Changes in production methods of a biological product may necessitate an assessment of comparability to ensure that these manufacturing changes have not affected the safety, identity, purity, or efficacy of the product. Depending on the nature of the protein or the change, this assessment consists of a hierarchy of sequential tests in analytical testing, preclinical animal studies and clinical studies. Differences in analytical test results between pre- and post-change products may require functional testing to establish the biological or clinical significance of the observed difference. An underlying principle of comparability is that under certain conditions, protein products may be considered comparable on the basis of analytical testing results alone. However, the ability to compare biological materials is solely dependent on the tests used, since no single analytical method is able to compare every aspect of protein structure or function. The advantages and disadvantages of any given method depends on the protein property being characterized.
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Johnson, I.S. The trials and tribulations of producing the first genetically engineered drug. Nat. Rev. Drug Discov. 2, 747–751 (2003).
Schmelzer, A.E. & Miller, W.M. Hyperosmotic stress and elevated pCO2 alter monoclonal antibody charge distribution and monosaccharide content. Biotechnol. Prog. 18, 346–353 (2002).
Okamoto, M. et al. Purification and characterization of three forms of differently glycosylated recombinant human granulocyte-macrophage colony-stimulating factor. Arch. Biochem. Biophys. 286, 562–568 (1991).
Macdougall, I.C. Optimizing the use of erythropoietic agents—pharmacokinetic and pharmacodynamic considerations. Nephrol. Dial. Transplant. 17 suppl. 5, 66–70 (2002).
Van Den Hamer, C.J., Morell, A.G., Scheinberg, I.H., Hickman, J. & Ashwell, G. Physical and chemical studies on ceruloplasmin. IX. The role of galactosyl residues in the clearance of ceruloplasmin from the circulation. J. Biol. Chem. 245, 4397–4402 (1970).
Stockert, R.J. The asialoglycoprotein receptor: relationships between structure, function, and expression. Physiol. Rev. 75, 591–609 (1995).
Wright, A. et al. In vivo trafficking and catabolism of IgG1 antibodies with Fc associated carbohydrates of differing structure. Glycobiology 10, 1347–1355 (2000).
Wright, A. & Morrison, S.L. Effect of glycosylation on antibody function: implications for genetic engineering. Trends Biotechnol. 15, 26–32 (1997).
Jefferis, R. Glycosylation of human IgG antibodies: relevance to therapeutic applications. Biopharm 14, 19–26 (2001).
Boyd, P.N., Lines, A.C. & Patel, A.K. The effect of the removal of sialic acid, galactose and total carbohydrate on the functional activity of Campath-1H. Mol. Immunol. 32, 1311–1318 (1995).
Wright, A. & Morrison, S.L. Effect of C2-associated carbohydrate structure on Ig effector function: studies with chimeric mouse-human IgG1 antibodies in glycosylation mutants of Chinese hamster ovary cells. J. Immunol. 160, 3393–3402 (1998).
Idusogie, E.E. et al. Mapping of the C1q binding site on rituxan, a chimeric antibody with a human IgG1 Fc. J. Immunol. 164, 4178–4184 (2000).
Shields, R.L. et al. Lack of fucose on human IgG1 N-linked oligosaccharide improves binding to human Fcgamma RIII and antibody-dependent cellular toxicity. J. Biol. Chem. 277, 26733–26740 (2002).
Shinkawa, T. et al. The absence of fucose but not the presence of galactose or bisecting N-acetylglucosamine of human IgG1 complex-type oligosaccharides shows the critical role of enhancing antibody-dependent cellular cytotoxicity. J. Biol. Chem. 278, 3466–3473 (2003).
Li, J. et al. Thrombocytopenia caused by the development of antibodies to thrombopoietin. Blood 98, 3241–3248 (2001).
Casadevall, N. et al. Pure red-cell aplasia and antierythropoietin antibodies in patients treated with recombinant erythropoietin. N. Engl. J. Med. 346, 469–475 (2002).
Koren, E., Zuckerman, L.A. & Mire-Sluis, A.R. Immune responses to therapeutic proteins in humans—clinical significance, assessment and prediction. Curr. Pharm. Biotechnol. 3, 349–360 (2002).
Schellekens, H. Bioequivalence and the immunogenicity of biopharmaceuticals. Nat. Rev. Drug Discov. 1, 457–462 (2002).
Chirino, A.J., Ary, M.L. & Marshall, S.A. Minimizing the immunogenicity of protein therapeutics. Drug Discov. Today 9, 82–90 (2004).
Cleland, J.L., Powell, M.F. & Shire, S.J. The development of stable protein formulations: a close look at protein aggregation, deamidation, and oxidation. Crit. Rev. Ther. Drug Carrier Syst. 10, 307–377 (1993).
Ryff, J.C. Clinical investigation of the immunogenicity of interferon-alpha 2a. J. Interferon Cytokine Res. 17 suppl. 1, S29–S33 (1997).
Josic, D. et al. Degradation products of factor VIII which can lead to increased immunogenicity. Vox Sang. 77 suppl. 1, 90–99 (1999).
Miller, L.L. et al. Abrogation of the hematological and biological activities of the interleukin-3/granulocyte-macrophage colony-stimulating factor fusion protein PIXY321 by neutralizing anti-PIXY321 antibodies in cancer patients receiving high-dose carboplatin. Blood 93, 3250–3258 (1999).
Wadhwa, M. et al. Immunogenicity of granulocyte-macrophage colony-stimulating factor (GM-CSF) products in patients undergoing combination therapy with GM-CSF. Clin. Cancer Res. 5, 1353–1361 (1999).
Braun, A., Kwee, L., Labow, M.A. & Alsenz, J. Protein aggregates seem to play a key role among the parameters influencing the antigenicity of interferon alpha (IFN-alpha) in normal and transgenic mice. Pharm. Res. 14, 1472–1478 (1997).
Bertolotto, A. et al. Interferon beta neutralizing antibodies in multiple sclerosis: neutralizing activity and cross-reactivity with three different preparations. Immunopharmacology 48, 95–100 (2000).
Bertolotto, A. et al. Differential effects of three interferon betas on neutralising antibodies in patients with multiple sclerosis: a follow up study in an independent laboratory. J. Neurol. Neurosurg. Psychiatry 73, 148–153 (2002).
Luo, P. et al. Development of a cytokine analog with enhanced stability using computational ultrahigh throughput screening. Protein Sci. 11, 1218–1226 (2002).
Hu, S. & Dovichi, N.J. Capillary electrophoresis for the analysis of biopolymers. Anal. Chem. 74, 2833–2850 (2002).
Ma, S. & Nashabeh, W. Analysis of protein therapeutics by capillary electrophoresis. Chromatographia 53, S75–S89 (2001).
Ma, S. & Nashabeh, W. Carbohydrate analysis of a chimeric recombinant monoclonal antibody by capillary electrophoresis with laser-induced fluorescence detection. Anal. Chem. 71, 5185–5192 (1999).
Lu, W., Han, D.S., Yuan, J. & Andrieu, J.M. Multi-target PCR analysis by capillary electrophoresis and laser-induced fluorescence. Nature 368, 269–271 (1994).
Chen, F.T., Dobashi, T.S. & Evangelista, R.A. Quantitative analysis of sugar constituents of glycoproteins by capillary electrophoresis. Glycobiology 8, 1045–1052 (1998).
Raju, T.S., Briggs, J.B., Borge, S.M. & Jones, A.J. Species-specific variation in glycosylation of IgG: evidence for the species-specific sialylation and branch-specific galactosylation and importance for engineering recombinant glycoprotein therapeutics. Glycobiology 10, 477–486 (2000).
Lee, T.T. & Yeung, E.S. High-sensitivity laser-induced fluorescence detection of native proteins in capillary electrophoresis. J. Chromatogr. 595, 319–325 (1992).
Colyer, C. Noncovalent labeling of proteins in capillary electrophoresis with laser-induced fluorescence detection. Cell Biochem. Biophys. 33, 323–337 (2000).
Lin, Y.W., Chiu, T.C. & Chang, H.T. Laser-induced fluorescence technique for DNA and proteins separated by capillary electrophoresis. J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 793, 37–48 (2003).
Rudd, P.M. & Dwek, R.A. Rapid, sensitive sequencing of oligosaccharides from glycoproteins. Curr. Opin. Biotechnol. 8, 488–497 (1997).
Harvey, D.J. Matrix-assisted laser desorption/ionization mass spectrometry of carbohydrates and glycoconjugates. Int. J. Mass Spectrom. 226, 1–35 (2003).
Moorhouse, K.G. et al. Validation of an HPLC method for the analysis of the charge heterogeneity of the recombinant monoclonal antibody IDEC-C2B8 after papain digestion. J. Pharm. Biomed. Anal. 16, 593–603 (1997).
Gitlin, G. et al. Isolation and characterization of a monomethioninesulfoxide variant of interferon alpha-2b. Pharm. Res. 13, 762–769 (1996).
Paranandi, M.V., Guzzetta, A.W., Hancock, W.S. & Aswad, D.W. Deamidation and isoaspartate formation during in vitro aging of recombinant tissue plasminogen activator. J. Biol. Chem. 269, 243–253 (1994).
Renlund, S. et al. Peptide mapping on HIV polypeptides expressed in Escherichia coli. Quality control of different batches and identification of tryptic fragments containing residues of aromatic amino acids or cysteine. J. Chromatogr. 512, 325–335 (1990).
Zhang, W., Czupryn, J.M., Boyle, P.T., Jr & Amari, J. Characterization of asparagine deamidation and aspartate isomerization in recombinant human interleukin-11. Pharm. Res. 19, 1223–1231 (2002).
Harris, R.J. et al. Assessing genetic heterogeneity in production cell lines: detection by peptide mapping of a low level Tyr to Gln sequence variant in a recombinant antibody. Bio/Technology 11, 1293–1297 (1993).
Besman, M.J. & Shiba, D. Evaluation of genetic stability of recombinant human factor VIII by peptide mapping and on-line mass spectrometric analysis. Pharm. Res. 14, 1092–1098 (1997).
Lopez Garcia, F., Zahn, R., Riek, R. & Wuthrich, K. NMR structure of the bovine prion protein. Proc. Natl. Acad. Sci. USA 97, 8334–8339 (2000).
Watson, E., Bhide, A. & van Halbeek, H. Structure determination of the intact major sialylated oligosaccharide chains of recombinant human erythropoietin expressed in Chinese hamster ovary cells. Glycobiology 4, 227–237 (1994).
Jacobsen, N.E. et al. Structure of the saponin adjuvant QS-21 and its base-catalyzed isomerization product by 1H and natural abundance 13C NMR spectroscopy. Carbohydr. Res. 280, 1–14 (1996).
Delmas, C. et al. Comparative structural study of the mannosylated-lipoarabinomannans from Mycobacterium bovis BCG vaccine strains: characterization and localization of succinates. Glycobiology 7, 811–817 (1997).
Clore, G.M. & Gronenborn, A.M. NMR structure determination of proteins and protein complexes larger than 20 kDa. Curr. Opin. Chem. Biol. 2, 564–570 (1998).
Kelly, S.M. & Price, N.C. The use of circular dichroism in the investigation of protein structure and function. Curr. Protein Pept. Sci. 1, 349–384 (2000).
Remmele, R.L. Jr., Nightlinger, N.S., Srinivasan, S. & Gombotz, W.R. Interleukin-1 receptor (IL-1R) liquid formulation development using differential scanning calorimetry. Pharm. Res. 15, 200–208 (1998).
Krishnamurthy, R. & Manning, M.C. The stability factor: importance in formulation development. Curr. Pharm. Biotechnol. 3, 361–371 (2002).
Wyatt, P.J. Light scattering and the absolute characterization of macromolecules. Anal. Chim. Acta 272, 1–40 (1993).
Bohidar, H.B. Light scattering and viscosity study of heat aggregation of insulin. Biopolymers 45, 1–8 (1998).
Laue, T.M. & Stafford, W.F., III. Modern applications of analytical ultracentrifugation. Annu. Rev. Biophys. Biomol. Struct. 28, 75–100 (1999).
Lebowitz, J., Lewis, M.S. & Schuck, P. Modern analytical ultracentrifugation in protein science: a tutorial review. Protein Sci. 11, 2067–2079 (2002).
Philo, J.S. A method for directly fitting the time derivative of sedimentation velocity data and an alternative algorithm for calculating sedimentation coefficient distribution functions. Anal. Biochem. 279, 151–163 (2000).
Schuck, P., Perugini, M.A., Gonzales, N.R., Howlett, G.J. & Schubert, D. Size-distribution analysis of proteins by analytical ultracentrifugation: strategies and application to model systems. Biophys. J. 82, 1096–1111 (2002).
Varley, P.G., Brown, A.J., Dawkes, H.C. & Burns, N.R. A case study and use of sedimentation equilibrium analytical ultracentrifugation as a tool for biopharmaceutical development. Eur. Biophys. J. 25, 437–443 (1997).
Avanzi, G.C. et al. M-07e human leukemic factor-dependent cell line provides a rapid and sensitive bioassay for the human cytokines GM-CSF and IL-3. J. Cell. Physiol. 145, 458–464 (1990).
Meager, A., Leung, H. & Woolley, J. Assays for tumour necrosis factor and related cytokines. J. Immunol. Methods 116, 1–17 (1989).
Sadick, M.D. et al. Kinase receptor activation (KIRA): a rapid and accurate alternative to end-point bioassays. J. Pharm. Biomed. Anal. 19, 883–891 (1999).
Cook, E.B. et al. Simultaneous measurement of six cytokines in a single sample of human tears using microparticle-based flow cytometry: allergics vs. non-allergics. J. Immunol. Methods 254, 109–118 (2001).
Spiro, A., Lowe, M. & Brown, D. A bead-based method for multiplexed identification and quantitation of DNA sequences using flow cytometry. Appl. Environ. Microbiol. 66, 4258–4265 (2000).
Johnson, L.R., McCormack, S.A., Yang, C.H., Pfeffer, S.R. & Pfeffer, L.M. EGF induces nuclear translocation of STAT2 without tyrosine phosphorylation in intestinal epithelial cells. Am. J. Physiol. 276, C419–C425 (1999).
Burrone, O.R., Kefford, R.F., Gilmore, D. & Milstein, C. Stimulation of HLA-A,B,C by IFN-alpha. The derivation of Molt 4 variants and the differential expression of HLA-A,B,C subsets. EMBO J. 4, 2855–2860 (1985).
Musco, M.L. et al. Comparison of flow cytometry and laser scanning cytometry for the intracellular evaluation of adenoviral infectivity and p53 protein expression in gene therapy. Cytometry 33, 290–296 (1998).
Grace, M.J. et al. The use of laser scanning cytometry to assess depth of penetration of adenovirus p53 gene therapy in human xenograft biopsies. Am. J. Pathol. 155, 1869–1878 (1999).
Chackerian, B., Lenz, P., Lowy, D.R. & Schiller, J.T. Determinants of autoantibody induction by conjugated papillomavirus virus-like particles. J. Immunol. 169, 6120–6126 (2002).
Guermonprez, P., Valladeau, J., Zitvogel, L., Thery, C. & Amigorena, S. Antigen presentation and T cell stimulation by dendritic cells. Annu. Rev. Immunol. 20, 621–667 (2002).
McHeyzer-Williams, M. et al. Helper T-cell-regulated B-cell immunity. Microbes Infect. 5, 205–212 (2003).
Schultes, B.C. & Whiteside, T.L. Monitoring of immune responses to CA125 with an IFN-gamma ELISPOT assay. J. Immunol. Methods 279, 1–15 (2003).
Barbosa, M.D.F.S., Tran, C-A. & Chirino, A.J. Testing MHC-binding epitopes: in vitro vaccination (IVV) (abstr. 9394) in 90th Meeting of the American Society of Immunologists. (American Society of Immunologists, Denver, Colorado, 2003).
Wadhwa, M., Bird, C., Dilger, P., Gaines-Das, R. & Thorpe, R. Strategies for detection, measurement and characterization of unwanted antibodies induced by therapeutic biologicals. J. Immunol. Methods 278, 1–17 (2003).
Mire-Sluis, A.R. et al. Recommendations for the design and optimization of immunoassays used in the detection of host antibodies against biotechnology products. J. Immunol. Methods 289, 1–16 (2004).
Lucas, C. et al. A sensitive radioimmunoprecipitation assay for the detection and quantitation of antibodies to the envelope glycoprotein gp120 of the human immunodeficiency virus (HIV-1). AIDS Res. Hum. Retroviruses 6, 357–370 (1990).
Takacs, M.A., Jacobs, S.J., Bordens, R.M. & Swanson, S.J. Detection and characterization of antibodies to PEG-IFN-alpha2b using surface plasmon resonance. J. Interferon Cytokine Res. 19, 781–789 (1999).
Swanson, S.J., Ferbas, J., Mayeux, P. & Casadevall, N. Evaluation of methods to detect and characterize antibodies against recombinant human erythropoietin. Nephron Clin. Pract. 96, c88–c95 (2004).
Rich, R.L. & Myszka, D.G. Advances in surface plasmon resonance biosensor analysis. Curr. Opin. Biotechnol. 11, 54–61 (2000).
Wierda, D., Smith, H.W. & Zwickl, C.M. Immunogenicity of biopharmaceuticals in laboratory animals. Toxicology 158, 71–74 (2001).
Soukharev, S. et al. Expression of factor VIII in recombinant and transgenic systems. Blood Cells Mol. Dis. 28, 234–248 (2002).
Reid, G.E. & McLuckey, S.A. 'Top down' protein characterization via tandem mass spectrometry. J. Mass Spectrom. 37, 663–675 (2002).
Harris, R.J. Processing of C-terminal lysine and arginine residues of proteins isolated from mammalian cell culture. J. Chromatogr. A. 705, 129–134 (1995).
Harris, R.J. et al. Identification of multiple sources of charge heterogeneity in a recombinant antibody. J. Chromatogr. B Biomed. Sci. Appl. 752, 233–245 (2001).
Lam, X.M., Yang, J.Y. & Cleland, J.L. Antioxidants for prevention of methionine oxidation in recombinant monoclonal antibody HER2. J. Pharm. Sci. 86, 1250–1255 (1997).
Abuchowski, A., McCoy, J.R., Palczuk, N.C., van Es, T. & Davis, F.F. Effect of covalent attachment of polyethylene glycol on immunogenicity and circulating life of bovine liver catalase. J. Biol. Chem. 252, 3582–3586 (1977).
Katre, N.V. Immunogenicity of recombinant IL-2 modified by covalent attachment of polyethylene glycol. J. Immunol. 144, 209–213 (1990).
Bailon, P. et al. Rational design of a potent, long-lasting form of interferon: a 40 kDa branched polyethylene glycol-conjugated interferon alpha-2a for the treatment of hepatitis C. Bioconjug. Chem. 12, 195–202 (2001).
Chapman, A.P. PEGylated antibodies and antibody fragments for improved therapy: a review. Adv. Drug Deliv. Rev. 54, 531–545 (2002).
Members of international regulatory authorities, industry and academia met at an FDA-sponsored meeting held at the National Institutes of Health, Bethesda, MD, June 10–13, 2003 to discuss how current analytical technologies are able to characterize biological products and contribute to the comparability process. The authors would like to thank fellow meeting participants Laura Bass, Marta Czupryn, William Egan, Darón Freedberg, Mike Grace, Martin Green, Reed Harris, Steven Indelicato, Eugene Koren, Stacey Ma, Mike Mulkerrin, Leland Paul, John Philo, Zorina Pitkin, Vytas Reipa, Amy Rosenberg, Joseph Siemiatkoski, Stephanie Simek, Steve Swanson, Guillermo Tous and Meenu Wadhwa for advice. The authors would also like to thank Bassil Dahiyat, John Desjarlais, Joyce Morrison and David Szymkowski for critical reading of the manuscript, and Marie Ary for valuable editorial advice.
The authors declare no competing financial interests.
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Chirino, A., Mire-Sluis, A. Characterizing biological products and assessing comparability following manufacturing changes. Nat Biotechnol 22, 1383–1391 (2004). https://doi.org/10.1038/nbt1030
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