Overview of Drug Product Development for Protein Therapeutics: Present Strategies and Future Perspectives

Transcription

1 Overview of Drug Product Development for Protein Therapeutics: Present Strategies and Future Perspectives Formulation development of protein therapeutics poses a unique set of technical challenges. Proteins constitute an extraordinarily diverse group of molecules, including antibodies, antibody fragments, enzymes, interferons, insulins, cytokines, fusion proteins, PEGylated proteins, and other modified proteins. These therapeutic proteins are derived from diverse process streams, such as E. coli, yeast, mammalian, insect, and plant expression systems, and exhibit a broad range of process-related impurity profile characteristics. They exhibit diverse and often poorly-understood degradation pathways and product-related variants. The size of proteins may range from a few kilodaltons to >500 kilodaltons, and their structures range from relatively unordered to complex, highly ordered, multimeric states. Formulators may be faced with glycosylation states ranging from unglycosylated to complex, highly glycosylated profiles and the frequent presence of structurally labile, solvent exposed active sites or effector sites. Finally, therapeutic proteins exhibit a diverse range of functions, with activity assays ranging from relatively simple enzymatic activity assays to highly complex cell-based or whole animal-based bioassays. Pooja Arora and Rajesh Gandhi Bristol Myers Squibb Tim Kelly KBI Biopharma Inc. Protein Stability The stability profile of a therapeutic protein is a critical determinant of the product shelf-life, and is dictated by the type and kinetics of degradation pathways that it may undergo throughout its lifecycle including expression, purification, storage and administration. These degradation pathways can be primarily divided into two main categories: physical and chemical. The chemical degradation pathways involve formation or breaking of covalent bonds, whereas physical degradation pathways do not. One of the most common physical instability challenges that protein formulators experience is aggregation, which typically involves non-covalent interaction between two or more protein molecules resulting in formation of higher molecular weight (HMW) species, and possibly sub-visible particles that can negatively impact the drug s safety profile. For reversible aggregates, the dissociation kinetics of these species following dilution in an IV administration fluid, subcutaneous injection, etc. should be considered while evaluating the safety implications. Since the loss of native, three-dimensional structure due to protein unfolding (either partial or full) often acts as a precursor to formation of HMW aggregates, the formulation development efforts should be driven towards maximizing the protein s thermodynamic stability. In addition to aggregation, proteins are also susceptible to adsorption 1 December 2011

2 «PROTEIN SCIENCE on surfaces and interfaces, resulting in loss of soluble protein that can impact the therapeutic efficacy. Other physical stability challenges include precipitation or formation of visible particles due to solubility limitations of monomer or HMW aggregate and phase separation (often observed for high concentration/viscosity formulations). Depending on the amino acid sequence and three dimensional structure of a protein, it can undergo chemical degradation reactions such as deamidation (asparagine, less often glutamine), isomerization (aspartic acid), oxidation (methionine, tryptphan, tyrosine), etc. The peptide bonds between amino acid residues can undergo hydrolysis resulting in formation of fragments that are not biologically active. Hydrolysis of peptide bonds can occur due to the presence of residual proteases, flexibility in the three-dimensional structure, extreme ph conditions, etc. Some of the other commonly observed protein chemical degradation pathways include loss of sugar moieties for glycosylated proteins, loss of disulfide bonds under reducing conditions and formation of non-native disulfide bonds. Although not common, amino acids in a protein can lose their stereochemistry via racemization that involves formation of a thermodynamically unstable carbanion. The acceptable upper limit specification for both physical and chemical degradant species should be determined based on safety and efficacy data from the non-clinical and clinical studies. In discussing protein stability, we often focus on a few wellstudied degradation mechanisms such as fragmentation and aggregation. In fact, there are many facets of protein stability for the formulator to consider, and it is useful to clearly define these aspects. Physical and chemical stability aspects for a protein are defined as discussed above. Thermal stability may be considered the maintenance of an optimal transition point (Tm) or unfolding temperature. Conformational stability conveys the resistance to unfolding and perturbations in protein structure. Finally, biological stability may be considered as an indication for the maintenance of native activity. Conformational stability is a key consideration since maintenance of native, three dimensional globular structure will generally improve the chemical, physical, and biological stability profile, and therefore the efficacy of a protein. Unfolding of proteins exposes hydrophobic amino acid residues which are otherwise protected inside the protein s structural core. These residues may then be subjected to chemical degradation processes. In addition, the hydrophobic residues may interact with each other to form aggregates and ultimately particulates (physical instability). Biophysical, Analytical and Binding Methodologies for Protein Characterization The biophysical methodologies most often used to support initial formulation screening studies include differential scanning calorimetry (DSC) and dynamic light scattering (DLS). These techniques are often used in combination since they provide orthogonal information regarding thermal / conformation stability (DSC) and physical stability (DLS). The methods also have the advantage of being relatively high throughput, quantitative, and platform-able generic methods can usually be employed without molecule-specific development or optimization. DSC measures the differential absorption of heat by protein and buffer samples. The apparent mid-point of endothermic transitions for protein unfolding corresponds to the Tm, defined as the temperature at which protein is equally distributed in its folded and unfolded forms. Tm values often vary as a function of ph, buffer and excipient type, so DSC may be used to differentiate among candidate formulations and select the conditions optimal for thermal stability. In DLS, the intensity fluctuations of the scattering light are analyzed. The fluctuation rate is related to the diffusion coefficient of the scattering species. If the diffusion coefficient is measured and the viscosity of the solvent is known, then the hydrodynamic radius for species such as monomer and aggregates can be calculated. DLS data can be used to assess physical stability of proteins via values such as the Z-average diameter, the intensity weighed mean hydrodynamic size of the population, and the polydispersity index, which accounts for the number and width of all the species in a population. Secondary and/or tertiary structure of proteins can be evaluated using biophysical techniques such as circular dichroism (CD), Fourier transform infrared spectroscopy (FTIR), and fluorescence spectroscopy. Each of these techniques may be informative for preformulation development depending on the unique structural characteristics of the protein in question, but these methods are not readily quantitative, making data interpretation challenging. For quantitative purity determination and real time or accelerated stability evaluation, analytical techniques such as high performance liquid chromatography (HPLC) and capillary electrophoresis (CE) are most useful. Both of these technologies rely primarily on UV or fluorescence-based detection, taking advantage of the inherent spectral properties of proteins. Commonly used HPLC methods include size exclusion (SE), where molecules are passed though a porous matrix and are separated on the basis of size, with smaller molecules eluting later than larger molecules. This method is therefore used to quantitate aggregates and fragments. SE-HPLC may be employed in combination with multiple angle light scattering (MALS) and refractive index (RI) detection to further characterize the molecular weight, hydrodynamic size, and conformation of detected species. Ion exchange (IEX) HPLC is commonly used to separate species on the basis of charge and quantitate charge variants such as deamidated species. Reverse phase (RP) and hydrophobic interaction (HIC) HPLC may also be used for quantitative purity assessment, where species are separated on the basis of differences in hydrophobicity. In peptide mapping, proteins are digested into defined peptides by sitespecific enzymes and the resulting peptides are separated by RP-HPLC. This is a highly sensitive analytical technique capable of confirming the nature and sites of chemical modifications such as oxidation and deamidation, particularly when coupled to mass spectrometry. However, peptide mapping is less readily quantitative than other HPLC methods, reducing its utility for stability studies. The primary CE methods used for protein stability studies are SDS- CGE, where proteins are separated by size in a manner analogous December

3 to traditional SDS-PAGE, and cief, where protein are separated by charge, similar to IEF gels. CE-based method are greatly preferred over their traditional slab gel electrophoresis counterparts because they are much more quantitative and capable of substantially higher throughput. The output of the CE analysis is an electropherogram based on inherent spectral properties of proteins which may be integrated similarly to a HPLC chromatogram, whereas densitometric analysis of stained stab gels poses many limitations and challenges. As mentioned previously, methods for evaluation of protein activity are nearly as diverse as proteins themselves. Enzymes often permit relatively straightforward substrate-based activity assays. For proteins where activity is based on its ability to bind a ligand, various methodologies are available to evaluate the affinity and/or kinetics of the binding interaction, such as ELISA, surface plasmon resonance, and bio-layer interferometry. Many protein therapeutics require complex cell-based bioassays to measure the activity of the protein to elicit a particular cellular response, such as proliferation, apoptosis, and cytokine release. Ligand-binding and cell-based bioassays are often not particularly useful to drive formulation development decisions since the inherent method variability generally precludes the ability to assess modest differences between lead candidate formulations. Preformulation and Formulation Development Acitivities There are many possible approaches to formulation development which may be employed depending on the molecule type, dosage form, and availability of platform processes. A typical workflow includes preformulation characterization, forced degradation, and formulation / final dosage form development. Preformulation development often begins with a research phase in which general information about the protein and dosage form are gathered to frame the approach for subsequent laboratory studies. The sequence, molecular weight, and especially isoelectric point provide important input to the study design. The goals for the dosage form, such as liquid versus lyophilized powder, and route of administration, such as intravenous versus subcutaneous, will also impact the selection of buffers and excipients for evaluation. Biophysical characterization techniques may be used to quickly screen a wide range of buffer, ph, ionic strength, and excipients conditions for their impact on the thermal, conformational, and physical stability of the protein in solution. The impact of these formulation factors on protein solubility is also typically evaluated during preformulation, along with the impact of surfactants to enhance physical stability as a consequence of shear stress. Ideally, these initial screening studies provide the basis to rule out suboptimal buffer/ph/excipient conditions and identify a more narrow range of conditions for further evaluation in a design of experiments (DoE) accelerated stability study. DoE provides an efficient means to identify the main effects of formulation factors on protein stability and the presence of interactions between factors, while assigning statistical significance to all the effects observed. As such, it is an extraordinarily valuable tool for preformulation development and protein design space characterization. Once lead candidate formulations have been identified based on preformulation development, forced degradation and formulation development studies are utilized to further characterize the candidates and arrive at the final selected composition. Forced degradation studies may include additional stress conditions such as photolysis, acid/base hydrolysis, agitation, freeze/thaw, forced oxidation and deamidation. The design of formulation development studies will vary depending on the dosage form and route of administration, and may include TFF process development, lyophilization process development, and materials compatibility studies (e.g., filters, pumps and tubing, container/closure systems, syringe/needle configurations). The preformulation development activities for a protein are primarily focused towards mitigation of one or more stability challenges discussed above. One of the primary goals is to develop a product with the shelf life that meets marketing and distribution requirements. Ready-to-Use (RTU) solution product is strongly desirable due to cost benefits, ease of manufacturability and administration, competitive landscape, and patient compliance. However, this target product profile may not be attainable for proteins with significant instability challenges, which cannot be mitigated to an acceptable level via formulation development studies. Freezing of the product may alleviate some of these stability concerns, but can in turn result in stringent cold chain requirements for storage, shipping and distribution. In these scenarios, development of a lyophilized or freeze-dried product can provide an alternative strategy to deliver a product with acceptable stability and shelf-life. The process of lyophilization or freeze-drying involves removal of bulk water (>95%) from the product, and therefore significantly reduces the rates of degradation that occur in the aqueous phase. Lyophilization comprises of three main steps: freezing, primary drying and secondary drying. During the freezing process, bulk water undergoes phase separation via formation of ice crystals. In order to result in shorter and consistent drying times, the rate and duration of freezing should be optimized to minimize supercooling. As the freezing process continues, the increase in concentrations of various ingredients (excipients and protein) due to removal of bulk water can accelerate kinetics of some of the degradation reactions. Following freezing, the temperature is increased and vacuum is applied to initiate the primary drying process during which ice is sublimed to remove most of the bulk water (80-95%). The product temperature is always maintained below the collapse temperature, which is closely related to the glass transition temperature (Tg ) for amorphous excipients, and the eutectic temperature (Teu) for crystalline excipients. For secondary drying, the product temperature is gradually increased to remove water molecules not present as ice that are closely associated with the protein, such that residual moisture in the final product is < 3%. Lyophilization cycles also often include an annealing step during freezing. Annealing facilitates complete crystallization of crystalline excipients and growth of ice crystals (to minimize supercooling), resulting in a homogenous product and a consistent, efficient process. After cycle completion, the vials are fully stoppered and unloaded. Crystalline bulking agents such as mannitol and glycine provide a mechanically strong, pharmaceutically elegant cake but cannot stabilize the protein during the critical freeze concentration phase since they are removed from the amorphous 3 December 2011

4 «PROTEIN SCIENCE environment of the protein upon crystallization. Amorphous bulking agents / lyoprotectants such as sucrose and trehalose remain in the amorphous phase and can protect the protein via hydrogen-bonding as the water is removed during freezing, but generally result in less uniform, weaker cake structure. Therefore, formulators often utilize a combination of crystalline and amorphous excipients to provide cake structure and enable shorter cycle duration while protecting the protein during the lyophilization process. For a lyophilized product, the scalability and robustness of the cycle is a critical component for the technical transfer for commercial manufacturing. Primary Packaging Components Selection of a suitable container closure for long term storage of the product is an integral part of the formulation development studies. Type I borosilicate glass is often used in combination with stoppers that are coated with a fluorotec polymer to minimize unfavorable interaction with the formulation components. For early clinical programs, a glass vial/rubber stopper presentation presents a flexible option to enable wide dose ranges following dilution in an IV fluid before administration. For Phase 3 and beyond, selection of the final container closure is significantly dictated by final dose requirements, patient compliance, ease of use and marketing preferences. A RTU product in a pre-filled syringe (PFS) presents an excellent option based on these criteria, but can often result in new stability challenges due to the presence of silicone oil as a lubricant, residual tungsten from the pins used for staked needles and leachables/extractables profile for glass barrel and plunger stopper. To support a change in container closure from vial to PFS, stability studies should be performed to evaluate these parameters, and make necessary formulation changes, as needed. For lyophilized products, the two components (freezedried cake and reconstitution diluent) can be commercialized as either a multi-component presentation or a single unit as dual chamber syringe that allows introduction of diluent into the lyophile chamber just before administration. Selection of needle gauge for a parenteral injection is dependent on acceptable injection time, product viscosity and shear sensitivity of the protein. The Container Closure Integrity (CCI) for the final presentation should be confirmed using representatives units. Finally, additional safety/convenience features may be included in the marketed product using a device such as an autoinjector, flange extender and safety syringe. Product Manufacturing Considerations In order to enable a simple DP manufacturing process, a formulated Bulk Drug Substance (BDS) requiring only filling and finishing operations is highly desirable, provided sufficient stability of the protein in the selected composition is established. Some of the factors to be considered during selection of BDS composition include stability profile, shipping convenience, cold chain requirements and thawing procedure. During the DP manufacturing process, appropriate inprocess analytical controls are included to ensure acceptable critical product and process attributes. The acceptable ranges for the various formulation and process components during the DP manufacturing process are determined based upon design space and stability results from the ruggedness and robustness studies. Additionally, prior to technical transfer, process development studies should be conducted to optimize various unit operations such as thawing of BDS, mixing/dissolving of solid excipients during buffer preparation/ compounding, filling, filtration, etc. Other critical features to be evaluated during development of DP manufacturing process include determination of stability under room temperature/light conditions, yield and scalability. These unit operations can be especially critical for a lyophilized product whose stability profile may be significantly process-driven. Both lyophilized and RTU products are visually inspected prior to packaging to identify defects such as container closure issues, cracks, deformation of components, presence of visible particles, etc. Dosage Form Development Proteins are mostly administered via the parenteral, or injectible route. IV administration following dilution into an appropriate diluent (e.g., saline, 5% Dextrose) is often implemented during the early clinical studies to allow flexibility while therapeutic doses are being determined. During the later clinical studies, a product for subcutaneous (SC) administration may be developed to enable increased patient compliance and convenience, accurate dosing, and self-administration using primary packaging components such as pre-filled syringes. Due to the limited volume requirements (typically <1.5mL) for a convenient SC injection, these products often necessitate relatively high protein concentration. The increased concentration can introduce development challenges including solubility limitations, physical instability due to aggregation, increased viscosity, long injection time, selection of suitable needle gauge and hypertonicity of the final formulation. The use time stability studies are required to evaluate the stability of the product under normal administration conditions for both IV and SC products. Future Perspectives The development activities for a growing number of therapeutic protein candidates in various stages of clinical development can be efficiently supported via incorporation of biophysical and analytical tools with automation platforms to increase the throughput. These units can enable evaluation of a wider experimental design space with minimal material, time and resources. The significant size for resulting datasets will require additional management tools to compile, analyze and interpret the results. Collaboration with external vendors to implement novel, alternative stabilization technologies may enable mitigation of challenges associated with product stability and drug delivery. Computational modeling tools can be incorporated at various stages of development to improve design of therapeutic December

5 candidates and targets during discovery phase, optimize formulation components, evaluate formulation/process ruggedness, and optimize process parameters. The availability of stability results from an increasing number of proteins facilitates creation of knowledge databases and identification of trends to inform the development approach for new products. These databases can be further extended by leveraging the experience of CROs when the projects are outsourced. Implementation of lessons learned from the previous molecules can enable more efficient design of experiments. If data permits, this approach may be further extended to design platform formulations that may be implemented for similar protein prototypes. The formulation development studies rely on relative comparison of stability between various prototypes, and therefore timely availability of stability indicating methods is critical to enable such selections. Considering the intricate balance of various stabilizing forces in protein s three dimensional structure, using multiple orthogonal methods, and not a single method, is strongly recommended while comparing various formulation prototypes to ensure an overall stability profile is evaluated. Due to potential safety and immunogenicity concerns, quantitation and characterization of subvisible particles in drug products is required. USP<788> method relies on the light obscuration technique, and has been conventionally used to determine the number of particles with sizes 10 and 25µm. Recent changes in the regulatory expectations necessitate monitoring of particles smaller than 10µm, and the analytical methodologies are constantly evolving to meet these requirements. Development of drug products to enable various clinical studies requires strong cross-functional area collaboration with aligned expectations and defined goals. The interactions between groups can be facilitated using project management tools to outline the timelines for various project milestones. The constantly evolving regulatory expectations impact the need for acquiring the required data within suitable timelines. Statistical tools such as DoE and QbD can be used to support robust selection of various product and process components. Overall, drug product and process development can be suitably designed based upon past experiences, combined with appropriate incorporation of innovative analytical methodologies and technologies (for e.g., disposable manufacturing components) based upon a supporting robust dataset. Author Biographies Pooja Arora is a Sr. Research Investigator in the Drug Product Science and Technology Department at Bristol- Myers Squibb. Pooja has more than ten years of experience in protein biophysical and analytical characterization. Her responsibilities at BMS include development of drug product presentation for protein therapeutics. These activities include identification of optimum formulation conditions that impart stability to achieve the desired shelf-life, support selection of primary packaging components, etc. Prior to BMS, Pooja was a KBI Biopharma, Inc. where she was the technical lead for projects that focused on preformulation development, stability studies, and biophysical and analytical characterization. Pooja earned her Ph.D. in Chemistry from Duke University. Dr. Rajesh Gandhi is Director in Drug Product Science and Technology at Bristol Myers Squibb Co. in New Brunswick, NJ. He received his Bachelors degree in Chemistry and Pharmacy from University of Bombay, India and a Masters degree in Pharmaceutics from Northeast Louisiana University. He graduated with a Doctorate degree in Pharmaceutics from University of Wisconsin-Madison, School of Pharmacy in His industrial experience spans in the areas of discovery support, preformulation, characterization of NCE s for small molecules and biologics, exploratory oral formulations of small molecules and exploratory and commercial parenteral formulations of biologics. Currently, he is leading a group of 30 scientists providing strategic and technical directions to support design, development, scale up and technology transfer of formulations and processes for clinical and commercial use for parenteral products. He has authored 14 publications and is the inventor of Stable Protein Formulations of Abatacept and D4T Polymorphic Form I Process patents. He has moderated several symposia at AAPS National Biotechnology and AAPS National meetings. He has served as a lecturer for a graduate course at the University of Sciences at Philadelphia. Tim Kelly is Vice President of Biopharmaceutical Development at KBI Biopharma, Inc. Tim has over 15 years of experience in protein and nucleic acid characterization and product development. His responsibilities at KBI Biopharma include formulation and analytical development, as well as cgmp laboratory operations. At KBI Biopharma, Tim has overseen formulation development programs for over 80 protein and peptide products, with a particular emphasis on monoclonal antibodies. Tim earned his Ph.D. in Biochemistry from Georgia State University. This article was an online exclusive in the December 2011 issue of American Pharmaceutical Review. Copyright rests with the publisher. For more information about American Pharmaceutical Review and to read similar articles, visit and subscribe for free. 5 December 2011