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Determining product quality is a critical measure of a proposed biosimilar product. Appropriate analytical methods are needed to assess quality as well as for applying quality control (QC). One of the key challenges in developing or selecting an assay to determine biosimilar product quality is determining which critical quality attributes (CQAs) are the most important for a given stage of the drug development lifecycle, notes Stephen Shakespeare, product manager, Sartorius. 

	“For example, a binding assay may be more relevant during clone selection than a mechanism-of-action-bioassay, which would be more suited for the final stages of development,” Shakespeare says.  

	Titer is another critical process parameter, and to ensure final product quality, it is necessary to measure the product concentration throughout a bioreactor run, adds Laura Madia, sales and marketing manager at IDEX Health & Science.

	Another point of consideration is the kind of information that is required from the assay. “Is it selective and specific for the product? Does it address the CQAs that have been identified from analysis of the innovator?” says Shakespeare, who points out that the use of orthogonal methods provides a host of information on the product that is critical for ensuring the quality.

	“There are a number of challenges facing biopharma production, and their impact (overall production, development timelines, cost, etc.) can be more significant for companies developing biosimilars,” includes Madia. “One challenge in particular is the drive for improving process efficiencies. Biosimilars are considered to be cost-effective alternatives to expensive biologics. There is a significant pressure placed on reducing production costs through improved manufacturing processes to improve yields without introducing product variation that might require additional processing or testing.”

	The tradeoffs between time and cost are particularly important when developing biosimilars given the limited development time, Madia continues, saying that “biosimilars do not benefit from the same patent protection as novel biologics so there is typically more pressure on prices immediately after approval.”

	Another question to consider is what are the most effective assays currently used to test for biosimilar product quality? When assessing a biosimilar, Shakespeare explains, it is critical to take what he calls an orthogonal approach to looking at antibody fragment (Fab) binding and fragment crystallization (Fc) binding using a variety of techniques. 

	“In Fc functional assays, for example, antibody-dependent cellular cytotoxicity (ADCC) can be addressed in numerous ways using peripheral blood mononuclear cells (PBMCs) or natural killer (NK) cells isolated from blood. Meanwhile, reporter gene assays can be validated for submission to the regulators. The sensitivity of each of these methods provides key information on the quality of the product at different stages of development,” Shakespeare notes.

	He adds that there is an array of methods, from enzyme-linked immunosorbent assay (ELISA) and flow cytometry to surface plasmon resonance (SPR), that can be used to determine Fab binding.

	Another example of an orthogonal approach is the use of size exclusion and ion exchange analytical methods, which can show differences in aggregation/fragmentation and charge profile, respectively. These attributes can be important when assessing the stability and safety of a biosimilar, Shakespeare says.

	Understanding the innovator biologic is also critical to understanding the CQAs of the biosimilar product, Shakespeare also notes. Understanding the innovator CQAs allows the biosimilar developer to determine the quality target product profile (QTPP) design space. Design-of-experiment software (e.g., Umetrics software, Sartorius) enables biomanufacturers to monitor the production process and track whether certain biosimilar CQAs are drifting, allowing them to respond accordingly.

	“Being able to monitor different aspects of a biosimilar production in real-time, such as binding and function, enables you to control CQAs and minimize any potential downstream issues with delivering consistent quality product,” Shakespeare states. 

	There are situations where a process depends on a quick measurement to move forward, Madia remarks. “Measuring titer is not considered simple nor quick. Titer is often run on a high-performance liquid chromatography (HPLC) in the QC lab due to the high level of skill required for method development and day-to-day operation. It is not uncommon for upstream process development or manufacturing teams to wait 24 hours or longer to see the results of a HPLC titer analysis because they had to send it to a separate lab,” Madia explains. As a result, titer is often underutilized or batched for retrospective insights, rather than using titer for real-time decision making, Madia points out.

	Since the manufacture of the first biosimilars, there have been many innovations in analytical techniques and practices. In recent years, for example, high throughput (HTP) flow cytometers (e.g., iQue screener, Sartorius) have enhanced the speed with which data can be generated, Shakespeare highlights. HTP flow cytometry, coupled with live cell imaging (e.g., Incucyte, Sartorius), enhances the breadth of information that is collected and allows for more informed decisions to be made faster, he explains. 

	Other recent analytical innovations include capillary electrophoresis techniques, which have replaced slab gel techniques, resulting in assays that have high resolution, are highly reproducible, and are applicable to a wide range of protein molecules-not just immunoglobulins and biosimilars. “The use of capillary isoelectric focusing (cIEF) and capillary electrophoresis sodium dodecyl sulfate (CE–SDS) techniques are now almost universally applied for CQA determination, stability studies, and lot release testing,” Shakespeare says.

	Manufacturers, meanwhile, are continuing to implement liquid chromatography–mass spectrometry (LC–MS) techniques for investigating glycan structure, verifying sequences, analyzing intact mass, and analyzing post-translational modifications of the innovator and of biosimilar candidates, Shakespeare adds. “By introducing simple and affordable kits, manufacturers are getting greater instrument resolution, improvements in software processing and databases, and fast sample preparations,” he explains.

	“In a similar vein, the ongoing reduction in particle size to < 3 µm in ultra-high-performance liquid chromatography (UHPLC) columns continues to enable reduced run times and higher throughput while maintaining peak resolution,” Shakespeare also adds. 

	Having an innovative new product that measures titer on demand is also a beneficial advancement, Madia adds. Drawing on experiences with the Tridex Analyzer (IDEX), Madia highlights how innovative technology can enable development and facilitate experimentation by providing trend data quickly and easily. With the trend data, it may be possible to determine product loss during the evaluation of filtration options or to monitor breakthrough with a new piece of equipment. “Ultimately, a full analysis will be required as part of the product acceptance, but by eliminating options early, it is only necessary to run a full analytical evaluation on the strongest candidates,” Madia says.

	The most common application for antibody titer is in determining concentration in the bioreactor tank at the end of a cell culture run, Madia emphasizes. Once a process moves to production, current analytical tools are too large or complex to be co-located near the tank or in the manufacturing suite, she explains. 

	“During the crucial transition from upstream to downstream purification, manufacturing teams require the final titer data in order to load the purification columns. Once the tank is emptied and the filtration step is complete, production stops. Product is put in a holding tank while the team waits for results. Eliminating downtime while waiting for titer could shave hours or days and can also reduce the risk of moving product between areas. Using real-time titer to streamline this transition can make a real impact at manufacturing scale,” Madia says.

	In May 2019, FDA proposed a draft guidance for the development of biosimilars (1) in which it outlines comparative analytical studies for determining quality criteria. The guidance itself recommends using orthogonal techniques and not relying on one analytical method when analyzing biosimilars, notes Shakespeare. 

	“For example, consider analyzing the charge profile of a sample using ion exchange on a UHPLC as well as by using cIEF on a capillary electrophoresis instrument. Both techniques will show the difference in charge profiles between the innovator and biosimilar, but when a manufacturer is gathering evidence of the biosimilarity of their molecule compared to the innovator, there is strength in using multiple techniques that incorporate different instruments,” Shakespeare says.

	Meanwhile, BioAgilytix, a bioanalytical lab service provider, encourages biosimilar developers to engage with their chemistry, manufacturing, and controls (CMC) team because it is important to discuss the analytical characterization of both the biosimilar and innovator biologic and note any observed differences. The company has pointed out that differences observed in an anti-drug antibody (ADA) assay, for example, “may reflect previously undiscovered analytical differences between the biosimilar and the originator” (2). It is advisable that a discussion be held with regulatory authorities if there are unexplained differences between the biosimilar and the innovator biologic, and that these discussions occur prior to moving on to the next stage of biosimilar drug development. 

	BioAgilytix supports a single assay approach, such as an ADA assay or antigenic assay, to support biosimilar development. When developing an ADA assay, the company advises building it in a systematic, stepwise manner. This will support the build-up of knowledge needed to instill confidence in the assay’s ability to detect antibodies against the biosimilar and the innovator biologic. To that end, the focus should be on reducing the risk for potential variability and developing a meaningful interpretation of data derived from the assay (2).

	If applying a single assay approach, BioAgilytix recommends using biosimilar-based reagents for both capture and detection in the assay. This should include an evaluation and comparison of specific parameters (e.g., antigenic equivalence) involving both an unlabelled biosimilar and an unlabelled originator biologic. The company has stated that the “[u]se of biosimilar-based capture/detection reagents ensures that antibodies against the biosimilar are reliably detected and limits residual uncertainty that the biosimilar is not equally or less immunogenic than the originator” (2).

Draft Guidance for Industry: Development of Therapeutic Protein Biosimilars: Comparative Analytical Assessment and Other Quality-Related Considerations

 (CDER, CBER, May 2019).
	2. T. Lester, “Questions on Immunogenicity Testing for Biosimilars Answered: Part 1,” 


	Vol. 32, No. 7
	July 2020
	Pages: 27–28, 32

	When referring to this article, please cite it as F. Mirasol, “Analytical Assays Determine Biosimilar Product Quality,” 

Analytics

Appropriate analytical assays are needed to determine and ensure that biosimilar critical quality parameters are on track.

Pharmaceutical Technology Europe-07-01-2020

Pharmaceutical Technology Europe, July 2020 Issue (PDF)

Analytical Assays Determine Biosimilar Product Quality