Biologic drug manufacturing has been focused on development of monoclonal antibody (mAb) drugs over the last couple of decades. As more mAb drugs are approved for therapeutic use, it poses a number of challenges in formulation development for drug manufacturers, including contract manufacturer organizations (CMO), since subcutaneous injections – which are gaining wider acceptance as an alternative to intravenous injections – typically require higher concentrations of product so as to achieve smaller injection volumes to alleviate patient pain. Though more stable than recombinant proteins, mAbs present unique challenges to formulation development when such preparations are highly concentrated (>100 mg/mL)1, including irreversible aggregation, irreversible precipitation, and/or high viscosity. The practical feasibility of whether proteins can be concentrated beyond protein and water weight ratios of 1:3 (about 350 mg/ml), due to volumetric contributions of the protein, is unclear. Even if stability could be managed at such high concentration, bulk viscosity would exceed the capabilities of current manufacturing practices or available parenteral delivery methods. This article details the challenges of high concentration protein/antibody formulations from a formulation development, drug substance formulation and analytical perspective, and reviews ways to mitigate high concentration development, formulation, and testing.
Developing high concentration mAb formulations present several developmental challenges that formulators must consider. With high concentration formulations, material consumption can be problematic. Some antibody concentrations routinely achieve greater than 150 mg/mL, and the amount of material required for a robust development strategy may be limited. Though small centrifugation concentrators are used during concentration and diafiltration activities to mitigate high material consumption during development, these concentrators can introduce product aggregation since sample concentrations need to be great than 2-fold to formulate product in these devices. Preferably, diafiltration or tangential flow filtration is used to reduce overconcentration affect, however these may require high mass needs. To address this, the use of high throughput equipment that combines analytical testing tools will ensure robust development, while keeping material requirements low.
Stability and aggregation are concerns for high concentration mAb formulation. Protein-protein interactions become more favorable as product concentrations increase and can, under certain conditions, increase product instability and aggregation. Two types of stabilities are related to the generation of antibody aggregates: conformational stability and colloidal stability. Conformational stability corresponds to the free energy difference between antibodies in native and denatured states (ΔG). The larger the value of ΔG becomes, the higher conformational stability an antibody takes. Thus, ΔG measurements are important for product stability. The measurements of TM, Ton and ΔG can be achieved using differential scanning fluorometry (DSF), differential scanning calorimetry (DSC), dynamic light scattering and should be in the analytical tool set of developers.
Colloidal stability corresponds to the dispersion state of antibody. Higher colloidal stability means that antibody molecules in a solution have the ability to exist as stable monomers in native states or partially denatured states, because of repulsive forces between molecules. Measurements of the second virial coefficient B22 and Kdiff (concentration dependence of the apparent diffusion constant) are performed by dynamic and static light scattering and can be used to identify stable formulations.
Ideally, analytical methods for characterizing high concentration samples should maximize the efficiency of screening, and also, minimize the total amount of sample required for analysis. For example, a method which can support the simultaneous processing and measurement of 50 to 100 samples, i.e. 96 plate format with minimal sample volume of less than 100 μL would be ideal. However, many degradant and formulation properties require product-specific methods developed to detect specific stability issues. Lengthy analytical times (i.e. HPLC) and larger volume sample requirement, (i.e. subvisible particle counting or viscosity measurements) may be unavoidable for some proteins.
Understanding the mechanisms of protein degradation is essential for selecting the analytical methods for characterizing formulation stability. Chemical degradation involves the breaking or formation of protein chemical bonds, i.e. deamidation of asparagine and glutamine residues, isomerization of asparagine residue, oxidation of methionine and tryptophan residues, and racemization of serine and cysteine residues, etc. Excipients may also degrade, causing the formation of degradants that react with proteins. For example, Polysorbate is known to form reactive oxygen species, which may damage API. Monitoring of chemical degradation is possible using well established bioanalytical methods, such as peptide mapping by LC-MS. Modifications are pseudo first-order reactions, thus, monitoring modifications at different time points at 40 °C, which is typically below the starting temperature of protein denaturation, can be useful for predicting the extent of modification and for the optimization of formulations.
Physical degradations can include conformation changes, denaturation, and undesirable adsorption to surfaces. When chemical degradation is mostly independent of protein concentration due to the pseudo-first order of the modification reactions, physical degradation occurs through intramolecular interactions, and, therefore, is concentration-dependent. Another aspect to be considered is the possible non-equivalency of physical behavior of protein molecules in high and low concentration formulations. Physical stability can be assessed by a thermal denaturation experiments using differential scanning fluorometry (DSF), calorimetry (DSC), static and dynamic light scattering.
Drug Substance Formulation/Scaling Challenges
Formulation development doesn’t end after a stable formulation is determined. Facility fit considerations should be evaluated. As a CMO, product concentrations from clients may vary significantly. When a CMO facility moves from a low concentration to high concentration product, equipment limitations should be assessed since containment vessels and mixers for low concentration formulations may not be suitable for high concentrations. For example, mixing plays a major role in the success of formulating high concentration products. A mixing vessel sized for a low concentration solution may not be appropriate for high concentration formulations. This may require additional capital expenditure and at-scale, flexible equipment strategy for CMOs to adjust to varying antibody concentrations.
Equipment sheer stress should be assessed for more temperamental products. Traditionally, peristaltic pumps have been used in CMO facilities because of their ease of use, disposable paths and low cost. However, these pumps can be problematic for less stable products, and a less sheer pump, such as diaphragm pump, may be required. Lastly, high concentration formulations can be a challenge particularly in terms of membrane fouling during ultrafiltration and diafiltration operations. Maximizing flux rates in development may decrease operation times, but will also increase high protein wall concentrations that cause membrane fouling. Lower flux rates may be needed to decrease fouling potential.
In summary, high concentration mAb formulations are a mainstay in formulation development. Critical to any high concentration formulation development is an understanding of the development, analytical and formulation scaling issues. However, with the appropriate analytical tools, high through put formulation methodologies and careful considerations of scaling, contract manufacturers can navigate through the formulation development pitfalls for successful development of high concentration formulations.
Brad Johnson is manager, purification development at Patheon and Alex Rostovtsev is principal scientist, analytical development at Patheon
- Wang, W., Singh, S., Zeng, D., King, K., Nema, S. Antibody Structure, Instability, and Formulation. Journal of Pharm Sci, VOL 96, NO. 1, 1-26 (2007)
Filed Under: Drug Discovery