During the (early) preclinical drug development process as well as manufacturing of biopharmaceutical (protein) products, analysis and characterization are crucial in gaining a better understanding of the physical and chemical properties of various materials. These properties can have an impact on the manufacturability as well as the performance, potential for metabolism, stability and appearance of a specific medicinal product. Hence, properly characterizing these products is essential for a drug candidate to move from drug development to regulatory approval and, finally, the clinic.
In recent years, complex biopharmaceutical drugs and biologics have evolved into mainstream therapeutics. The manufacturing of these compounds, including monoclonal antibodies, bispecifics, antibody-drug conjugates (ADCs), recombinant and other therapeutic proteins, require extensive analytical and comprehensive characterization using a variety of techniques, including non-compendial, and sometimes an intricate quality control methodology, to confirm manufacturing consistency and product quality.
Because biopharmaceuticals and biologics exhibit highly diverse structures and broad biological activities, a study of these agents is a relatively complex process requiring sophisticated analytical techniques. Furthermore, in addition to these complexities, regulatory expectations to better understand product impurities and degradants in biopharmaceutical products continue to increase.
As a result, many drug developers may find that their current global chemistry, manufacturing, and control (CMC) systems are quickly becoming obsolete. Consequently, new, highly sensitive and specific technologies are becoming the new normal.
Keywords: Biopharmaceutical analysis, Characterization, Protein therapeutics, Bioanalytical methods, Structure and function, Physical and Chemical properties
2.0 New Analytical Approaches
The field of monoclonal antibodies, launched with Köhler and Milstein’s initial study published in 1975 of a method to produce fully intact murine IgG antibodies, has created a new area of the development of novel medicinal products.  In the more than three decades since the initial development of monoclonal antibodies, chimerization, humanization and fully human antibody technology followed. 
Subsequent to the growth of antibody-based products, new technologies have emerged for creating modified forms of antibodies, including antibody fragments, antibody-drug conjugates or ADCs as well as bi- and multi-specific antibodies.
In the development of these next-generation medicinal compounds, a better understanding of currently approved ADCs and novel site-specific bio-conjugation technologies is required. For example, a better analytical understanding of the structure-activity relationship accelerates the discovery and development of the next-generation ADCs with defined and homogeneous compositions.
Analytical methods and characterization for novel biopharmaceuticals and biologics involve complex, multi-faceted procedures stretching from early (pre-) clinical drug discovery to clinical development, regulatory approval and, finally, market entry.
Most of this work takes place during the early development phase, and is vital to help understand the influence of process changes, measured against an established reference standard.
3.0 Protein Therapeutics
Biopharmaceutical therapeutics are inherently challenging to characterize because of their complexity and natural heterogeneity. Therefore, appropriate and complete analysis ensures meaningful and reliable characterization, and provides the data required to satisfy regulatory requirements concerning product identity, (im)purity, concentration, potency, stability, safety and overall quality.
Methods used to characterize primary and higher-order structures (including techniques to determine protein sequence, posttranslational modifications, folding and aggregation) and protein concentration (including amino acid analysis, intrinsic protein absorbance and colorimetric methods) are vital to avoid aberrant results for key attributes that could, potentially, raise quality issues.
In addition, characterization and analysis of biopharmaceutical proteins also involves product- and process-related determination of impurities, which may compromise the safety of the protein therapeutics. This includes various assays (including bioassays and noncell-based binding assays) for determining the functional activity of proteins, which may be indicative of potency.
Overall, a complete approach to characterization helps developers to be confident that their product meets regulatory requirements as well as product quality and safety standards.
4.0 Changing Technologies
While spectrophotometric analyses of proteins are commonly used, there may be a number of important reasons to change analytical methods and characterization techniques.[a]
The reasons may include:
- New techniques may allow for better characterization, making it possible to follow the stability of specific molecules and proteins, as well as contribute to deeper understanding of them. New techniques may include imaging, capillary-electrophoresis, ultra-high-resolution mass spectrometry, micro-flow imaging (MFI), etc.; (Figure 1.0)
- Improved technologies to replace legacy methods. Examples include using ultra-high-performance liquid chromatography (UHPLC), a relatively new technique giving new possibilities in liquid chromatography, instead of high-performance liquid chromatography (HPLC) and Capillary Western (WES), a quantitative western blot produced by a protein simple, which offers increased precision and specificity versus ELISA; (Table 1.0)
- Formulation and process changes may occur in the early stages of drug development. Even through Clinical Trial phase I and phase II, there may be formulation or process changes, which may require additional or new analytical methods;
- There may an interfering compound within the formulation. One example is the use of surfactants[b], such as Polysorbate 80[c](also known as PS80) which may interfere with the reverse-phase method. To be certain about stability, when observing new degradants, it may be required to use a new method that will resolve and quantify the new analytes;
- There are specific regulatory requirements that apply to approved products, including the expectation of periodic method assessment for improvement;
- Many techniques allow for strategic business decisions, resulting in high throughput with low costs. This largely depends on how many lots and stability studies are necessary. In turn, this may directly impact the costs associated with the regulatory approval process of products being developed.
5.0 Regulatory Implications
The regulatory process established by the U.S. Food and Drug Administration (FDA) requires that each New Drug Applications (NDA) and Abbreviated New Drug Application (ANDA) includes the analytical procedures necessary to ensure the identity, strength, quality, purity and potency of the drug substance and drug product.  Furthermore, each Therapeutic Biologic Application (BLA) needs to include a full description of the manufacturing process. This includes analytical procedures that demonstrate that the manufactured product meets prescribed standards of identity, quality, safety, purity and potency. 
The analytical procedures and methods validation for drugs and biologics, Guidance for Industry, states that, over the life cycle of a medicinal product, new information (e.g., a better understanding of product characteristics) may warrant the development and validation of a new or alternative analytical method. 
But analytical methods should not be considered to be “locked down” or validated once clinical trial phase I or phase II is reached. To fully understand the biopharmaceutical products involved, the FDA requires scientists to consider new or alternative analytical technologies, even after completion of the drug approval process.
The FDA also requires that drug developers and manufacturers periodically evaluate the appropriateness of an analytical method and consider new or alternative methods. To make this process simpler and more robust, and in anticipation of life cycle changes in the analytical process, an appropriate number of drug samples should be archived to allow for comparative studies. These samples must not only be put away for stability studies, but a reasonable number of samples should be archived at the proper temperature (typically at -80 degrees for a biopharmaceutical sample) to be used for crossover and comparability studies. This is critical to smoothing the pathway for change from one analytical method to another. 
6.0 Regulatory Reporting Requirements
Establishing a regulatory framework, the FDA sets “safety reporting requirements for human drugs and biological products” that include mandatory reporting of any change in analytical methodology, and describes—among other things—a developer’s responsibilities for reviewing information relevant to the safety of an investigational drug and their responsibilities for notifying FDA. These reporting guidelines cover minor, medium and major changes. (See: Table 2.0)
6.1 Minor Changes
Minor changes are those within the “validated change of the analytical method.” For example, when a validated chromatography method for a column temperature range of 10° to 40° change from a nominal of 30° to 35°, this would be considered to be a minor change. While this change can be submitted as part of the annual report, it is still required that the applicant reports the change to the agency. 
The Guideline for Industry detailing the requirements for the annual report stipulates that properly reporting post-approval manufacturing changes must be made in compliance with current Good Manufacturing Practice (cGMP). 
6.2 Moderate Changes
At the moderate level, the validated range is exceeded in certain parameters. Such a change may have an adverse effect on the identity, strength, quality, purity or potency of the drug product. Using chromatography as an example, this could be a change in mobile phase from acetonitrile to methanol, or a change in the actual gradient of a method. Such a change has more stringent requirements and required validation of this new method in additional comparability studies.
6.3 Major Changes
Major changes include modifications that establish a new analytical method, eliminate a current method (substituting one method for another rather than adding a new method), or delete or change the acceptance criteria for a stability protocol.
At the major level, there are substantial changes to the analytical method. For example, a major change includes switching from UV detection to mass spec (MS-) detection. Such a change must be validated with a formal, highly statistical comparability study designed to show any differences, or lack thereof.
In case of a major change, developers are also required to submit and receive FDA approval of a supplemental application to the original NDA or ANDA. In what is known as a Prior Approval Supplement (PAS), a major change needs to be reported and include a detailed description of the proposed change, which products were involved, a description of the new method, the validation protocols and data, a description of the changes to evaluate the effect of the change, a comparability report, a description of the statistical method of evaluation and a final study report. 
While a PAS is generally required for approved drugs, it also sets expectations for early-phase products. Although they are not covered under formal CFR regulations, the FDA does, in fact, expect at least a similar study to be performed when a drug is in clinical trial phase I, II or III.
7.0 Comparability Study
The comparability process is critical. The FDA requires that a manufacturer carefully assess manufacturing changes and evaluate the product resulting from these changes for comparability to the pre-existing product. In such a case, the goal is to show that a new analytical method is superior to the original method. 
Based on the guideline for industry, determinations of product comparability may be based on chemical, physical and biological assays and, in some cases, other nonclinical data. This requires referring to archived samples from historical batches, and whether those are included in the Investigational New Drug (IND) submission, clinical or registration batches. 
This is a critical part of the process, because developers need to show that a new method is more sensitive or selective, and is therefore detecting and quantifying impurities or degradants that were always present, but not seen by the current (existing) method and, as a result of a change of methodology, can now be better monitored.
8.0 Comparability Design
A well-planned comparability design will assess the effect of CMC changes, allowing the FDA to determine if a specified change can be reported in a category lower than the category for the same change. Appropriate samples should be included, allowing a comparison of the ability of the new and original method to detect relevant product variants and degradation. This approach provides sufficient information for the FDA to determine whether the potential for an adverse effect on the product can be adequately evaluated.  
To be adequate, the number of batches should be statistically relevant. The guidance to industry emphasizes the use of a trained statistician. The reason is that, while the FDA recognizes that a comparability design is less complicated than a clinical trial, it requires a statistician to design a robust program clearly showing differences between methods. 
There are a number of concerns associated with the development and the implementation of new methods designed to replace a current (existing) method. The biggest question is whether the results of the analytical methods will be different.
In general, the expectation is that by changing analytical methods, there is indeed a fairly high probability of getting different results. Hence, if there is a change to an improved method, the ideal scenario is a change in sensitivity or specificity, which would therefore show an additional or higher level of impurities or degradants.
Another concern is assay bias. For the statistical analysis of data, it is important that both the new and old data are within specification. Based on the guidelines to industry, the cause of bias must be examined to see if such bias has an effect on the data. Hence, analyzing archived samples to show that impurities and degradants were always present is crucial.
For products that have already been marketed, there is a concern that new impurities may result in the requirement for new, additional, clinical work. If there are archived samples to show that the materials were always there, the clinical data will still prove that the drug is safe and efficacious, and that the newly measured impurities and degradants could not be measured with the previous method.
If such is the case, statistical analysis is still necessary to justify the bias; however, there is no need for additional clinical work. The new process is just implemented to compare and show an improved method. 
Preclinical drug discovery and development process, as well as manufacturing of biopharmaceutical products, involves a complicated process including rigorous (experimental) scientific study. By following regulatory guidelines, successful advancement of novel drug candidates requires early planning, setting aside archived samples, having a very tight validation report and study and, finally, having a well-planned, statistically rigorous comparability study.
If these steps are present, there is a high probability of a smooth regulatory process. Drug developers may expect to receive approval to use the new analytical method for a marketed product. And if the product is in a pre-approval process, the expectation is that there is no need for additional questions from the agency.
[a]UV-VIS spectroscopy (ultraviolet and visible spectroscopy) is typically used for the determination of protein concentration by either a dye-binding assay or by determining the absorption of a solution of a protein at one or more wavelengths in the near UV region (260-280 nm). Circular dichroism is another spectroscopic method used in the early-phase characterization of biopharmaceuticals (proteins).
[b]Surfactants are compounds that lower the surface or interfacial tension between two liquids.
[d] UHPLC and UPLC (Waters Corp.) allow for better separation of peptide mapping
[e]CBE-30 is similar to Changes Being Effected (CBE) and involves a filing with the FDA to gain approval of a moderate change (this may include a change that has a moderate potential to have an adverse effect on the identity, strength, quality, purity or potency of the drug product, as these factors may relate to the safety or effectiveness of the drug product. Based on the CBE-30, the FDA has 30 days to respond prior to implementation of any change. If a filer does not receive a reply from the FDA within 30 days, it is assumed that a change is approved.
[f]Chemistry, Manufacturing and Controls (CMC) is renamed to Pharmaceutical Quality/CMC
October 21, 2016 | Corresponding Author: Glenn Petrie | doi: 10.14229/jadc.2016.10.21.001
Received: August 19, 2016 | Published online October 21, 2016 | This article has been submitted for peer reviewed by an independent editorial review board.