OR WAIT null SECS
More than two-thirds of recombinant biopharmaceutical products on the market are glycoproteins, and every stage of their manufacture is carefully monitored and tested to ensure consistency in quality, safety, and effectiveness (1). Of the various aspects of biopharmaceutical production (such as yield, protein folding, and post-translational modifications), the host cell’s biosynthesis of attached oligosaccharides (glycans) is often the most difficult to control. Selected expression systems and even slight changes in process conditions can alter the synthesis of glycans and as a consequence, the physicochemical properties (e.g., serum half-life), safety, efficacy, and immunogenicity of the end product. Regulatory agencies worldwide require state-of-the-art glycan analyses and the demands placed on these methods have steadily increased as better technologies have been developed. Ultimately, robust, information-rich, and reproducible methods for glycan analysis must be included in regulatory filings for glycoprotein-based biotherapeutics to ensure accuracy and consistency. Method simplification and standardization will provide additional assurance that the glycan-analysis methods used are transferrable between testing sites both within and outside (e.g., contract research organizations) of the organization, ensuring better quality and efficiency in manufacturing.
Glycans face new scrutiny
By 2008, the biotechnology company Genzyme had developed and marketed the drug Myozyme (alglucosidase alfa) for the treatment of Pompe disease, a rare and progressively debilitating disorder characterized by deficiency of lysosomal enzyme alpha-glucosidase (GAA). The company was preparing to expand the targeted treatment population from primarily children to adults. Its 160-L production facility was working at capacity, so $53 million was invested to build a 2000-L facility for Myozyme in Allston, MA (2). The company was ready to launch, but FDA rejected Genzyme’s application to sell the drug from the 2000-L plant. According to regulators, the version made in the 2000-L tank was no longer the same drug as the one produced in the 160-L tank. FDA argued that the differences in glycosylation-specifically in this case, the composition of mannose-6-phosphate-meant that the drug was no longer the biological equivalent of the original material produced in the 160-L bioreactor, and may in fact introduce unknown clinical variables. Genzyme argued that it had already conducted a clinical trial on the larger batch material, demonstrating safety and effectiveness. Ultimately, Genzyme had to market the product from the larger bioreactor under a different name.
The incident was a watershed moment in the biopharmaceutical industry, marking the emergence of new challenges (1). First, regulatory authorities were beginning to scrutinize the glycan structures of biopharmaceutical products more carefully based on established technical guidelines (e.g., ICH Q5E, ICH Q6B, and FDA’s Guidance for Industry, PAT-A Framework for Innovative Pharmaceutical Development, Manufacturing, and Quality Assurance), yet there remained inconsistencies in how FDA, the European Medicines Agency (EMA), and Japanese regulators determined what is “biosimilar”. Second, products with complex glycosylation patterns have the potential to easily fall out of specification with changes in biomanufacturing processes and scale-up, so to meet the new regulatory demands, manufacturers had to start carefully characterizing product glycosylation and its relation to the biological and clinical activity of a medication, and begin monitoring relevant glycan characteristics during production (3, 4, 5).
In the years following FDA’s decision on Myozyme, the attention given to glycan structure in biopharmaceuticals has only increased, reflecting improvement in analytical technology and a greater understanding of the role these structures play in the physical characteristics, stability, biological activity, and the clinical safety and effectiveness of a drug (6, 7). The technical guidelines for characterizing and monitoring glycans have changed little since 2008; manufacturers refer mainly to International Conference on Harmonization (ICH) documents Q5E and Q6B (3, 4). These documents list the following recommendations on characterizing glycans:
“For glycoproteins, the carbohydrate content (neutral sugars, amino sugars, and sialic acids) is determined. In addition, the structure of the carbohydrate chains, the oligosaccharide pattern (antennary profile), and the glycosylation site(s) of the polypeptide chain is analyzed, to the extent possible.”
Other guidelines exist, setting expectations for glycan analysis, such as FDA’s Guidance for Industry, Immunogenicity Assessment for Therapeutic Protein Products, and EMA’s 2007 monograph on the characterization of monoclonal antibodies (8). The monograph says the following on glycans:
“Glycan structures should be characterized, and particular attention should be paid to their degree of mannosylation, galactosylation, fucosylation, and sialylation. The distribution of the main glycan structures present (often G0, G1, and G2) should be determined.”
These documents, however, present few details on how to set specification limits on glycans, or recommend technologies and procedures for consistent analytical results. The consequences for this long-standing ambiguity are that manufacturers and regulators sometimes end up with different ideas as to what constitutes a necessary specification for a glycan structure. Furthermore, companies submit reports to regulatory authorities with widely different analytical approaches. Procedures may vary even within the same organization, potentially leading to inconsistent results, analytical testing failures, and ultimately, regulatory delays.
Quality by Design vs. quality in practice
In 2002, in response to an increasing burden on FDA of regulating product manufacturing, and a perception among companies that regulatory requirements were limiting flexibility in process optimization, FDA implemented changes through its Pharmaceutical cGMP 21st Century Initiative and the release of FDA’s process analytical technology guidance (PAT) (5). The new approach placed greater responsibility on the manufacturers to monitor quality control through timely measurements and corrections during processing.
Around the same time, ICH published two guidance documents: ICH Q8 Pharmaceutical Development (7), ICH Q9 Quality Risk Management (8), and ICH Q10 Quality Systems Approach to Pharmaceutical cGMP Regulations (9, 10, 11). These documents helped to further define current scientific and risk-based approaches to pharmaceutical quality control.
The concept of quality by design (QbD) was incorporated into FDA review in 2004, which together with the aforementioned guidelines, emphasized a greater understanding of the product and its manufacturing process, and designing quality control into the process, rather than testing it after the fact (12). This approach is particularly well-suited to glycan analysis, which is typically associated with a complex set of critical quality attributes (CQAs) (such as sialylation, antennary structure, or glycan structure heterogeneity) that are important to the biological or clinical activity of the drug. The CQAs must be identified, measured during process development, and maintained within required parameters (i.e., the design space) during production.
In the case of glycans, the measurement itself may introduce uncertainty and risk, due to a high variability of outcomes when characterizing oligosaccharide chains. An interlaboratory study presenting 11 industrial, regulatory, and academic labs with the same set of four released N-glycans demonstrated that results were not consistent between the laboratories when comparing analyses of sialylation and antennary structure (13). This particular study did not address the potentially added variability caused by sample preparation. The variability in outcomes may be due in part to the availability of numerous analytical approaches and differences between labs as to the selection of approach and limitations of available equipment. Inconsistent levels of training and expertise in glycan analysis may also have had an impact.
Basic requirements for standardized protocols of glycan analysis
The establishment of a robust protocol for glycan analysis can help extract the maximum benefit from QbD practice; give manufacturers greater control over product quality and comparability between batches and process modifications; and ensure consistency and quality in regulatory submissions. Such a protocol should have the following features.
Well-characterized reference standards
A selection of known glycoproteins, glycopeptides, released glycans, and monosaccharides will help calibrate and validate any system of glycoprofiling used in the initial characterization of the product or monitoring of the manufacturing process.
Well-characterized sample standards
Isolated product with a known clinical safety and efficacy profile provides a reference point for comparing glycan structure of batch products under different process conditions and times.
Comprehensive identification of critical glycan attributes
Structural features of glycans have been linked to circulating half-life of the glycoprotein in the blood (sialylation); placental transport (galactosylation); antibody-dependent cell-mediated cytotoxicity (core fucosylation); and a wide range of effector functions, bioavailability, and safety characteristics (14, 15). Critical attributes may include:
Establishment of ranges of acceptable variation in complex glycosylation patterns
Many glycoproteins, particularly those with multiple glycosylation sites, do not exist as a single species, but as a mixture of glycoforms. The natural complexity and heterogeneity of glycan structures can have important functional relevance for a protein, and even minor, low-abundance glycoform species can be crucial. For clinical purposes, each product may have a different tolerance or requirement for glycoform distribution. In particular, clarity on the extent to which low-abundance glycoforms should be identified and monitored is essential.
Adherence to best practices in sample preparation
Selecting the most appropriate method from the wide range of published and commercial sample preparation methods can be daunting. For example, purification of glycans after release from protein may be performed by solvent precipitation, solid-phase extraction, or size-exclusion, hydrophobic-interaction, or hydrophilic-interaction chromatography. Some methods may lead to a non-stoichiometric recovery of oligosaccharides, skewing the results of glycan profiling. Recent developments in sample preparation have allowed for a reduction in preparation times and improved quantitative yields of both high- and low-abundance glycoforms (16).
Selection of glycoanalysis technologies, methods, and strategy
There is a wide array of technologies that can be applied to glycan analysis (see Tables I–III). A series of detailed optimal workflows and best practices would help to harmonize analytical procedures between and within organizations that submit regulatory reports. Workflows would cover initial characterization through to routine monitoring and quality control. Considerations should be made with respect to the simplicity and time of analysis, as long as the required levels of accuracy and reproducibility are not compromised.
The use of orthogonal and complementary methods of analysis help compensate for systematic errors in measurement. These methods typically isolate molecules and their fragments based on different physical properties (e.g., high-performance capillary electrophoresis [HPCE] vs. hydrophilic interaction liquid chromatography [HILIC]) or analytical treatment (e.g., electrospray ionization-mass spectrometry [ESI-MS] vs. matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry [MALDI–TOF–MS]), and are compared to compensate for potential bias introduced by each analytical method.
Pharmaceutical regulatory agencies worldwide have laid out the general principles of quality control and risk management in biopharmaceutical manufacturing. Of the many CQAs that require consideration, the variation of the N-linked and O-linked glycosylation profiles of biotherapeutic glycoproteins is one of the most complex to assess. Currently, there are numerous methods used to elucidate these structures with varying degrees of accuracy and precision. In addition, the use of these somewhat disparate methodologies makes it not always possible to directly compare results between laboratories. To meet regulatory requirements for consistent process and quality control, it would be beneficial to establish more specific and standardized guidelines for glycan analysis performance with respect to reproducibility, accuracy, and sensitivity for the characterization and routine monitoring of critical glycoforms, including those of low abundance. While such guidelines are within purview of national regulatory bodies and international consensus organizations (such as ICH), no such guidelines have been released to date. The requirements for glycan analysis described in this article could address many of the issues related to process and quality control in glycoprotein manufacturing.
1. G. Walsh, Nature Biotech. 28 (9), pp. 917-924 (2010).
2. G. Mack, Nature Biotech. 26 (6), pp. 592 (2008).
3. ICH, Q5E Specifications: Test Procedures and Acceptance Criteria for Biotechnological/Biological Products, EMA Document CPMP/ICH/5721/03 (Geneva, 2003).
4. ICH Q6B Specifications: Test Procedures and Acceptance Criteria for Biotechnological/Biological Products, EMA Document CPMP/ICH/365/96 (Geneva, 1999).
5. FDA, Guidance for Industry, PAT--A Framework for Innovative Pharmaceutical Development, Manufacturing, and Quality Assurance (Rockville, MD, Sept. 2004).
6. R. Jefferis, Biotechnol. Prog. 21, pp. 11-16 (2005).
7. S.A. Brooks, Mol. Biotechnol. 28 (3), pp. 241-255 (2004).
8. EMA, Guideline on Development, Production, Characterization and Specifications for Monoclonal Antibodies and Related Products, EMEA/CHMP/BWP/157653/2007 (London, 2007).
9. FDA, Guidance for Industry, Q8 Pharmaceutical Development (Rockville, MD, May 2006).
10. FDA, Guidance for Industry, Q9 Quality Risk Management, (Rockville, MD, June 2006).
11. A.S. Rathore, A. Sharma, and D. Chillin, BioPharm Int. 19, pp. 48-57 (2006).
12. A.S. Rathore, Trends Biotechnol. 27 (9), pp. 546-553 (2009).
13. S. Thobhani et al., Glycobiology, 19 (3), pp. 201-211 (2009).
14. T. Kibe et al., J. Clin. Biochem. Nutr. 21 (1), pp. 57-63 (1996).
15. A. Okazaki et al., J. Mol. Biol. 336 (5), pp. 1239-1249 (2004).
16. M.A. Lauber et al., Anal. Chem. 87 (10), pp. 5401-5409 (2015).
About the Author
Jennifer Fournier is product marketing manager, consumables business unit-ASR, at Waters Corporation.
Article DetailsBioPharm International
Vol. 28, No. 10
Citation: When referring to this article, please cite it as J. Fournier, "A Review of Glycan Analysis Requirements," BioPharm International 28 (10) 2015.