormone.

It has been previously shown, by equilibrium denaturation, that human growth hormone (hGH) folds by a cooperative two-state process. This is in contrast to the folding pathways of other nonhuman growth hormones that contain stable monomeric and multimeric equilibrium intermediates. We have reinvestigated the equilibrium denaturation of hGH at higher protein concentrations and found smooth transitions from the native to denatured state, but the calculated free energy for unfolding, delta G, decreases with increasing protein concentration.