Mark L. Brader

Fragmentation of a highly purified monoclonal antibody attributed to residual CHO cell protease activity.

Monoclonal antibody (mAb) fragmentation can be a widespread problem across the biotechnology industry and there is a current need to better understand the underlying principles. Here, we report an example of a high-purity human IgG1 mAb prepared from CHO cells exhibiting fragmentation that can be attributed to residual proteolytic enzyme activity. The concomitant occurrence of proteolytic and non-proteolytic peptide bond cleavage is shown and the respective fragmentation patterns characterized using high-resolution LC-MS. Fragmentation rates are monitored by SE-HPLC and SDS-PAGE over the pH range 4-6 and characterized in the presence and absence of pepstatin A, an inhibitor of acidic proteases. After 20 days at 40°C, pH 4, ∼60% decrease in BIIB-mAb monomer peak occurred attributed to residual proteolytic activity. At pH 5, this value was ∼13%. These results have implications for formulation design studies and the interpretation of accelerated stability data. A simple method to screen for acidic protease activity using the proteolytic enzyme inhibitor pepstatin A is described.

Examination of Thermal Unfolding and Aggregation Profiles of a Series of Developable Therapeutic Monoclonal Antibodies

Screening for pharmaceutically viable stability from measurements of thermally induced protein unfolding and short-term accelerated stress underpins much molecule design, selection, and formulation in the pharmaceutical biotechnology industry. However, the interrelationships among intrinsic protein conformational stability, thermal denaturation, and pharmaceutical stability are complex. There are few publications in which predictions from thermal unfolding-based screening methods are examined together with pharmaceutically relevant long-term storage stability performance. We have studied eight developable therapeutic IgG molecules under solution conditions optimized for large-scale commercial production and delivery. Thermal unfolding profiles were characterized by differential scanning calorimetry (DSC) and intrinsic fluorescence recorded simultaneously with static light scattering (SLS). These molecules exhibit a variety of thermal unfolding profiles under common reference buffer conditions and under individually optimized formulation conditions. Aggregation profiles by SE-HPLC and bioactivity upon long-term storage at 5, 25, and 40 °C establish that IgG molecules possessing a relatively wide range of conformational stabilities and thermal unfolding profiles can be formulated to achieve pharmaceutically stable drug products. Our data suggest that a formulation design strategy that increases the thermal unfolding temperature of the Fab transition may be a better general approach to improving pharmaceutical storage stability than one focused on increasing Tonset or Tm of the first unfolding transition.

Orthogonal Methods for Characterizing the Unfolding of Therapeutic Monoclonal Antibodies: Differential Scanning Calorimetry, Isothermal Chemical Denaturation, and Intrinsic Fluorescence with Concomitant Static Light Scattering.

Evaluating prospective protein pharmaceutical stability from accelerated screening is a critical challenge in biotherapeutic discovery and development. Measurements of protein unfolding transitions are widely employed for comparing candidate molecules and formulations; however, the interrelationships between intrinsic protein conformational stability and pharmaceutical robustness are complex and thermal unfolding measurements can be misleading. Beyond the discovery phase of drug development, astute formulation design is one of the most crucial factors enabling the protein to resist damage to its higher order structure-initially from bioprocessing stresses, then from stresses encountered during its journey from the product manufacturing site to the bloodstream of the patient. Therapeutic monoclonal antibodies are multidomain proteins that represent a large and growing segment of the biotechnology pipeline. In this chapter, we describe how differential scanning calorimetry may be leveraged synergistically with isothermal chemical denaturation and intrinsic fluorescence with concomitant static light scattering to elucidate characteristics of mAb unfolding and aggregation that are helpful toward understanding and designing optimal pharmaceutical compositions for these molecules.

Effects of Submicron Particles on Formation of Micron-Sized Particles During Long-Term Storage of an Interferon-Beta-1a Solution

We present evidence that homogeneous submicron particles can influence the growth rate of larger particles upon long-term storage in a temperature-dependent manner. Interferon-beta-1a was thermally stressed at 50°C for 6 h and characterized using nanoparticle tracking analysis (NTA), microflow digital imaging (MFI), and circular dichroism (CD) spectroscopy. This study showed selective formation of submicron particles exhibiting a perturbed protein conformation. These thermally induced submicron particles were spiked into an unstressed solution at three levels, and then monitored for micron-sized particle formation upon storage at 5°C and 25°C for 12 months. The resulting particle growth effects were temperature dependent. NTA and MFI results at 5°C showed little evidence that initial submicron particle levels impacted particle growth across the range ~0.03-25 μm. In contrast, MFI results at 25°C indicated that particle growth in the 1-10 μm size range correlated strongly with initial submicron particle levels, and particle counts in the 10-25 μm size range were highest after 12 months for the samples with highest initial submicron particle content.

Calcium Binding to a Factor IX Fc Fusion Protein and Effects on Higher‐Order Structure

There is significant scope for more meaningful evaluation of higher-order structure in defining the quality of biopharmaceutical products [Bush L. 2010. Biopharm Int 23(4):14]. We have used isothermal titration calorimetry (ITC) to characterize the Ca(2+) -binding isotherm of a recombinant human factor IX Fc fusion protein (rFIXFc) and the parent recombinant human factor IX molecule (rFIX). Circular dichroism, intrinsic fluorescence, and Fourier transform infrared spectroscopies detected characteristic spectral changes that appear qualitatively consistent with the previously characterized behavior of the factor IX molecule. Sedimentation velocity and dynamic light scattering measurements were recorded in the presence and absence of Ca(2+) over the protein concentration range 1-10 mg/mL. ITC of Ca(2+) binding to rFIXFc reveals a distinctive exothermic-binding isotherm, which is interpreted as consistent with two high-affinity and approximately 14 lower-affinity Ca(2+) sites reported in the literature for human factor IX (Schmidt AE, Bajaj SP. 2003. Trends Cardiovasc Med 13(1):39-45). Analysis of accelerated degradation samples showed significant alterations in Ca(2+) binding, which correlates with significant loss of biopotency and fragmentation by gel chip capillary electrophoresis. Collectively, these data establish a close correspondence in the Ca(2+) -binding characteristics of rFIXFc and its parent rFIX molecule. The utility of ITC to provide a highly pertinent and selective biophysical signature of structure-function for a therapeutic factor protein is discussed.

Evaluation of Incremental Siliconization Levels on Soluble Aggregates, Submicron and Subvisible Particles in a Prefilled Syringe Product

The evaluation of stability with respect to particles in prefilled syringes is complicated by the presence of silicone oil. The mobility, colloidal characteristics, and kinetic instability of silicone oil in contact with a protein formulation may be influenced in unpredictable ways by pharmaceutical variables, storage, and handling conditions. To provide insight into the impact of these variables on silicone oil originating specifically from the siliconized prefillable syringe (PFS), a series of studies were conducted at incremental syringe barrel siliconization levels. Size-exclusion chromatography and particle counting methods were used to quantitate soluble aggregates and submicron and subvisible particles in peginterferon beta-1a in a PFS siliconized with a fixed nozzle spray-on siliconization process. The effect of silicone oil on the peginterferon beta-1a molecule was examined under pharmaceutically relevant conditions, accelerated degradation, and under denaturing conditions. Resonant mass measurement was used to discriminate silicone oil from protein particles establishing that silicone oil does not mask adverse trends in non-silicone oil particles. The peginterferon beta-1a molecule was shown to be stable in the presence of silicone oil and robust with respect to the formation of soluble aggregates and submicron and subvisible particles in its PFS siliconized over the range of 0-1.2 mg silicone oil per syringe barrel.

The Value of UV, Fluorescence, and FTIR Spectroscopy in Biopharmaceutical Development

Optical spectroscopy may be viewed as a variety of clever ways to beam light through samples to enable deductions about the materials with which the collected light interacted. This chapter gives an overview of three spectroscopic techniques commonly employed in the biopharmaceutical development laboratory: ultraviolet (UV) absorption spectroscopy, fluorescence spectroscopy, and Fourier Transform infrared spectroscopy (FT-IR). Each of these techniques is applied in a multitude of incarnations across the entire breadth of therapeutic protein discovery, development, and product quality control. The emerging field of biosimilars and the impact of robotic-based high-throughput screening instrumentation are two major factors that are currently driving a renewed embrace of these spectroscopic methods in support of a protein pharmaceutical development.