, Volume 53, Issue 1–3, pp 121–125 | Cite as

Modifications of therapeutic proteins: challenges and prospects

  • Nigel Jenkins
NICB special Issue


The production of therapeutic proteins is one of the fastest growing sectors of the pharmaceutical industry. However, most proteins used in drug therapy require complex post-translational modifications for efficient secretion, drug efficacy and stability. Common protein modifications include variable glycosylation, misfolding and aggregation, oxidation of methionine, deamidation of asparagine and glutamine, and proteolysis. These modifications not only pose challenges for accurate and consistent bioprocessing, but also may have consequences for the patient in that incorrect modifications or aggregation may lead to an immune response to the protein therapeutic. This review provides examples of analytical and preventative advances that have been devised to meet these challenges, and insights into how further advances can improve the efficiency and safety in manufacturing recombinant proteins.


Recombinant protein Cell culture Aggregation Folding Oxidation Deamidation Glycosylation Therapeutics 



antibody-dependent cellular cytotoxicity


Chinese hamster ovary




electrospray ionization mass spectrometry


high pressure liquid chromatography


matrix-assisted laser desorption-ionization


post-translational modification




  1. Baker KN, Rendall MH, Hills AE, Hoare M, Freedman RB, James DC (2001) Metabolic control of recombinant protein N-glycan processing in NS0 and CHO cells. Biotechnol Bioeng 73:188–202CrossRefGoogle Scholar
  2. Bertolotti A, Zhang Y, Hendershot LM, Harding HP, Ron D (2000) Dynamic interaction of BiP and ER stress transducers in the unfolded-protein response. Nat Cell Biol 2:326–332CrossRefGoogle Scholar
  3. Bragonzi A, Distefano G, Buckberry LD, Acerbis G, Foglieni C, Lamotte D, Campi G, Marc A, Soria MR, Jenkins N et al (2000) A new Chinese hamster ovary cell line expressing alpha2,6-sialyltransferase used as universal host for the production of human-like sialylated recombinant glycoproteins. Biochim Biophys Acta 1474:273–282Google Scholar
  4. Capelle MA, Gurny R, Arvinte T (2007) High throughput screening of protein formulation stability: Practical considerations. Eur J Pharm Biopharm 65:131–148Google Scholar
  5. Chakravarthi S, Jessop CE, Bulleid NJ (2006) The role of glutathione in disulphide bond formation and endoplasmic-reticulum-generated oxidative stress. EMBO Rep 7:271–275CrossRefGoogle Scholar
  6. Chelius D, Rehder DS, Bondarenko PV (2005) Identification and characterization of deamidation sites in the conserved regions of human immunoglobulin gamma antibodies. Anal Chem 77:6004–6011CrossRefGoogle Scholar
  7. Chirino AJ, Mire-Sluis A (2004) Characterizing biological products and assessing comparability following manufacturing changes. Nat Biotechnol 22:1383–1391CrossRefGoogle Scholar
  8. Cleland JL, Lam X, Kendrick B, Yang J, Yang TH, Overcashier D, Brooks D, Hsu C, Carpenter JF (2001) A specific molar ratio of stabilizer to protein is required for storage stability of a lyophilized monoclonal antibody. J Pharm Sci 90:310–321CrossRefGoogle Scholar
  9. Cudna RE, Dickson AJ (2003) Endoplasmic reticulum signaling as a determinant of recombinant protein expression. Biotechnol Bioeng 81:56–65CrossRefGoogle Scholar
  10. De Groot AS (2006) Immunomics: discovering new targets for vaccines and therapeutics. Drug Discov Today 11:203–209CrossRefGoogle Scholar
  11. Demeule B, Lawrence MJ, Drake AF, Gurny R, Arvinte T (2007) Characterization of protein aggregation: the case of a therapeutic immunoglobulin. Biochim Biophys Acta 1774:146–153Google Scholar
  12. Egrie JC, Dwyer E, Browne JK, Hitz A, Lykos MA (2003) Darbepoetin alfa has a longer circulating half-life and greater in vivo potency than recombinant human erythropoietin. Exp Hematol 31:290–299CrossRefGoogle Scholar
  13. Elliott S, Egrie J, Browne J, Lorenzini T, Busse L, Rogers N, Ponting I (2004) Control of rHuEPO biological activity: the role of carbohydrate. Exp Hematol 32:1146–1155CrossRefGoogle Scholar
  14. Ferrara C, Brunker P, Suter T, Moser S, Puntener U, Umana P (2006) Modulation of therapeutic antibody effector functions by glycosylation engineering: influence of Golgi enzyme localization domain and co-expression of heterologous beta1, 4-N-acetylglucosaminyltransferase III and Golgi alpha-mannosidase II. Biotechnol Bioeng 93:851–861CrossRefGoogle Scholar
  15. Gu X, Wang DI (1998) Improvement of interferon-gamma sialylation in Chinese hamster ovary cell culture by feeding of N-acetylmannosamine. Biotechnol Bioeng 58:642–648CrossRefGoogle Scholar
  16. Harris RJ (2005) Heterogeneity of recombinant antibodies: linking structure to function. Dev Biol (Basel) 122:117–127Google Scholar
  17. Harris RJ, Kabakoff B, Macchi FD, Shen FJ, Kwong M, Andya JD, Shire SJ, Bjork N, Totpal K, Chen AB (2001) Identification of multiple sources of charge heterogeneity in a recombinant antibody. J Chromatogr B Biomed Sci Appl 752:233–245CrossRefGoogle Scholar
  18. Hermeling S, Crommelin DJ, Schellekens H, Jiskoot W (2004) Structure-immunogenicity relationships of therapeutic proteins. Pharm Res 21:897–903CrossRefGoogle Scholar
  19. Houde D, Kauppinen P, Mhatre R, Lyubarskaya Y (2006) Determination of protein oxidation by mass spectrometry and method transfer to quality control. J Chromatogr A 1123:189–198CrossRefGoogle Scholar
  20. Jenkins N, Parekh RB, James DC (1996) Getting the glycosylation right: implications for the biotechnology industry. Nat Biotechnol 14:975–981CrossRefGoogle Scholar
  21. Koopmann JO, Blackburn J (2003) High affinity capture surface for matrix-assisted laser desorption/ionisation compatible protein microarrays. Rapid Commun Mass Spectrom 17:455–462CrossRefGoogle Scholar
  22. Lin JJ, Meyer JD, Carpenter JF, Manning MC (2000) Stability of human serum albumin during bioprocessing: denaturation and aggregation during processing of albumin paste. Pharm Res 17:391–396CrossRefGoogle Scholar
  23. Muthing J, Kemminer SE, Conradt HS, Sagi D, Nimtz M, Karst U, Peter-Katalinic J (2003) Effects of buffering conditions and culture pH on production rates and glycosylation of clinical phase I anti-melanoma mouse IgG3 monoclonal antibody R24. Biotechnol Bioeng 83:321–334CrossRefGoogle Scholar
  24. Purohit VS, Middaugh CR, Balasubramanian SV (2006) Influence of aggregation on immunogenicity of recombinant human Factor VIII in hemophilia A mice. J Pharm Sci 95:358–371CrossRefGoogle Scholar
  25. Roach P, Woodworth JR (2002) Clinical pharmacokinetics and pharmacodynamics of insulin lispro mixtures. Clin Pharmacokinet 41:1043–1057CrossRefGoogle Scholar
  26. Schroder M, Schafer R, Friedl P (2002) Induction of protein aggregation in an early secretory compartment by elevation of expression level. Biotechnol Bioeng 78:131–140CrossRefGoogle Scholar
  27. Serrato JA, Palomares LA, Meneses-Acosta A, Ramirez OT (2004) Heterogeneous conditions in dissolved oxygen affect N-glycosylation but not productivity of a monoclonal antibody in hybridoma cultures. Biotechnol Bioeng 88:176–188CrossRefGoogle Scholar
  28. Sheridan C (2007) Commercial interest grows in glycan analysis. Nat Biotechnol 25:145–146CrossRefGoogle Scholar
  29. Soenderkaer S, Carpenter JF, van de Weert M, Hansen LL, Flink J, Frokjaer S (2004) Effects of sucrose on rFVIIa aggregation and methionine oxidation. Eur J Pharm Sci 21:597–606CrossRefGoogle Scholar
  30. Taggart C, Cervantes-Laurean D, Kim G, McElvaney NG, Wehr N, Moss J, Levine RL (2000) Oxidation of either methionine 351 or methionine 358 in alpha 1-antitrypsin causes loss of anti-neutrophil elastase activity. J Biol Chem 275:27258–27265Google Scholar
  31. Tsumoto K, Ejima D, Kita Y, Arakawa T (2005) Review: Why is arginine effective in suppressing aggregation? Protein Pept Lett 12:613–619CrossRefGoogle Scholar
  32. Walsh G, Jefferis R (2006) Post-translational modifications in the context of therapeutic proteins. Nat Biotechnol 24:1241–1252CrossRefGoogle Scholar
  33. Warnock D, Bai X, Autote K, Gonzales J, Kinealy K, Yan B, Qian J, Stevenson T, Zopf D, Bayer RJ (2005) In vitro galactosylation of human IgG at 1 kg scale using recombinant galactosyltransferase. Biotechnol Bioeng 92:831–842CrossRefGoogle Scholar
  34. Yang M, Butler M (2002) Effects of ammonia and glucosamine on the heterogeneity of erythropoietin glycoforms. Biotechnol Prog 18:129–138CrossRefGoogle Scholar
  35. Ye H (2006) Simultaneous determination of protein aggregation, degradation, and absolute molecular weight by size exclusion chromatography-multiangle laser light scattering. Anal Biochem 356:76–85CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2007

Authors and Affiliations

  1. 1.National Institute for Bioprocessing Research and TrainingUniversity College DublinBelfield, DublinIreland

Personalised recommendations