If used correctly, these new analytical methods can reduce analysis and product development time.
ABSTRACT
Monoclonal antibodies represent a significant portion of sales in the biopharmaceuticals market. Ever-growing biotechnology pipelines have greatly increased demand for higher throughput, multi-product protein characterization methods. By taking a holistic approach to improving the analytical process, one can identify strategies to increase throughput and reduce development times and the costs of analytical methods. This article discusses recent advances in high-throughput and multi-product liquid chromatography and electrophoretic methods for protein characterization. In addition to advances in analytical methodology, technological advancements in sample preparation throughput and data handling automation add value and time savings to the overall characterization process, which will enable increased productivity and faster product development.
Monoclonal antibodies (MAbs) are an important class of therapeutic proteins in biotechnology and have been developed to treat a variety of indications to fill significant unmet medical needs.1 MAbs generally are target-specific and well tolerated with a relatively long half-life, contributing to their success in drug development. Of the classes of immunoglobulins, IgG1 is the most commonly used for pharmaceutical and biomedical purposes.2
In recent years, increased understanding of disease and advances in drug discovery have resulted in continuously growing pipelines for potential therapeutic antibody products. To keep up with the increasing demand for protein characterization, high-throughput and multi-product characterization methods are being developed. If used correctly, these methods can reduce analysis and development time and thus accelerate the progress of therapeutic proteins toward the clinic.
REGULATORY EXPECTATIONS FOR THE ANALYSIS OF MABS
Several guidance documents have been issued by regulatory agencies recommending approaches for protein characterization.3,4 Although guidance documents are valuable to the industry, other publications have emerged that more clearly identify methods that are useful for characterizing MAbs.5 These orthogonal assays include potency, identity, and impurity assays that evaluate critical quality attributes (CQAs) such as size and charge heterogeneity.5 These CQAs are part of the overall target product profile, which is based on the desired clinical performance. Appropriate control of CQAs is a common review concern for both investigational new drug (IND) and license applications.6,7 Product quality characteristics encompass a wide variety of product variants that include product-related species and product-related impurities.8 For example, aggregation is a carefully monitored product variant from the earliest stages of clinical development because of the possibility of eliciting an immunogenic response in the patient.
Many of the recommended protein characterization assays are based on liquid chromatography methods such as ion exchange chromatography (IEC) for charge heterogeneity analysis, size exclusion chromatography (SEC) for size heterogeneity, and reverse-phase high performance liquid chromatography (RP-HPLC) for peptide mapping.9 This article primarily will focus on multi-product and high-throughput charge-based methods for analyzing charge heterogeneity and size exclusion methods for quantifying size heterogeneity, in particular aggregates or high molecular weight species (HMWS). These methods are among the most frequently used tests for lot release of drug substance and drug product,5 as well as during formulation and process development. Several multi-product analytical methods for MAb size and charge assessment are shown in Table 1.
Before an analytical method can be incorporated into a characterization platform or a quality control system, it must first demonstrate that the method is suitable for its intended purpose. Guidelines for validating analytical methods have been published in the US Pharmacopeia,10 by the US Food and Drug Administration,11,12 and in published reviews.13 The guidelines published by the International Conference on Harmonization (ICH) have established a uniform understanding of the performance characteristics that are evaluated in the course of validation.14 Although high-throughput and multi-product methods can save time and add value to a business process, these methods also must be evaluated considering regulatory requirements and validation procedures. In other words, the validatability of these methods must be assessed before implementation.
HOLISTIC VIEW OF SAMPLE ANALYSIS FOR PROTEIN CHARACTERIZATION
A generalized sample analysis workflow is shown in Figure 1. Typically, protein samples are submitted, prepared, and analyzed, and then results are reviewed and reported. When developing high throughput or multi-product analytical methods, it is important to keep a holistic viewpoint of the sample analysis workflow to optimize the total process. For example, if a new method is developed with reduced run time but with increased sample preparation steps, then the value of reduced run time could easily be negated.
When exploring high-throughput and multi-product methods, investigation into cost savings is aided by a holistic view of the organization. Methods that use the existing analytical equipment in an organization may be more advantageous to develop than methods that require extensive capital investment on new instruments and consumables. In addition to capital costs, analyst experience should be leveraged when implementing new methods. Many biotechnology and pharmaceutical companies are well equipped with many types of liquid chromatography instruments and analysts that are trained to use them. Thus, the development and implementation of high-throughput and multi-product liquid chromatography methods that leverage existing facilities may result in greater time and cost savings compared to novel methods that do not use existing resources. Over-engineering a solution should be avoided; an existing technology should not be overhauled when a minor update would suffice to meet business needs.