Mary Cromwell

Protein aggregation and bioprocessing

Protein aggregation is a common issue encountered during manufacture of biotherapeutics. It is possible to influence the amount of aggregate produced during the cell culture and purification process by carefully controlling the environment (eg, media components) and implementing appro-priate strategies to minimize the extent of aggregation. Steps to remove aggregates have been successfully used at a manufacturing scale. Care should be taken when developing a process to monitor the compatibility of the equipment and process with the protein to ensure that potential aggregation is minimized.

An Industry Perspective on the Monitoring of Subvisible Particles as a Quality Attribute for Protein Therapeutics

Concern around the lack of monitoring of proteinaceous subvisible particulates in the 0.1-10 microm range has been heightened (Carpenter et al., 2009, J Pharm Sci 98: 1202-1205), primarily due to uncertainty around the potential immunogenicity risk from these particles. This article, representing the opinions of a number of industry scientists, aims to further the discussion by developing a common understanding around the technical capabilities, limitations, as well as utility of monitoring this size range; reiterating that the link between aggregation and clinical immunogenicity has not been unequivocally established; and emphasizing that such particles are present in marketed products which remain safe and efficacious despite the lack of monitoring. Measurement of subvisible particulates in the <10 microm size range has value as an aid in product development and characterization. Limitations in measurement technologies, variability from container/closure, concentration, viscosity, history, and inherent batch heterogeneity, make these measurements unsuitable as specification for release and stability or for comparability, at the present time. Such particles constitute microgram levels of protein with currently monitored sizes >or=10 microm representing the largest fraction. These levels are well below what is detected or reported for other product quality attributes. Subvisible particles remain a product quality attribute that is also qualified in clinical trials.

Effect of Individual Fc Methionine Oxidation on FcRn Binding: Met252 Oxidation Impairs FcRn Binding More Profoundly than Met428 Oxidation

The long serum half-lives of mAbs are conferred by pH-dependent binding of IgG-Fc to the neonatal Fc receptor (FcRn). The Fc region of human IgG1 has three conserved methionine residues, Met252, Met358, and Met428. Recent studies showed oxidation of these Met residues impairs FcRn binding and consequently affects pharmacokinetics of therapeutic antibodies. However, the quantitative effect of individual Met oxidation on Fc-FcRn binding has not been addressed. This information is valuable for defining critical quality attributes. In the present study, two sets of homodimeric site-directed IgG1 mutations were generated to understand how individual Fc Met oxidation affects FcRn binding. The first approach used Met to Leu mutants to block site-specific Met oxidation. In the other approach, Met to Gln mutants were designed to mimic site-specific Met oxidation. Both mutagenesis approaches show that either Met252 or Met428 oxidation alone significantly impairs Fc-FcRn binding. Met252 oxidation has a more deleterious effect on FcRn binding than M428 oxidation, whereas Met428 oxidation has a bigger destabilization effect on the thermal stability. Our results also show that Met358 oxidation does not affect FcRn binding. In addition, our study suggests that Met to Gln mutation may serve as an important tool to understand Met oxidation.

Establishing a control system using QbD principles.

Quality by design (QbD) is a global regulatory initiative with the goal of enhancing pharmaceutical development through the proactive design of pharmaceutical manufacturing process and controls to consistently deliver the intended performance of the product. The principles of pharmaceutical development relevant to QbD are described in the ICH guidance documents (ICHQ8-11). An integrated set of risk assessments and their related elements developed at Roche/Genentech were designed to provide an overview of product and process knowledge for the production of a recombinant monoclonal antibody. This chapter describes the elements and tools used to establish acceptance criteria and an attribute testing strategy (ATS) for product variants and process related impurities. The acceptable ranges for CQAs are set based on their potential impact on efficacy and safety/immunogenicity. This approach is focused on the management of patient impacts, rather than simply maintaining a consistent analytical profile. The ATS tools were designed to identify quality attributes that required process and/or testing controls, or that could be captured in a monitoring system to enable lifecycle management of the control strategy.

The effect of chemical modifications on octanol/water partition (log D) and permeabilities across Caco-2 monolayers

Abstract In the past octanol/water partition coefficients (log D 7.4 ) have been used extensively to predict intestinal absorption. More recently flux measurements across confluent monolayers of Caco-2 cells, a human colonic carcinoma cell line, have been introduced to estimate oral absorption. Recently we reported that dexamethasone-β- d -glucuronide (sodium salt) had a permeability coefficient that was one order of magnitude higher than dexamethasone-β- d -glucoside despite a two orders of magnitude lower log D 7.4 , i.e., the compound displaying the free acid moiety had a higher permeability than the uncharged dexamethasone-β- d -glucoside [1]. This finding is inconsistent with the dogma that a higher log D 7.4 should translate into a higher permeability coefficient. Therefore, the objective of the present study was to examine whether structures unrelated to steroids containing free acid moieties and their respective ethyl esters (uncharged) demonstrate similar characteristics. Using reversed-phase HPLC we have determined the octanol/water partition coefficient (log D 7.4 ) and permeability coefficients across Caco-2 monolayers of a limited series of bioactive compounds based on a benzodiazepine-2,5-dione scaffold, displaying a free acid moiety and the respective ethyl ester. Our results indicate: i . ester prodrugs have approximately two orders of magnitude higher octanol/water partition coefficient (log D 7.4 ) than the parents (free acids) and ii . the permeability coefficients of the prodrugs were lower than for the parents. We conclude log D 7.4 may not be a good predictor of the permeability coefficient and oral absorption.

Structural Elements Which Govern the Resistance of Intestinal Tissues to Compound Transport

The series of cyclized RGD peptides in this study demonstrated a very low partition into octanol as judged by HPLC. Thus, these molecules are likely to move predominantly through the paracellular pathway. Permeability across Caco-2 monolayers was determined using reversed phase HPLC and found to be restricted by molecular weight and possibly charge-charge interactions between the solute and charged moieties within the paracellular shunt. When normalized for molecular weight, molecules with a net charge between -1 and -2 demonstrated the highest permeabilities, which suggests an optimal net charge with respect to permeability.

Integration of QbD risk assessment tools and overall risk management

Quality by design (QbD) is a global regulatory initiative with the goal of enhancing pharmaceutical development through the proactive design of pharmaceutical manufacturing process and controls to consistently deliver the intended performance of the product. The principles of pharmaceutical development relevant to QbD are described in the ICH guidance documents (ICHQ8-11). An integrated set of risk assessments and related elements developed at Roche/Genentech were designed to provide an overview of product and process knowledge for the production of a recombinant monoclonal antibody. This chapter describes how the risk assessments, logic and interactions of the tools are designed to connect the set of QbD tools and elements into an overarching risk management system. The tools allow comparisons of options based on elective decisions that the sponsor could take and reflect relative values of these options. The overall risk management strategy assures product quality from this enhanced set of assessments and employs a science and risk based approach resulting in a consistent and transparent set of process and product controls and a rational monitoring system.

Protein Aggregation in Frozen Trehalose Formulations: Effects of Composition, Cooling Rate, and Storage Temperature

This study was designed to assess the effects of cooling rate, storage temperature, and formulation composition on trehalose phase distribution and protein stability in frozen solutions. The data demonstrate that faster cooling rates (>100°C/min) result in trehalose crystallization and protein aggregation as determined by Fourier Transform Near-Infrared (FT-NIR) spectroscopy and size-exclusion chromatography, respectively. Conversely, at slower cooling rates (≤1°C/min), trehalose remains predominantly amorphous and there is no effect on protein stability. Evaluation of storage temperatures demonstrates that aggregation increases more rapidly at -14°C compared with higher (-8°C) and lower (-20°C) storage temperatures; however, a relatively higher amount of cumulative aggregation was observed at lower (-20°C) temperature compared with higher storage temperatures (-14°C and -8°C). Further evaluation of the effects of formulation composition suggests that the phase distribution of amorphous and crystallized trehalose dihydrate in frozen solutions depends on the ratio of trehalose to mAb. The results identify an optimal range of trehalose-mAb (w/w) ratio, 0.2-2.4, capable of physically stabilizing mAb formulations during long-term frozen storage-even for fast cooled (>100°C/min) formulations.

Concluding summary: Proceedings of the AAPS Biotec Open Forum on “Aggregation of Protein Therapeutics”

With the advent of recombinant DNA technology and improvements in large-scale bioprocessing, it has been possible to produce large quantities of proteins to be used as therapeutic agents. One of the key challenges is that proteins can degrade both by chemical and physical degradation pathways. One of the most important pathways for protein physical degradation is the generation of protein aggregates. The ability of proteins to aggregate has been recognized from the early beginnings in protein biochemistry. A recent article in the AAPS Newsmagazine (June 2006) discusses the general impact of protein aggregation on biopharmaceutical development as well as the creation of the AAPS Protein Aggregation and Immunogenicity Focus group, which is affi liated with the Biotec Section. In June 2005, preceding the AAPS National Biotechnology Conference (NBC), the AAPS Biotec Section organized an Open Forum Meeting on “ Aggregation of Protein Therapeutics. ” This issue of The AAPS Journal presents selected papers that resulted from the presentations at this meeting. This Open Forum also included time for panel discussions with the presenters and the questions and responses were transcribed and are included in these proceedings.

Biophysical Analysis in Support of Development of Protein Pharmaceuticals

Development of proteins as pharmaceuticals is more challenging compared to traditional small molecule drugs because of the increased complexity of the chemical and physical stability of protein pharmaceuticals. This necessitates the use of both analytical and biophysical methods to investigate the stability of proteins. The choice of which biophysical methods to use is dictated by the types of studies and specific requirements that support protein drug development. This chapter discusses the various types of biophysical studies that are often performed during protein formulation development including (1) early screening assessments, (2) intense characterization, and (3) confirmatory studies. Case studies with representative examples from Genentech are presented for each of the above topics.