Advertisement

Assessing the Quality of Recombinant Products Made in Yeast

  • Karola Vorauer-UhlEmail author
  • Gabriele Lhota
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1923)

Abstract

The product quality of recombinant proteins is of major importance for their intended purpose. The initial characterization of both simple and complex products should be performed as soon as practical. However, to comply with this high standard, appropriate selection of complementary methods is required. Therefore, conventional and sophisticated techniques are available, providing diverse information about the product quality.

In this chapter methods are presented, which enable the determination of the overall protein quality, their aggregation and peptide composition. Methods applied for the determination of posttranslational modifications such as glycan analysis are not described. In this regard, chromatographic, high-resolution technologies for the integrity of proteins as well as Western blot with specific detection methods are introduced, and individual strengths and perceived limitations are highlighted.

Key words

Protein quality Size exclusion chromatography Peptide map Charge variations Electrophoresis Western blot 

References

  1. 1.
    Lin-Cereghino GP, Lin-Cereghino J, Ilgen C, Cregg JM (2002) Production of recombinant proteins in fermenter cultures of the yeast Pichia pastoris. Curr Opin Biotechnol 13:329–332.  https://doi.org/10.1016/S0958166902003300CrossRefGoogle Scholar
  2. 2.
    Ferrer-Miralles N, Domingo-Espín J, Corchero J et al (2009) Microbial factories for recombinant pharmaceuticals. Microb Cell Factories 8:17.  https://doi.org/10.1186/1475-2859-8-17CrossRefGoogle Scholar
  3. 3.
    Martinez JLL, Liu L, Petranovic D, Nielsen J (2012) Pharmaceutical protein production by yeast: towards production of human blood proteins by microbial fermentation. Curr Opin Biotechnol 23(6):965–971.  https://doi.org/10.1016/j.copbio.2012.03.011CrossRefPubMedGoogle Scholar
  4. 4.
    Hong P, Koza S, Bouvier ESP (2012) A review size-exclusion chromatography for the analysis of protein biotherapeutics and their aggregates. J Liq Chromatogr Relat Technol 35:2923–2950.  https://doi.org/10.1080/10826076.2012.743724CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Synge RL (1950) Fractionation of hydrolysis products of amylose by electrokinetic ultrafiltration in an agaragar jelly. Biochem J 24:41–42Google Scholar
  6. 6.
    Lindqvist B, Storgards T (1975) Molecular-sieving properties of starch. Nature 175:511–512CrossRefGoogle Scholar
  7. 7.
    Coutinho FMB, Rcia M, Lica A et al (1997) Porous structure and swelling properties of styrene– divinylbenzene copolymers for size exclusion chromatography. J Appl Polym Sci 65:1257–1262.  https://doi.org/10.1002/(SICI)1097-4628(19970815)65CrossRefGoogle Scholar
  8. 8.
    Li Y, Tolley HD, Lee ML (2010) Size-exclusion separation of proteins using a biocompatible polymeric monolithic capillary column with mesoporosity. J Chromatogr A 1217:8181–8185.  https://doi.org/10.1016/j.chroma.2010.10.067CrossRefPubMedGoogle Scholar
  9. 9.
    Striegel AM (2016) Viscometric detection in size-exclusion chromatography: principles and select applications. Chromatographia 79:945–960.  https://doi.org/10.1007/s10337-016-3078-0CrossRefGoogle Scholar
  10. 10.
    Manning MC, Manning RR, Holcomb RE et al (2014) Review of orthogonal methods to SEC for quantitation and characterization of protein aggregates. BioPharm Int 27:1–2Google Scholar
  11. 11.
    Folta-Stogniew E (2006) Oligomeric states of proteins determined by size-exclusion chromatography coupled with light scattering, absorbance, and refractive index detectors. In: New and Emerging Proteomic Techniques. Humana Press, Totowa, NJ, pp 97–112.  https://doi.org/10.1385/1-59745-026-X:97CrossRefGoogle Scholar
  12. 12.
    Folta-Stogniew E, Williams KR (1999) Determination of molecular masses of proteins in solution: implementation of an HPLC size exclusion chromatography and laser light scattering service in a core laboratory. J Biomol Tech 10:51–63PubMedPubMedCentralGoogle Scholar
  13. 13.
    Tarazona MP, Saiz E (2003) Combination of SEC/MALS experimental procedures and theoretical analysis for studying the solution properties of macromolecules. J Biochem Biophys Methods 56:95–116.  https://doi.org/10.1016/S0165-022X(03)00075-7CrossRefPubMedGoogle Scholar
  14. 14.
    Ambrogelly A, Liu YH, Li H et al (2012) Characterization of antibody variants during process development: the tale of incomplete processing of N-terminal secretion peptide. MAbs 4:701–709.  https://doi.org/10.4161/mabs.21614CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Bouvier ESP, Koza SM (2014) Advances in size-exclusion separations of proteins and polymers by UHPLC. Trends Anal Chem 63:85–94.  https://doi.org/10.1016/j.trac.2014.08.002CrossRefGoogle Scholar
  16. 16.
    Gulati D, Bongers J, Burman S (1999) RP-HPLC tryptic mapping of IgG1 proteins with post-column fluorescence derivatization. J Pharm Biomed Anal 21:887–893.  https://doi.org/10.1016/S0731-7085(99)00119-3CrossRefPubMedGoogle Scholar
  17. 17.
    Lee GF, Anderson DC (1991) Reversed-phase high-pressure liquid chromatographic tryptic peptide mapping for the comparison and study of monoclonal antibodies. Bioconjug Chem 2:367–374.  https://doi.org/10.1021/bc00011a012CrossRefPubMedGoogle Scholar
  18. 18.
    Lundell N, Schreitmüller T (1999) Sample preparation for peptide mapping – a pharmaceutical quality-control perspective. Anal Biochem 266:31–47.  https://doi.org/10.1006/abio.1998.2919CrossRefPubMedGoogle Scholar
  19. 19.
    Wang Y, Li H, Shameem M, Xu W (2016) Development of a sample preparation method for monitoring correct disulfide linkages of monoclonal antibodies by liquid chromatography-mass spectrometry. Anal Biochem 495:21–28.  https://doi.org/10.1016/j.ab.2015.11.010CrossRefPubMedGoogle Scholar
  20. 20.
    Hennessy TP, Boysen RI, Huber MI et al (2003) Peptide mapping by reversed-phase high-performance liquid chromatography employing silica rod monoliths. J Chromatogr A 1009:15–28.  https://doi.org/10.1016/S0021-9673(03)00445-XCrossRefPubMedGoogle Scholar
  21. 21.
    Krokhin OV, Craig R, Spicer V et al (2004) An improved model for prediction of retention times of tryptic peptides in ion pair reversed-phase HPLC. Mol Cell Proteomics 3:908–919.  https://doi.org/10.1074/mcp.M400031-MCP200CrossRefPubMedGoogle Scholar
  22. 22.
    Chakraborty AB, Berger SJ (2005) Optimization of reversed-phase peptide liquid chromatography ultraviolet mass spectrometry analyses using an automated blending methodology. J Biomol Tech 16(4):327–335PubMedPubMedCentralGoogle Scholar
  23. 23.
    Wagner K, Miliotis T, Marko-Varga G et al (2002) An automated on-line multidimensional HPLC system for protein and peptide mapping with integrated sample preparation. Anal Chem 74:809–820.  https://doi.org/10.1021/ac010627fCrossRefPubMedGoogle Scholar
  24. 24.
    Gong B, Burnina I, Stadheim TA, Li H (2013) Glycosylation characterization of recombinant human erythropoietin produced in glycoengineered Pichia pastoris by mass spectrometry. J Mass Spectrom 48:1308–1317.  https://doi.org/10.1002/jms.3291CrossRefPubMedGoogle Scholar
  25. 25.
    Xie H, Gilar M, Gebler JC (2009) Characterization of Protein Impurities by Peptide Mapping with UPLC/MSE Application Note 1–6, Waters Application Note, Waters Corporation, http://www.waters.com/webassets/cms/library/docs/720002809en.pdf
  26. 26.
    Leblanc Y, Ramon C, Bihoreau N, Chevreux G (2017) Charge variants characterization of a monoclonal antibody by ion exchange chromatography coupled on-line to native mass spectrometry: case study after a long-term storage at +5 °C. J Chromatogr B 1048:130–139.  https://doi.org/10.1016/j.jchromb.2017.02.017CrossRefGoogle Scholar
  27. 27.
    Fekete S, Veuthey JL, Guillarme D (2017) Achievable separation performance and analysis time in current liquid chromatographic practice for monoclonal antibody separations. J Pharm Biomed Anal 141:59–69.  https://doi.org/10.1016/j.jpba.2017.04.004CrossRefPubMedGoogle Scholar
  28. 28.
    Ahamed T, Nfor BK, Verhaert PDEM et al (2007) pH-gradient ion-exchange chromatography: an analytical tool for design and optimization of protein separations. J Chromatogr A 1164:181–188.  https://doi.org/10.1016/j.chroma.2007.07.010CrossRefPubMedGoogle Scholar
  29. 29.
    Fekete S, Veuthey JL, Guillarme D (2015) Comparison of the most recent chromatographic approaches applied for fast and high resolution separations: theory and practice. J Chromatogr A 1408:1–14.  https://doi.org/10.1016/j.chroma.2015.07.014CrossRefPubMedGoogle Scholar
  30. 30.
    Rea JC, Moreno GT, Lou Y, Farnan D (2011) Validation of a pH gradient-based ion-exchange chromatography method for high-resolution monoclonal antibody charge variant separations. J Pharm Biomed Anal 54:317–323.  https://doi.org/10.1016/j.jpba.2010.08.030CrossRefPubMedGoogle Scholar
  31. 31.
    Ghosh R, Gilda JE, Gomes AV (2014) The necessity of and strategies for improving confidence in the accuracy of western blots. Expert Rev Proteomics 11:549–560.  https://doi.org/10.1586/14789450.2014.939635CrossRefPubMedPubMedCentralGoogle Scholar
  32. 32.
    Towbin H, Staehelin T, Gordon J (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci 76:4350–4354.  https://doi.org/10.1073/pnas.76.9.4350CrossRefPubMedGoogle Scholar
  33. 33.
    Blancher C, Jones A (2001) SDS-PAGE and Western blotting techniques. In: Metastasis research protocols. Humana Press, Totowa, NJ, pp 145–162.  https://doi.org/10.1385/1-59259-136-1:145CrossRefGoogle Scholar
  34. 34.
    Kurien BT, Scofield RH (2009) Introduction to protein blotting. Methods Mol Biol 536:9–22.  https://doi.org/10.1007/978-1-59745-542-8_3CrossRefPubMedGoogle Scholar
  35. 35.
    Mishra M, Tiwari S, Gomes AV (2017) Protein purification and analysis: next generation Western blotting techniques. Expert Rev Proteomics 14:1037–1053.  https://doi.org/10.1080/14789450.2017.1388167CrossRefPubMedGoogle Scholar
  36. 36.
    Bakalova R, Zhelev Z, Ohba H, Baba Y (2005) Quantum dot-based western blot technology for ultrasensitive detection of tracer proteins. J Am Chem Soc 127:9328–9329.  https://doi.org/10.1021/ja0510055CrossRefPubMedGoogle Scholar
  37. 37.
    Devine PL, Warren JA (1990) Glycoprotein detection on immobilon PVDF transfer membrane using the periodic acid/Schiff reagent. BioTechniques 8:492–495PubMedGoogle Scholar
  38. 38.
    Thornton DJ, Carlstedt I, Sheehan JK (1996) Identification of glycoproteins on nitrocellulose membranes and gels. Mol Biotechnol 5:171–176.  https://doi.org/10.1007/BF02789065CrossRefPubMedGoogle Scholar
  39. 39.
    Packer NH, Ball MS, Devine PL, Patton WF (2002) Detection of glycoproteins in gels and blots. In: Protein protocols handbook. Humana Press, Totowa, NJ, pp 761–772CrossRefGoogle Scholar
  40. 40.
    Roth Z, Yehezkel G, Khalaila I (2012) Identification and quantification of protein glycosylation. Int J Carbohydr Chem 2012:1–10.  https://doi.org/10.1155/2012/640923CrossRefGoogle Scholar
  41. 41.
    Gauci VJ, Wright EP, Coorssen JR (2011) Quantitative proteomics: assessing the spectrum of in-gel protein detection methods. J Chem Biol 4:3–29.  https://doi.org/10.1007/s12154-010-0043-5CrossRefPubMedGoogle Scholar
  42. 42.
    Sato T (2014) Lectin-probed Western blot analysis. Methods Mol Biol 1200:93–100.  https://doi.org/10.1007/978-1-4939-1292-6_8CrossRefPubMedGoogle Scholar
  43. 43.
    Badr HA, AlSadek DMM, Mathew MP et al (2015) Lectin staining and Western blot data showing differential sialylation of nutrient-deprived cancer cells to sialic acid supplementation. Data Brief 5:481–488.  https://doi.org/10.1016/j.dib.2015.09.043CrossRefPubMedPubMedCentralGoogle Scholar
  44. 44.
    Zachara NE, Vosseller K, Hart GW (2011) Detection and analysis of proteins modified by O-linked N -acetylglucosamine. Curr Protoc Mol Biol Chapter 17:Unit 17.6.  https://doi.org/10.1002/0471142727.mb1706s95CrossRefPubMedGoogle Scholar
  45. 45.
    Mandell JW (2003) Phosphorylation state-specific antibodies: applications in investigative and diagnostic pathology. Am J Pathol 163:1687–1698CrossRefGoogle Scholar
  46. 46.
    Candiano G, Bruschi M, Musante L et al (2004) Blue silver: a very sensitive colloidal Coomassie G-250 staining for proteome analysis. Electrophoresis 25:1327–1333.  https://doi.org/10.1002/elps.200305844CrossRefPubMedPubMedCentralGoogle Scholar
  47. 47.
    Gurramkonda C, Adnan A, Gäbel T et al (2009) Simple high-cell density fed-batch technique for high-level recombinant protein production with Pichia pastoris: application to intracellular production of Hepatitis B surface antigen. Microb Cell Factories 8:13:1–13:8.  https://doi.org/10.1186/1475-2859-8-13CrossRefGoogle Scholar
  48. 48.
    Gurramkonda C, Polez S, Skoko N et al (2010) Application of simple fed-batch technique to high-level secretory production of insulin precursor using Pichia pastoris with subsequent purification and conversion to human insulin. Microb Cell Factories 9(3):1–11.  https://doi.org/10.1186/1475-2859-9-31CrossRefGoogle Scholar
  49. 49.
    Morrissey JH (1981) Silver stain for proteins in polyacrylamide gels: a modified procedure with enhanced uniform sensitivity. Anal Biochem 117:307–310CrossRefGoogle Scholar
  50. 50.
    Neuhoff V, Stamm R, Eibl H (1985) Clear background and highly sensitive protein staining with Coomassie Blue dyes in polyacrylamide gels: a systematic analysis. Electrophoresis 6:427–448.  https://doi.org/10.1002/elps.1150060905CrossRefGoogle Scholar
  51. 51.
    Advion (2017) https://advion.com/. Accessed 14 Dec 2017

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Department of BiotechnologyUniversity of Natural Resources and Life Sciences Vienna (BOKU)ViennaAustria

Personalised recommendations