Role of Protein Characterization at Various Stages of Bioprocessing
Protein characterization data play different roles at various stages of drug discovery, development, and manufacturing. At
the discovery stage, these data form the basis for candidate screening and can provide valuable guidance on cell line and
strain selection and upstream operating conditions. Assays used at this stage should be focused on determining relative yields
from various cell lines or strains, examining post-translational modifications, identifying lead candidates with desirable
properties (e.g., high in vitro enzyme activity, solubility at physiological pH), and screening out molecules that raise red flags regarding potential immunogenicity
and half-life issues (e.g., readily oxidized or deamidated molecules).2 These analyses also can provide a preliminary assessment of short-term stability at the target pH, salt concentration, and
storage conditions.
As molecules progress through the development process, protein characterization techniques are used to guide process optimization—identifying
operating conditions that produce higher yields and increased purity, and limit aggregation. The data obtained also are used
to support formulation development, to develop product storage and handling instructions, and to define product specifications.3 Relevant analytical methods are used for in-process monitoring of intermediates, final product analysis, and reference standard
characterization in preparation for preclinical and clinical manufacturing. As biopharmaceutical products move into clinical
manufacturing, detailed analyses allow systematic collection of stability data, demonstrate comparability following scale
up or a manufacturing site change, and during process validation. Novel assays and increasingly sensitive analytical methods
can identify minor changes in protein structure, composition, or impurity profiles which could have significant effects on
in vivo immunogenicity, toxicity, and half life.4 The FDA recommends that manufacturers use orthogonal methods for analyzing biopharmaceutical product purity and identity,
so a combination of different techniques must be selected that provide a balance between regulatory compliance and commercialization.5
This balance can be difficult to define, however. The FDA has advocated the use of a risk-based approach for evaluating product
comparability subsequent to a manufacturing change (e.g., scale up, site transfer, process changes). This involves the manufacturer
engaging in a theoretical analysis of how the change could potentially affect both the impurity profile and the product itself,
and then designing a characterization program which enables detection of these "high probability" changes as well as any "high
impact" changes that would significantly affect product quality, safety, or efficacy should they occur.6 Companies can leverage their experience with biological products in general and with the specific molecule in particular
to conduct these comprehensive risk assessments. A change identified as high risk, i.e., likely to result in material changes
to the final product, would then lead to extensive testing to determine comparability between the product pre- and post-change.
Examples of high-risk changes would include moving from serum-containing to serum-free media upstream or moving from crystallization
to chromatography downstream. Low-risk changes such as using a new supplier of a common buffer would dictate a less comprehensive
evaluation.
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