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. Consequently, subvisible particle analysis has expanded beyond routine testing of finished dosage forms using traditional compendial methods. Over the past decade, advances have been made in the detection and understanding of subvisible particle formation. This article presents industry case studies to illustrate the implementation of strategies for subvisible particle analysis as a characterization tool to assess the nature of the particulate matter and applications in drug product development, stability studies and post-marketing 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. In this manuscript on perspectives on subvisible particles in protein therapeutic drug products, we focus on the tools available for detection, characterization, and quantification of these species and the strategy around their application.

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. We have developed an automated 96-well plate-based sample preparation platform for high-throughput N-glycan analysis using a liquid handling robotic system. Complete process automation includes monoclonal antibody (mAb) purification directly from bioreactor media, glycan release, fluorescent labeling, purification, and subsequent ultra-performance liquid chromatography (UPLC) analysis. The entire sample preparation and commencement of analysis is achieved within a 5-h timeframe. The automated sample preparation platform can easily be interfaced with other downstream analytical technologies, including mass spectrometry (MS) and capillary electrophoresis (CE), for rapid characterization of oligosaccharides present on therapeutic antibodies.

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. Here, we present a methodology combining in-source fragmentation (ISF) with CID-MS/MS to elucidate the PEGylated sites in PEGylated products. All PEGylated samples, either prepared in acidic solution, or collected from a RP-HPLC stream, were first ionized via ISF to produce products containing small PEG fragment attachment, and then those fragment ions obtained were sequenced via CID MS/MS to deduce the PEGylation site. The methodology was successfully applied to PEGylated glucagon and IgG4 antibody light chain, which demonstrated that the small PEG fragments attached were stable during the CID activation.

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. To demonstrate the utility of our methodology, we employed two approaches, epitope excision and epitope extraction, to study a model system consisting of a Fab antibody fragment with specificity toward the peptide abeta(1-40). In epitope excision, the Fab and abeta(1-40) complex was treated with proteolytic enzymes and the digested complexes were directly monitored by MS under native conditions. Mass differences between the Fab-abeta complex and the Fab control revealed the size of epitope peptides that were bound to the Fab. Using the epitope extraction approach, abeta(1-40) was first digested by Lys-C, and the fragment containing the epitope was selected by Fab binding. Data analysis allowed mapping of the epitope to abeta(16-27) which is in good agreement with previously unpublished data. The utility of the methodology was demonstrated by elucidating the binding epitopes for two full-length anti-abeta(1-40) mAbs.

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. The technique uses the benefits of ultra-performance liquid chromatography systems in conjunction with small-particle-size amide columns capable of generating a fluorescence glycan profile of a MAb in 30 min, reducing the current run time by a factor of 6. The method is also compatible with online electrospray mass spectrometry, permitting the identification of glycans present. Overall, this strategy allows the confident determination of N-glycans present on recombinant MAbs in a significantly reduced amount of time.

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. With these conditions, PEG is diethylmethyl- or triethyl-ammoniated instead of protonated while the protein or peptide remains protonated. The charges for PEG and the PEGylated compounds were greatly reduced, and there was no convolution among differently charged ions, even for 40 kDa PEG or tri-20 kDa PEGylated IgG4 heavy chain. Mass accuracies (<0.01%) obtained are similar to large molecular weight proteins. By selecting specific amines, such as DEMA, commercially available software was used to deconvolute the spectra composed of the diethylmethylammoniated PEG or the diethylmethylammoniated and protonated PEGylated peptide or protein to obtain accurate masses. The examples presented in this report demonstrate that the methodology can be used to elucidate different PEG structures and modifications, such as oxidation and maleimide ring opening of PEGylated peptides or proteins.

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. CSII devices were programmed for continuous infusion at 0.8 U/h (19.2 U/day), and three 6-U bolus doses were also manually delivered each day (18 U/day). Formulation infused from the devices was collected every 24 h over the 7-day study. The material obtained from each 24-h period was analyzed to assess the physicochemical properties of insulin lispro and the overall formulation. Insulin lispro potency, purity, high-molecular-weight protein (HMWP), and m-cresol content were determined using high-performance liquid chromatographic methods. Solution pH, delivered volume, and formulation physical appearance were also evaluated. RESULTS: The pH of the infused formulation remained unchanged throughout the entire study. Insulin lispro potency for each device remained within 95-105% of label claim each day of the stress test, indicating that adsorption or surface-induced denaturation of insulin lispro did not occur. Levels of insulin lispro deamidated at position 21 in the A-chain were essentially constant during the 7-day stress test. A time-dependent increase in HMWP and other insulin lispro chemical transformation products was observed over the testing period for each device. However, the levels of these known degradation products remained well below published specifications for these analytical properties. The concentration of the antimicrobial agent, m-cresol, was decreased because of absorption, but the levels remained sufficient to provide protection against microbial contamination. Total delivered volume results were in good agreement with expected values and confirmed that formulation viscosity remained unchanged. Samples collected from the infusion sets were clear and free of precipitates. Occlusion alarms did not occur, and no other electronic or mechanical malfunctions of the CSII devices were encountered. CONCLUSIONS: Insulin lispro demonstrates appropriate physicochemical stability for use in MiniMed 507c, H-TRONplus, and D-TRON CSII devices.

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 0.5 h; however, longer agitation times resulted in the formation of fibrillated structures with varying levels of higher-order assembly. Height, periodicity, and amplitude measurements of these structures allowed the identification of four distinct fiber types. The most elementary fiber form, designated a filament, self-associates in a specific wound fashion to produce protofibrils composed of two filaments. Subsequent self-assembly of these filaments and protofibrils leads to two well-defined fibrillar motifs, termed Type I and Type II. Atomic force microscopy imaging of pH 2.8 glucagon solutions not agitated or exposed to elevated temperature revealed the presence of amorphous aggregates before the formation of fibrillar structures similar to those seen at pH 2.0. Time-course solution Fourier transform infrared spectroscopy and thioflavin T binding studies suggested that glucagon aggregation and fibril formation were associated with the development of beta-sheet structure. The results of these studies are used to describe a possible mechanism for glucagon aggregation and fibrillation that is consistent with a hierarchical assembly model proposed for amyloid fibril formation.

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. The physicochemical properties of insulin demonstrate the numerous chemical reactions and physical transformations that are exclusive to proteins. These properties are heavily influenced by formulation conditions and must be carefully evaluated when process changes are made. In addition, physical and chemical testing performed on representative formulations can provide valuable insight when assessing the comparability between pre- and post-change materials. This paper reviews our experience with manufacturing changes involving insulin emphasizing the important role of formulation in the comparability exercise for protein biopharmaceuticals.

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 (83.7 and 97.5 bar), solution concentration (15 and 30 mg/mL), and solution flow rate (2 and 4 mL/min). Temperature (37 degrees C), CO2 mass flow rate (137 g/min), and volume of solution sprayed (20 mL) were held constant. High-performance liquid chromatography, circular dichroism spectroscopy, infrared and Raman spectroscopy, scanning electron microscopy, dry powder size distribution analysis, thermogravimetric analysis, and atomic absorption spectroscopy were used to characterize the processed insulin powder. The processed insulin retained its potency, was slightly degraded chemically, and exhibited reversible structural changes. The precipitated powder consisted of physical aggregates of 50-nm spheres. Through deagglomeration of these aggregates, it may be possible to obtain discrete uniform particles (1-5 microm) suitable for pulmonary therapy. Over the ranges of operating variables studied, the factors chosen for the experimental design had little effect on the product characteristics.