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The development of mAb formulations poses challenges at the manufacturing, stability, analytical, and administration levels.
Monoclonal antibodies (mAbs) have become popular therapeutic agents in the past couple of decades in the treatment of life-threatening conditions such as cancer, inflammatory, cardiovascular, respiratory, and infectious diseases. Inherent specificity coupled with their potential therapeutic activity have contributed to the rise of mAb therapies.
In general, subcutaneous injections of mAbs are preferred over intravenous administrations from patient compliance and ease-of-use perspectives. For mAb-based therapies, typically, high doses in the range of hundreds of milligrams to a gram are needed per dose to achieve the desired therapeutic concentration. To be able to administer such a high dose range in a volume less than 1.5 mL by the subcutaneous route, as required by FDA, these mAb formulations may need to be prepared at a concentration greater than 100 mg/mL (1). However, such highly concentrated mAb preparations can present challenges in the manufacturing process as well as stability and analytical testing, characterized by solubility, high viscosity, and aggregation issues. Table I lists some of the commercially available highly concentrated mAb products.
Table I. Highly concentrated commercial monoclonal antibody (mAb) products.
To formulate high concentration mAb solutions, the target mAb must be dissolved in the solvent to achieve the desired protein concentration. Typically, the solution should be visually clear and not sediment at 30,000 g centrifugation for 30 minutes (2). The solubility of mAbs, similarly to any protein, can be enhanced by the addition of non-reducing sugars, such as sucrose, and by kosmotropic salts, such as sodium chloride (3). Such agents are reported to enhance the solubility of proteins through changing the overall conformation of the target protein, as well as through the phenomenon of preferential hydration of the protein. The use of excessive amounts of such excipients, however, may lead to hypertonic preparations or changes in ionic strength of the formulation and related protein aggregation issues.
As a mAb formulation is concentrated using the commonly applied ultrafiltration/diafiltration technology (tangential flow filtration, TFF) to achieve the desired protein concentration, the viscosity of the solution may increase exponentially at high protein concentrations (>100 mg/mL) (4). However, higher viscosities lead to higher manufacturing losses, and subsequently, higher costs as well. Even a higher percentage of volume overage could be needed in finished product vials to make up for the vial, needle, and syringe loss. Secondly, high viscosities of mAb solutions can create back pressure on the TFF equipment, resulting in longer processing times and potential equipment failures. Thirdly, proteins may unfold and undergo aggregation from shear stresses and cavitation shocks due to mixing, pumping, or centrifugation (5). Proteins may undergo adsorption at the air-water interfaces and other hydrophobic surfaces, resulting in aggregation (6). These aggregates can clog the membrane pores during ultrafiltration/diafiltration and filtration during the concentrating procedure, and lead to production issues. It has been reported in literature that the viscosity of high concentration mAb preparations can be reduced by the proper selection of pH, buffers, salts, amino acids, and sugars (7-9). Also, an increase in processing temperature can facilitate reduction in viscosity of high-concentration mAb preparations that have sufficient thermal stability.
Formation of reversible non-covalent aggregates, by intermolecular interactions, is one of the major challenges of high-concentration mAb preparations. Both covalent and non-covalent aggregates can adversely impact the stability of the formulation during storage, and consequently, patients’ safety.
With an increase in mAb concentration, a reduced effective volume is available for the mAb molecules. This phenomenon facilitates formation of reversible non-covalent protein aggregates with resulting increased viscosity. Increased viscosity can be expected at a pH far away from the isoelectric point of the target mAb, resulting from excess net positive or negative charges formed from the ionization of amino or carboxylic groups on the mAb and the subsequent increase in the hydration radius (10). However, increased viscosities have been noted at the isoelectric point of some proteins at high concentrations. It has been hypothesized that at the isoelectric point, proteins can experience attractive forces resulting from hydrophobic, van der Waals, or localized attractive forces, all contributing to protein aggregation for high mAb concentrations near the isoelectric point (4,10,11).
Apart from non-covalent reversible aggregation, high-concentration mAb preparations can undergo hydrolytically driven chemical degradations, such as deamidation, isomerization, and cleavage and fragmentation of peptide bonds to form irreversible covalent aggregates. mAbs can form irreversible covalent aggregates from disulfide exchange of free thiol groups from unpaired cysteine residues, disulfide exchange from beta elimination, oxidation, and glycation. These aggregates can have different size and charge profiles from the native mAbs, thereby, potentially affecting the safety and efficacy of the product (12).
In their native state, mAbs have their hydrophobic amino acids buried in their interior to prevent any interaction with the aqueous environment. mAbs, however, can form irreversible non-covalent aggregates from hydrophobic amino-acid residues, once unfolded by thermal, mechanical, or chemical stresses (13).
The stability of high-concentration mAb preparations can be improved by the addition of excipients, such as sugars that prevent the formation of irreversible aggregates. These excipients, being more polar, prefer to interact with water molecules and are, therefore, excluded from the hydration shell of protein molecules. This phenomenon increases the partial molar free energy of the denatured protein relative to the native conformation, thus pushing the protein molecules to maintain their native conformation and reducing the formation of aggregates (14). However, excessive use of kosmotropic agents such as sugars can increase tonicity of highly concentrated mAb formulations.
These stability problems of high-concentration mAb preparations may be avoided if the target mAb is formulated at less than 100 mg/mL, lyophilized, and then finally reconstituted with a small volume of diluent prior to subcutaneous administration. In general, proteins are expected to be more stable towards physical and chemical degradation reactions in the dry lyophilized state than in the solution form. In the lyophilized form, the possibility of hydrolytic reactions is minimized due to the removal of water by drying. Moreover, the mobility of proteins is restricted in the amorphous lyophilized matrix, reducing the potential for aggregation.
High-concentration mAb products are heterogeneous in nature from variations in mAb expression, mAb recovery/purification, formulation preparation, and storage of the product. Such products must be characterized extensively, like other conventional protein preparations, using orthogonal analytical methods.
High-concentration mAbs can be inherently complex due to the presence of higher-order aggregates formed either though covalent linkages or non-covalent molecular associations. Capillary electrophoresis (CE) and size exclusion chromatography (SEC) can be applied to analyze the size variants caused by covalent modifications. CE-sodium dodecyl sulfate (SDS) non-reduced is the method of choice for evaluating mAb fragments, disulfide cross linkages, and the covalent or non-covalent nature of aggregates. CE-SDS reduced can be employed to assess thioether linkages between light and heavy chains resulting from the beta elimination process (12,15). Irreversible noncovalent species formed through interactions of hydrophobic amino acid residues normally buried in the interior of proteins can be resolved by SEC, analytical ultracentrifuge (AUC), and field flow fractionation (FFF). SEC still continues to be the primary analytical method for the analyses of non-covalent reversible aggregates formed through molecular association of mAb molecules. However, highly concentrated mAb samples often require dilution prior to analysis by capillary electrophoresis, mass spectroscopic methods, and SEC. Such dilution schemes may impact the conformation of the native mAbs, as well as the composition and conformation of the aggregates. In addition, SEC runs the risk of undesirable interactions of mAbs with the columns and of the associated changes in their hydrodynamic volume, thus resulting in poor resolution and irreproducible peaks (16). Salts may be added to the mobile phase to minimize undesirable ionic interactions with the column, provided the resulting elevated ionic strength does not affect the stability of the mAb adversely. Orthogonal analytical methods that are being used for analyzing reversible size variants of high-concentration mAbs products are static and dynamic light scattering and AUC. However, analytical methods based on AUC can be complex, highly dependent on instrument quality, and require fitting of data to complex models (17). Light scattering techniques (dynamic and static) do not provide the number of particles for a given size and are not suitable for polydisperse systems.
Charge variants resulting from deamidation of asparagine residues, glycation, N-terminal pyroglutamate formation, incomplete C-terminal lysine processing, or succinimide formation can be analyzed by isoelectric focusing (IEF), imaged capillary isoelectric focusing (iCIEF), and ion exchange chromatography (IEC). However, IEF determinations can at times be complex to interpret due to high charge heterogeneity.
For subvisible particulate matter determination, orthogonal analytical methods, such as light obscuration and microflow imaging, are commonly applied. Both light obscuration and microflow imaging techniques provide the size and the number particles without any information on the chemical composition of the particle. Microflow imaging technology provides additional information on the morphology and optical properties of the particles in addition to particulate count determination (18).
As the concentration of a mAb increases, the viscosity of the high concentration mAb formulations increases exponentially, as shown in Equation 1 (19):
[Eq. 1] h = h0 (1 + k1Cp + k2Cp2 + k3Cp3 + …)
where η is the formulation viscosity; η0 is the solvent viscosity; k1 is the intrinsic viscosity contributed from the individual solute molecules; k2 and higher order coefficients represent effects from interactions of two, three, or more protein molecules; and cp is the concentration of the protein in mg/mL. The higher order terms predominate when soluble reversible protein aggregates are formed for high-concentration mAb preparations, resulting in higher viscosity.
High viscosity can adversely affect the ease of withdrawing the high-concentration mAb solution in the syringes. The backpressure resulting from dispensing a highly viscous solution may necessitate higher gauge needles and cause more pain to the patient.
For high-concentration mAb preparations, syringeability studies evaluate the ease of withdrawal of a product in the syringe, as well as flow of the product through the needle. One should be cognizant of the potential loss of the product from sticking to the contact surface due to its high viscosity, and calculate the overage accordingly from vial, needle, and syringe loss. The injectability of high-concentration mAb formulations is generally evaluated in terms of the force and the time required to complete an injection at a constant flow. Compatibility studies should be performed to identify any incompatibility or stability issues of the product with the contact surface. Shear stresses from withdrawal or injection of the formulations may impact the stability of the high concentration mAb products. The presence of surfactant in the products to prevent adsorption or aggregation of mAbs may cause foaming issues during administration of the product.
High-concentration mAb formulations have witnessed lots of interest in recent years due to their therapeutic potential and ease of administration to patients. The development of these products, however, still poses challenges at manufacturing, stability, analytical, and administration levels. Robust manufacturing processes, stable formulations, and orthogonal analytical methods are often required to resolve these challenges and produce a commercially viable high concentration mAb product.
Nilanjana Das, PhD, is a senior research investigator at Bristol-Myers Squibb, 1 Squibb Drive, New Brunswick, New Jersey 08903, US, email@example.com.
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Vol. 29, No. 11
When referring to this article, please cite as N. Das, "Commercializing High-Concentration mAbs," BioPharm International 29 (11) 2016.