Opalescence Measurements: Improvements in Fundamental Knowledge, Identifying Sources of Analytical Biases, and Advanced Applications for the Development of Therapeutic Proteins.

Opalescence of biopharmaceutical solutions can indicate suboptimal colloidal stability and is therefore a generally undesirable attribute that requires investigation and potentially remediation. While there are numerous instrumentation options available for measuring opalescence, cross-instrument comparisons and detailed knowledge of analytical biases have been limited.  Here, we highlight key findings from a multi-instrument investigation where differences in reported opalescence values are explained with particular emphasis on how the optical configuration and detector properties of each instrument affect the response of the sample and the primary formazin standards required for instrument calibration. In doing so, the particle size distribution, angular-dependent light scattering properties and refractive index of the primary formazin standard material are characterized and presented. Finally, the advanced application of a 90° angle light scattering instrument is presented as a suitable approach for making low volume, temperature controlled, nephelometric measurements of opalescence.  Moreover, we demonstrate how this approach enables the simultaneous evaluation of key physical properties, such as hydrodynamic size, that are pertinent to investigations of opalescent biopharmaceuticals but have historically required the use of separate instrumentation.  The findings reported here address key knowledge gaps and provide opportunities for improving the efficiency and inter-laboratory comparability of opalescence measurements for biopharmaceuticals.

Investigation of anomalous charge variant profile reveals discrete pH-dependent conformations and conformation-dependent charge states within the CDR3 loop of a therapeutic mAb.

During the development of a therapeutic monoclonal antibody (mAb-1), the charge variant profile obtained by pH-gradient cation exchange chromatography (CEX) contained two main peaks, each of which exhibited a unique intrinsic fluorescence profile and demonstrated inter-convertibility upon reinjection of isolated peak fractions. Domain analysis of mAb-1 by CEX and liquid chromatography-mass spectrometry indicated that the antigen-binding fragment chromatographed as two separate peaks that had identical mass. Surface plasmon resonance binding analysis to antigen demonstrated comparable kinetics/affinity between these fractionated peaks and unfractionated starting material. Subsequent molecular modeling studies revealed that the relatively long and flexible complementarity-determining region 3 (CDR3) loop on the heavy chain could adopt two discrete pH-dependent conformations: an "open" conformation at neutral pH where the HC-CDR3 is largely solvent exposed, and a "closed" conformation at lower pH where the solvent exposure of a neighboring tryptophan in the light chain is reduced and two aspartic acid residues near the ends of the HC-CDR3 loop have atypical pKa values. The pH-dependent equilibrium between "open" and "closed" conformations of the HC-CDR3, and its proposed role in the anomalous charge variant profile of mAb-1, were supported by further CEX and hydrophobic interaction chromatography studies. This work is an example of how pH-dependent conformational changes and conformation-dependent changes to net charge can unexpectedly contribute to perceived instability and require thorough analytical, biophysical, and functional characterization during biopharmaceutical drug product development.

Cyclization of N-Terminal Glutamic Acid to pyro-Glutamic Acid Impacts Monoclonal Antibody Charge Heterogeneity Despite Its Appearance as a Neutral Transformation.

Pyroglutamic acid (pyroGlu) is commonly observed at the N-terminus of therapeutic monoclonal antibodies. Notably, the term "pyroGlu" refers to a single product that could originate from the cyclization of either an N-terminal glutamine or an N-terminal glutamic acid. This is an important and easily overlooked distinction that has major implications on the charge variant nature of a pyroGlu relative to its uncyclized form. Cyclization of an N-terminal glutamine for instance clearly produces an acidic variant with a lower isoelectric point owing to the loss of the positively charged N-terminal amine. In this report, we demonstrate that cyclization of an N-terminal glutamic acid on the other hand produces a basic variant with a higher isoelectric point contrary to the typical assumption that the simultaneous loss of the N-terminal amine and the carboxylic acid side-chain would negate the formation of a charge variant. The results of our investigation demonstrate the need to consider the relative strengths of the acidic and basic functional groups which are altered when assessing whether the product will be a charge variant. This study also adds new knowledge and experimental evidence to understand charge heterogeneity in monoclonal antibodies.

Comparison of imaged capillary isoelectric focusing and cation exchange chromatography for monitoring dextrose-mediated glycation of monoclonal antibodies in infusion solutions.

Intravenous (IV) infusion of therapeutic proteins typically involves dilution of the formulated product into infusion media such as normal saline or dextrose, 5% m/v in water. We report results from a rigorous evaluation of imaged capillary isoelectric focusing (iCIEF) for monitoring dextrose-mediated glycation of proteins in IV infusion solutions. In addition to detecting stable Amadori glycation products, iCIEF was able to detect the labile Schiff base (SB) glycation adducts since the equilibrium with free dextrose is maintained on capillary. Method parameters such as sample dilution factor and ampholyte composition (but not urea) were found to influence the observed level of SB glycation adducts. The impacts of dextrose and urea on the apparent pI values are also reported. iCIEF results were compared with results from cation exchange chromatography, which was found to preferentially detect the more stable Amadori glycation products due to the on-column decomposition of the SB adducts resulting from the separation of the protein from free dextrose which in turn altered the SB adduct- free dextrose equilibrium. These results demonstrate the need for careful consideration when selecting the analytical methodology to investigate protein sensitivity to dextrose and to monitor protein stability in dextrose-containing infusion solutions.

Identification of leachables observed in the size exclusion chromatograms of a low concentration product stored in prefilled syringes.

Requisite leachables testing of pharmaceutical products is commonly conducted with pre-defined analytical methods on a subset of materials intended to be representative of the marketed product. Throughout product development, leachables may occasionally be detected in other methods not specifically intended for monitoring such impurities. We have identified two leachables, ethyl 4-ethoxybenzoate (E4E) and 2,6-di(t-butyl)-4-hydroxy-4-methyl-2,5-cyclohexadien-1-one (BHT-OH) in a low concentration product stored in prefilled syringes (PFS). The leachables were initially detected by size exclusion chromatography (SEC) as late-eluting impurity peaks. Syringe component extraction studies indicated that the impurities were related to the syringe stoppers. Positive identification of E4E was accomplished by reversed phase liquid chromatography- tandem mass spectrometry (RPLC-MS/MS). Positive identification of BHT-OH required RPLC-solid phase extraction-cryoflow NMR (RPLC-SPE-NMR), as initial RPLC-MS/MS investigations were unsuccessful in elucidating the structure. We focus specifically on the efforts required to identify the leachables, and the fortuitous mixed mode separation mechanism and low concentration nature of the product, which were the main factors contributing to the unlikely detection of the leachables by SEC. We note that our investigations were conducted independently of formal leachables and extractables (L&E) studies and we discuss challenges with designing and conducting such studies in a manner that captures the comprehensive L&E profile of a product.

Drug-to-antibody determination for an antibody-drug-conjugate utilizing cathepsin B digestion coupled with reversed-phase high-pressure liquid chromatography analysis.

Antibody drug conjugates or ADCs are currently being evaluated for their effectiveness as targeted chemotherapeutic agents across the pharmaceutical industry. Due to the complexity arising from the choice of antibody, drug and linker; analytical methods for release and stability testing are required to provide a detailed understanding of both the antibody and the drug during manufacturing and storage. The ADC analyzed in this work consists of a tubulysin drug analogue that is randomly conjugated to lysine residues in a human IgG1 antibody. The drug is attached to the lysine residue through a peptidic, hydrolytically stable, cathepsin B cleavable linker. The random lysine conjugation produces a heterogeneous mixture of conjugated species with a variable drug-to-antibody ratio (DAR), therefore, the average amount of drug attached to the antibody is a critical parameter that needs to be monitored. In this work we have developed a universal method for determining DAR in ADCs that employ a cathepsin B cleavable linker. The ADC is first cleaved at the hinge region and then mildly reduced prior to treatment with the cathepsin B enzyme to release the drug from the antibody fragments. This pre-treatment allows the cathepsin B enzyme unrestricted access to the cleavage sites and ensures optimal conditions for the cathepsin B to cleave all the drug from the ADC molecule. The cleaved drug is then separated from the protein components by reversed phase high performance liquid chromatography (RP-HPLC) and quantitated using UV absorbance. This method affords superior cleavage efficiency to other methods that only employ a cathepsin digestion step as confirmed by mass spectrometry analysis. This method was shown to be accurate and precise for the quantitation of the DAR for two different random lysine conjugated ADC molecules.

Cooperative ordering at liquid crystal interfaces and its role in orientational memory.

Orientational memory in interfacial liquid crystal films occurs when cells heated above the isotropic transition temperature return to their initial ordered texture upon cooling. First observed over 80 years ago, the origins of orientational memory, which is sometimes called the surface memory effect, remain poorly understood. In this study, films of the thermotropic liquid crystal 4'-octyl-4-cyanobiphenyl on graphite were studied by scanning tunneling and polarizing optical microscopy. Strong orientational memory was observed despite relatively weak molecule-surface interactions of the kind previously thought to be responsible for this effect. By preparing cells in a uniformly oriented initial reference state and separately measuring bulk and surface order parameters as systems were thermally disordered, cooperative interactions were found to play an important role, leading to the recovery of long-range order that neither the bulk nor surface layers alone retained. When the surface and bulk layers were partially decoupled using a magnetic field, orientational memory in the surface layer almost disappeared. The findings provide a new interpretation of the origins of orientational memory in liquid crystal films and underscore the potentially important role of cooperativity in bulk <--> interfacial liquid crystal interactions.

Combined application of high resolution and tandem mass spectrometers to characterize methionine oxidation in a parathyroid hormone formulation.

Identification and monitoring of degradation products is a critical aspect of drug product stability programs. This process can present unique challenges when working with complex biopharmaceutical formulations that do not readily lend themselves to straightforward HPLC analysis. The therapeutic 34 amino acid parathyroid hormone fragment (PTH1-34) contains methionine (Met) residues at positions 8 and 18. Oxidation of these Met residues results in reduced biological activity and thus efficacy of the potential drug product. Here, we present an effective approach for the identification of PTH1-34 oxidation products in a drug product formulation in which the stability indicating method used non-MS compatible HPLC conditions to separate excipients, drug substance and degradation products. High resolution and tandem mass spectrometers were used in conjunction with cyanogen bromide (CNBr) mediated digestion to accurately identify the oxidation products observed in an alternative MS compatible HPLC method used for drug substance analysis. All anticipated CNBr digested peptide fragments, including both oxidized and nonoxidized peptide fragments, were positively identified using TOF MS without the need for additional enzymatic digestion. Once identified, the oxidation products generated were injected onto the original non-MS compatible HPLC drug product stability indicating method and the respective retention times were confirmed. This allowed the oxidative stability of different formulations to be effectively monitored during the solid state stability program and during variant selection.

Screening for physical stability of a Pseudomonas amylase using self-interaction chromatography.

Formulation development is an integral step in the successful commercialization of protein-based products in both the biotechnology and pharmaceutical industries. As the number of these protein formulations increases, so does the need for innovative approaches to characterize physical and chemical product stability. In this study, the osmotic second virial coefficient (B) of a commercial amylase was evaluated by self-interaction chromatography (SIC) as an innovative approach to characterize physical protein stability. B was measured as a function of pH and several common formulation additives (cosolvents), including sodium chloride, sucrose, and sorbitol. Cosolvent- and pH-induced physical stabilization of amylase is discussed in terms of positive shifts in B. Liquid chromatographic measurements of total soluble amylase and enzymatic activity measurements correlated qualitatively with trends in B except near the pI of amylase, where physical stability was minimal.

Effect of buffer species on the thermally induced aggregation of interferon-tau.

It is now becoming apparent that a common pathway of protein aggregation involves the unimolecular structural rearrangement from the native state to a slightly expanded aggregation-competent species. It is the goal of this study to understand the aggregation and the effects of buffer on the stability of IFN-tau. In this study, the thermally-induced aggregation of interferon-tau (IFN-tau) is described. By monitoring the aggregation rate in the presence of increasing amounts of sucrose, the relative change in surface area (Deltas) for conversion to the aggregation-competent state can be determined. Under conditions of pH 7 and in 20 mM buffer, the protein displays different aggregation rates depending on the nature of the buffer species. The protein aggregates mostly quickly in phosphate buffer, slower in the presence of Tris and slowest in the presence of histidine. The largest value for Deltas occurs for the histidine-containing samples, where aggregation proceeds via a slightly expanded aggregation competent state with a surface area increase of 7.6%. Furthermore, it appears that histidine binds to the native state of IFN-tau, thereby stabilizing the native state and retarding aggregation. Measurement of the second virial coefficient, B(22), for different formulations indicates that inclusion of histidine has only a small effect on repulsion between protein molecules, suggesting that colloidal stabilization is not the dominant mechanism for stabilization of IFN-tau. This study represents the first detailed biophysical study of specific buffer-induced stabilization, resulting in shifting the equilibrium towards the native state and away form the expanded aggregation-competent species.

Colloidal behavior of proteins: effects of the second virial coefficient on solubility, crystallization and aggregation of proteins in aqueous solution.

There has been an increasing awareness that proteins, like other biopolymers, are large enough to exhibit colloidal behavior in aqueous solution. Net attractive or repulsive forces have been found to govern important physical properties, such as solubility and aggregation. The extent of intermolecular interactions, usually expressed in terms of the osmotic second virial coefficient, B, is most often measured using static light scattering. More recently, self-interaction chromatography (SIC) has emerged as a method for rapid determination of B in actual formulations, as it uses much less protein and has higher throughput. This review will summarize the relationship of B to crystallization, solubility, and aggregation of proteins in aqueous solution. Moreover, the capability of SIC to obtain B values in a rapid and reproducible fashion will be described in detail. Finally, the use of miniaturized devices to measure B is presented.

Second virial coefficient studies of cosolvent-induced protein self-interaction.

Protein self-interaction is important in protein crystal growth, solubilization, and aggregation, both in vitro and in vivo, as with protein misfolding diseases, such as Alzheimer's. Although second virial coefficient studies can supply invaluable quantitative information, their emergence as a systematic approach to evaluating protein self-interaction has been slowed by the limitations of traditional measurement methods, such as static light scattering. Comparatively, self-interaction chromatography is an inexpensive, high-throughput method of evaluating the osmotic second virial coefficient (B) of proteins in solution. In this work, we used self-interaction chromatography to measure B of lysozyme in the presence of various cosolvents, including sucrose, trehalose, mannitol, glycine, arginine, and combinations of arginine and glutamic acid and arginine and sucrose in an effort to develop a better fundamental understanding of protein self-interaction in complex cosolvent systems. All of these cosolvents, alone or in combination, increased B, indicating a reduction in intermolecular attraction. However, the magnitude of cosolvent-induced changes in B was found to be largely dependent on the ability to control long-range electrostatic repulsion. To the best of our knowledge, this work represents the most comprehensive virial coefficient study to date focusing on complex cosolvent-induced effects on the self-interaction of lysozyme.