iBET

Recombinant proteins including monoclonal antibodies

Recombinant proteins produced in animal cells provide essential therapies in various diseases including anemia (erythropoietin), viral hepatitis (interferon), hemophilia (Factor VIII), cancer treatment (numerous monoclonal antibodies), among others. Animal cells are essential as they can provide these proteins with the necessary human-like post-translational modifications. We have been working on the development of recombinant protein production platforms using a wide variety of animal cells and different expression strategies - stable or transient gene expression, by transfection with plasmid vectors or using the baculovirus-insect cell system (BEVS). For upstream process development we have been using stirred-tank bioreactors with different capacities (from 200 ml up to 30 l), operated in batch, fed-batch or perfusion modes. Chromatography and membrane-based techniques, namely expanded bed and affinity chromatography have been developed in our group to purify recombinant proteins.

For more information please access our Protein Production Platform.

Selected Publications

Recombinant Proteins

 

  1. Sequeira DP, Correia R, Carrondo MJT, Roldao A, Teixeira AP, Alves PM.  2017Combining stable insect cell lines with baculovirus-mediated expression for multi-HA influenza VLP production.  Vaccine. pii: S0264-410X(17)30246-3. 
  2. Isidro IA, Portela RM, Clemente JJ, Cunha AE, Oliveira R.  2016Hybrid metabolic flux analysis and recombinant protein prediction in Pichia pastoris X-33 cultures expressing a single-chain antibody fragment.  Bioprocess Biosyst Eng.  39(9):1351-63. 
  3. Patrone M, Coroadinha AS, Teixeira AP, Alves PM. 2016Palmitoylation strengthens cholesterol-dependent multimerization and fusion activity of human cytomegalovirus gB. Journal of Biochemical Chemistry. 
  4. Alves, M., Lamego, J., Bandeiras, T., Castro, R., Tomas, H., Coroadinha, A.S., Costa, J., and Simplício, A.L. 2016. Human Carboxylesterase 2: studies on the role of glycosylation for enzymatic activity. Biochem Biophy Rep 5, 105-10. 
  5. Vlaming M, van Duijn E, Dillingh MR, Brands R, Windhorst AD, Hendrikse NH, Bosgra S, Burggraaf J, de Koning MC, Fidder A, Mocking J, Sandman H, de Ligt R, Fabriek BO, Pasman WJ, Seinen W, Alves T, Carrondo M, Peixoto C, Peeters P, Vaes W. 2015. Microdosing of a carbon-14 labeled protein in healthy volunteers accurately predicts its pharmacokinetics at therapeutic dosages. Clin Pharmacol Ther. Apr13. 
  6. Patrone M, Carinhas N, Sousa MQ, Peixoto C, Ciferri C, Carfì A, Alves PM. 2014. Enhanced expression of full-length human cytomegalovirus fusion protein in non-swelling baculovirus-infected cells with a minimal fed-batch strategy. PloS One.   
  7. Fernandes F, Dias MM, Vidigal J, Patrone M, Teixeira AP, Alves PM. 2014. Production of rotavirus core-like particles in Sf9 cells using recombinase-mediated cassette exchange. J Biotechnol. 71: 34–38 http://dx.doi.org/10.1016/j.jbiotec.2013.11.020
  8. Carinhas N, Duarte TM, Barreiro LC, Carrondo MJT, Alves PM, Teixeira AP. 2013. Metabolic signatures of GS‑CHO cell clones associated with butyrate treatment and culture phase transition. Biotechnol Bioeng, 110(12), 3244–3257. 
  9. Fernandes F, Teixeira AP, Carinhas N, Carrondo MJT, Alves PM. 2013. Insect cells as production platform of complex VLPs. Expert Rev Vaccines. Feb;12(2):225-36. 
  10. Simplício AL, Coroadinha AS, Gilmer JF, Lamego J. 2013. A methodology for detection and quantification of esterase activity. Methods Mol Biol. 2013;984:309-19. 
  11. Lamego J, Cunha B, Peixoto C, Sousa MF, Alves PM, Simplicio AL, Coroadinha AS, 2013. Carboxylesterase 2 production and characterization in human cells: new insights into enzyme oligomerization and activity. Appl Microbiol Biotechnol. Feb;97(3):1161-73.
  12. Fernandes F, Vidigal J, Dias M, Prather KL, Coroadinha AS, Teixeira AP, Alves PM, 2012. Flipase-mediated cassette exchange in Sf9 insect cells for stable gene expression. Biotechnol Bioeng, 109(11):2836-44. 
  13. Lamego J, Coroadinha AS, Simplicio AL, 2011. Detection and Quantification of Carboxylesterase 2 Activity by Capillary Electrophoresis. Anal Chem, 83(3):881–887. 
  14. Franco CF, Mellado MC, Alves PM, Coelho AV, 2010. Monitoring virus-like particle and viral protein production by intact cell MALDI-TOF mass spectrometry. Talanta, 80(4):1561-8. 
  15. Vieira HL, Pereira AC, Peixoto C, Moraes RH, Alves PM, Mendonça RZ, 2010. Improvement of recombinant protein production by an anti-apoptotic protein from hemolymph of Lonomia obliqua. Cytotechnology, 62(6):547-55. 
  16. Roldão A, Oliveira R, Carrondo MJ, Alves PM, 2009. Error Assessment in Recombinant Baculovirus Titration: Evaluation of Different Methods. J Virol Methods, 159:69-80. 
  17. Vieira HL, Cunha L, Goldmacher VS, Alves PM, 2009. The effect of the cell death suppressor vMIA on the production of a recombinant protein in the adenovirus-293 expression system. Protein Expr Purif, 64(2):179-84. 
  18. Gouveia RM, Gomes CM, Sousa MF, Alves, PM, Costa J, 2008. Kinetic analysis of L1 Homophilic Interaction: Role of the First Four Ig Domains and Implications on L1-L1 Binding Mechanism. J Biol Chem, 283(42):28038-47. 
  19. Mellado MC, Peixoto C, Cruz PE, Carrondo MJ, Alves PM, 2008. Purification of recombinant rotavirus VP7 for the study of in vitro rotavirus-like particles assembly. J Chromatogr B, 874:89-94. 
  20. Bento I, Peixoto C, Zaitsev VN, Lindley P, 2007. Ceruloplasmin revisited: structural and functional roles of various metal cation-binding sites. Acta Crystallogr D Biol Crystallogr, 63(2):240-48. 
  21. Teixeira A, Alves C, Alves PM, Carrondo MJ, Oliveira R, 2007. Hybrid elementary flux analysis/nonparametric modeling: application for bioprocess control. BMC Bioinformatics, 8(1):30. 
  22. Teixeira AP, Carinhas N, Dias JM, Cruz PE, Alves PM, Carrondo MJ, Oliveira R, 2007. Hybrid semi-parametric mathematical systems: bridging the gap between biology and process engineering. J Biotechnol, 132:418-25. 
  23. Wirth D, Gama-Norton L, Riemer P, Sandhu U, Schucht R, Hauser H, 2007. Road to precision: recombinase-based targeting technologies for genome engineering. Curr Opin Biotechnol, 18:411–19. 
  24. Gouveia RM, Morais VA, Peixoto C, Sousa MF, Regalla M, Alves PM, Costa J, 2007. Production and purification of functional truncated soluble forms of human recombinant L1 cell adhesion glycoprotein from Spodoptera frugiperda Sf9 cells. Protein Expr and Purif, 52:182-93. 
  25. Vieira HL, Pereira ACP, Carrondo MJ, Alves PM, 2006. Catalase effect on apoptosis for the improvement of recombinant protein production in baculovirus-insect cell system. Bioprocess Biosyst Eng, 29(5-6):409-14. 
  26. Teixeira AP, Clemente JJ, Cunha AE, Carrondo MJ, Oliveira R, 2006. Bioprocess iterative batch-to-batch optimization based on hybrid parametric/nonparametric models. Biotechnol Prog, 22(1):247-58. 
  27. Lameiro MH, Lopes A, Martins LO, Alves PM, Melo E, 2006. Incorporation of a Model Protein into Chitosan-Bile salt Microparticles. Int J Pharm, 312:119-30. 
  28. Teixeira A, Cunha AE, Clemente JJ, Moreira JL, Cruz HJ, Alves PM, Carrondo MJ, Oliveira R, 2005. Modelling and optimization of a recombinant BHK-21 cultivation process using hybrid grey-box systems. J Biotechnol, 118:290-303. 
  29. Carvalhal AV, Marcelino I, Carrondo MJ, 2003. Metabolic changes during cell growth inhibition by p27 overexpression. Appl Microbiol Biotechnol, 63:164-73. 
  30. Carvalhal A, Coroadinha AS, Alves PM, Moreira JL, Hauser H, Carrondo MJ, 2002. Metabolic changes during cell growth inhibition by the IRF-1 system. Enzyme Microb Technol, 30:95-109. 
  31. Cruz HJ, Conradt HS, Dunker R, Peixoto CM, Cunha AE, Thomaz M, Burger C, Dias EM, Clemente J, Moreira JL, Reike E, Carrondo MJ, 2002. Process development of a recombinant antibody/interleukin-2 fusion protein expressed in protein-free medium by BHK cells. J Biotechnol, 96:169-83. 
  32. Cruz PE, L Maranga, MJ Carrondo, 2002. Integrated process optimization: lessons from retrovirus and virus-like particle production. J Biotechnol, 99:199-214. 
  33. Maranga L, PE Cruz, JG Aunins, MJ Carrondo, 2002. Production of core and virus-like particles with baculovirus infected insect cells. Adv Biochem Eng Biotechnol, 74:183-206. 
  34. Maranga L, Rueda P, Antonis AF, Vela C, Langeveld JP, Casal JI, Carrondo MJ, 2002. Large scale production and downstream processing of a recombinant porcine parvovirus vaccine. Appl Microbiol Biotechnol, 59:45-50. 
  35. Carvalhal AV, Moreira JL, Carrondo MJ, 2001. Strategies to modulate BHK cell proliferation by the regulation of IRF-1 expression. J Biotechnol, 92:47-59. 
  36. Morais VA, Serpa J, Palma AS, Costa T, Maranga L, Costa J, Wu N, Ataai MM, 2001. Expression and characterization of recombinant human alpha-3/4-fucosyltransferase III from Spodoptera frugiperda (Sf9) and Trichoplusia ni (Tn) cells using the baculovirus expression system. Biochem J, 353:719-25. 
  37. Carvalhal AV, Moreira JL, Carrondo MJ, 2001. Strategies to modulate BHK cell proliferation by the regulation of IRF-1 expression. J Biotechnol, 92:47-59. 
  38. Cruz HJ, Dias EM, Peixoto CM, Moreira JL, Carrondo MJ, 2000. Product quality of a recombinant fusion protein expressed in immobilised baby hamster cells grown in protein-free medium. Biotechnol Lett, 22:677-82. 
  39. Cruz HJ, Peixoto CM, Nimtz M, Alves PM, Dias EM, Moreira JL, Carrondo MJ, 2000. Metabolic shifts do not influence the glycosylation patterns of a recombinant fusion protein expressed in BHK cells. Biotechnol Bioeng, 69:129-39. 
  40. Burger C, Carrondo MJ, Cruz H, Cuffe M, Dias E, Griffiths JB, Hayes K, Hauser H, Looby D, Mielke C, Moreira JL, Rieke E, Savage AV, Stacey GN, Welge T, 1999. An integrated strategy for the process development of a recombinant antibody-cytokine fusion protein expressed in BHK cells. Appl Microbiol Biotechnol, 52:345-53. 
  41. Cruz PE, Martins PC, Alves PM, Peixoto CC, Santos H, Moreira JL, Carrondo MJ,  1999. Proteolytic activity in infected and non infected insect cells: degradation of HIV-1 Pr55gag particles. Biotechnol Bioeng, 65:133-43. 
  42. Cruz HJ, Moreira JL, Stacey G, Dias EM, Hayes K, Looby D, Griffiths B, Carrondo MJ, 1998. Adaptation of BHK cells producing a recombinant protein to serum-free media and protein-free medium. Cytotechnology, 26:59-64. 
  43. Cruz PE, Peixoto CC, Moreira JL, Carrondo MJ, 1998. Optimization of the production of virus-like particles in insect cells. Biotechnol Bioeng, 60:408-18. 
  44. Cruz PE, Peixoto CC, Moreira JL, Carrondo MJ, 1998. Production and quality analysis of Pr55gag particles produced in baculovirus-infected insect cells. J Chem Technol Biotechnol, 72:149-58. 
  45. Racher AJ, Moreira JL, Alves PM, Wirth M, Weidle UH, Hauser H, Carrondo MJ, Griffiths JB, 1994. Expression of recombinant antibody and secreted alkaline phosphatase in mammalian cells. Influence of cell line and culture system upon production kinetics. Appl Microbiol Biotechnol, 40:851-6. 
  46. Moreira JL, Wirth M, Fitzek M, Hauser H, 1992. Evaluation of reporter genes in mammalian cell lines. Meth Mol Cell Biol, 3:23-2. 

Downstream Processing

  1. Sofia B. Carvalho, A. Raquel Fortuna, Michael Wolff, Cristina Peixoto, Paula M. Alves, Udo Reichl, Manuel J.T. Carrondo. 2017. Purification of influenza virus-like particles using sulfated cellulose membrane adsorbers. Journal of Membrane Science
  2. Cunha B, Aguiar T, Carvalho SB, Silva MM, Gomes RA, Carrondo MJT, Gomes-Alves P, Peixoto C, Serra M, Alves PM. 2017. Bioprocess integration for human mesenchymal stem cells: from up to downstream processing scale-up to cell proteome characterization.  J Biotechnol. S0168-1656(17)30031-7.   
  3. Carvalho SB, Freire JM, Moleirinho MG, Monteiro F, Gaspar D, Castanho MA, Carrondo MJ, Alves PM, Bernardes GJ, Peixoto C. 2016. Bioorthogonal Strategy for Bioprocessing of Specific-Site-Functionalized Enveloped Influenza-Virus-Like Particles. Bioconjugate Chemistry. 2386–2399.  
  4. Cunha B, Silva RJ, Aguiar T, Serra M, Daicic J, Maloisel JL, Clachan J, Åkerblom A, Carrondo MJ, Peixoto C, Alves PM. 2016. Improving washing strategies of human mesenchymal stem cells using negative mode expanded bed chromatography. J Chromatogr A. 2016 Jan 15;1429:292-303. 
  5. Silva R, Mota JP, Peixoto C, Alves PM, Carrondo MJT. 2015. Improving the downstream processing of vaccine and gene therapy vectors with continuous chromatography. Pharmaceutical Bioprocessing 3(8): 489-505. 
  6. Nestola P, Peixoto C, Villain L, Alves PM, Carrondo MJT, Mota JPB. 2015. Rational development of two flowthrough purification strategies for adenovirus type 5 and retrovirus-like particles, Journal of Chromatography A. 
  7. Cunha B, Aguiar T, Silva MM, Silva R, Sousa MF, Pineda E, Peixoto C, Carrondo MJ, Serra M, Alves PM. 2015. Exploring continuous and integrated strategies for the up- and downstream processing of human mesenchymal stem cells. Journal of Biotechnology - special issue on integrated continuous biomanufacturing   
  8. Cunha B, Peixoto C, Silva M, Carrondo MJT, Serra M, Alves PM 2015. Filtration methodologies for the clarification and concentration of human mesenchymal stem cells. Journal of Membrane Science, 478:117-129. 
  9. Nestola P, Peixoto C, Silva RJS, Alves PM, Mota JPB, Carrondo MJT 2015. Improved virus purification processes for vaccines and gene therapy. Biotechnology and Bioengineering, 112(5):843-57. 
  10. Nestola P, Silva RJS, Peixoto C, Alves PM, Carrondo MJT, Mota JPB 2015. Robust design of adenovirus purification by two-column, simulated countercurrent, size-exclusion chromatography. Journal of Biotechnology.
  11. Nestola P, Martins DL, Peixoto C, Roederstein S, Schleuss T, Alves PM, Mota JPB, Carrondo MJ. 2014. Evaluation of Novel Large Cut-Off Ultrafiltration Membranes for Adenovirus Serotype 5 (Ad5) Concentration. PLoS ONE, 9(12):-115802. 
  12. Vlaming M, van Duijn E, Dillingh MR, Brands R, Windhorst AD, Hendrikse NH, Bosgra S, Burggraaf J, de Koning MC, Fidder A, Mocking J, Sandman H, de Ligt R, Fabriek BO, Pasman WJ, Seinen W, Alves T, Carrondo M, Peixoto C, Peeters P, Vaes W. 2015. Microdosing of a carbon-14 labeled protein in healthy volunteers accurately predicts its pharmacokinetics at therapeutic dosages. Clin Pharmacol Ther. Apr13. 
  13. Gonçalves CSM, Sousa M, Martins DL, Barroso T, Pina AS, Peixoto C, Ricardo A, Roque CA. 2015. Biobased Monoliths for Adenovirus Purification. ACS Appl. Mater. Interfaces, 7 (12), pp 6605–6612. 
  14. Nestola P, Silva RJS, Peixoto C, Alves PM, Carrondo MJT, Mota JPB. 2014. Adenovirus purification by two-column, size-exclusion, simulated countercurrent chromatography. Journal of Chromatography A,1347: 111–121.  
  15. Nestola P, Villain L, Peixoto C, Martins DL, Alves PM, Carrondo MJT, Mota JPB. 2014. Impact of grafting on the design of new membrane adsorbers for adenovirus purification. Journal of Biotechnology, 181: 1–11. 
  16. Fernandes P, Peixoto C, Santiago VM, Kremer EJ, Coroadinha AS, Alves PM, 2013. Bioprocess development for canine adenovirus type 2 vectors. Gene Ther. Apr;20(4):353-60. 
  17. Bandeira V, Peixoto C, Rodrigues AF, Cruz PE, Alves PM, Coroadinha AS, Carrondo MJ, 2012. Downstream processing of lentiviral vectors: releasing bottlenecks. Hum Gene Ther Methods, 23(4):255-63. 
  18. Vicente T, Mota JP, Peixoto C, Alves PM, Carrondo MJ, 2011. Rational design and optimization of downstream processes of virus particles for biopharmaceutical applications: current advances. Biotechnol Adv, 29: 869-78. 
  19. Lesch HP, Laitinen A, Peixoto C, Vicente T, Makkonen ME, Laitinen L, Pikkarainen JT, Samaranayake H, Alves PM, Carrondo MJ, Ylä-Herttuala S, Airenne KJ, 2011. Production and purification of lentiviral vectors generated in 293T suspension cells with baculoviral vectors. Gene Ther, 18:531-38. 
  20. Vicente T, Roldão A, Peixoto C, Carrondo MJ, Alves PM, 2011. Large-scale production and purification of VLP-based vaccines. J Invertebr Pathol, 107:S42-S48. 
  21. Vicente T, Fáber R, Alves PM, Carrondo MJ, Mota JP, 2011. Impact of ligand density on the optimization of ion-exchange membrane chromatography for viral vector purification. Biotechnol Bioeng, 108(6):1347-59. 
  22. Roldão A, Vicente T, Peixoto C, Carrondo MJ, Alves PM, 2011. Quality control and analytical methods for baculovirus based products. J Invertebr Pathol, 107:S94-S105. 
  23. Vicente T, Mota JP, Peixoto C, Alves PM, Carrondo MJ, 2010. Analysis of adsorption of a baculovirus bioreaction bulk on an ion-exchange surface by surface plasmon resonance. J Biotechnol, 148(4):171-81. 
  24. Vicente T, Mota JP, Peixoto C, Alves PM, Carrondo MJ, 2010. Modeling protein binding and elution over a chromatographic surface probed by surface plasmon resonance. J Chromatogr A, 1217:2032-41. 
  25. Vicente T, Peixoto C, Alves PM, Carrondo MJ, 2010. Modeling electrostatic interactions of baculovirus vectors for ion-exchange process development. J Chromatogr A, 1217:3754-64. 
  26. Vicente T, Peixoto C, Carrondo MJ, Alves PM, 2009. Purification of recombinant baculoviruses for gene therapy using membrane processes. Gene Ther, 16:766-775. 
  27. Mellado MC, Peixoto C, Cruz PE, Carrondo MJ, Alves PM, 2008. Purification of recombinant rotavirus VP7 for the study of in vitro rotavirus-like particles assembly. J Chromatogr B, 874:89-94. 
  28. Peixoto C, Ferreira TB, Sousa MF, Carrondo MJ, Alves PM, 2008. Towards purification of adenovirus vectors based on membrane technology. Biotechnol Prog, 24:1290-96. 
  29. Rodrigues T, Alves A, Lopes A, Carrondo MJ, Alves PM, Cruz PE, 2008. Removal of envelope protein-free retroviral vectors by anion exchange chromatography to improve product quality. J Sep Sci, 31:3509-18. 
  30. Mellado MC, Franco C, Coelho AV, Alves PM, Simplício AL, 2008. Sodium dodecyl sulfate-capillary gel electrophoresis analysis of rotavirus-like particles. J Chromatogr A, 1192:166-72. 
  31. Vicente T, Sousa MF, Peixoto C, Mota JP, Alves PM, Carrondo MJ, 2008. Anion exchange membrane chromatography for purification of rotavirus-like particles. J Membr Sci, 311:270-83. 
  32. Peixoto C, Marcelino I, Amaral AI, Carrondo MJ, Alves PM, 2007. Purification by membrane technology of an intracellular Ehrlichia ruminantium candidate vaccine against heartwater. Proc Biochem, 42(7):1084-89. 
  33. Rodrigues T, Carvalho A, Carmo M, Carrondo MJ, Alves PM, Cruz PE, 2007. Scaleable purification process for gene therapy retroviral vectors. J Gene Med, 9(4):233-43. 
  34. Peixoto C, Sousa MF, Silva AC, Carrondo MJ, Alves PM, 2007. Downstream processing of triple-layered rotavirus like particles. J Biotechnol, 127:452-61 
  35. Rodrigues T, Carrondo MJ, Alves PM, Cruz PE, 2007. Purification of retroviral vectors for clinical application: biological implication and technological challenges. J Biotechnol, 127:520-41. 
  36. Rodrigues T, Carvalho A, Roldao A, Carrondo MJ, Alves PM, Cruz PE, 2006. Screening anion-exchange chromatographic matrices for isolation of onco-retroviral vectors. J Chromatogr B, 837(1-2):59-68.
  37. Peixoto C, Ferreira TB, Carrondo MJ, Cruz PE, Alves PM, 2006. Purification of adenoviral vectors using expanded bed chromatography. J Virol Methods, 132:121-6. 
  38. Peixoto C, Marcelino I, Vachiéry N, Bensaid A, Martinez D, Carrondo MJ, Alves PM, 2005. Quantification of Ehrlichia ruminantium by real time PCR. Vet Microbiol, 107:273-8. 
  39. Souza AP, Peixoto CC, Maranga L, Carvalhal AV, Moraes RH, Mendonça RZ, Pereira CA, Carrondo MJ, Mendonça RZ, 2005. Purification and characterization of an anti-apoptotic protein isolated from Lonomia obliqua Hemolymph. Biotechnol Prog, 21:99-105. 
  40. Carmo M,  Peixoto CC, Coroadinha AS, Alves PM, Cruz PE, Carrondo MJ, 2004. Quantitation of MLV-based retroviral vectors using real-time RT-PCR. J Virol Methods, 119:115-9. 
  41. Maranga L, Rueda P, Antonis AF, Vela C, Langeveld JP, Casal JI, Carrondo MJ, 2002. Large scale production and downstream processing of a recombinant porcine parvovirus vaccine. Appl Microbiol Biotechnol, 59:45-50. 
  42. Cruz PE, Peixoto CC, Devos K, Moreira JL, Saman E, Carrondo MJ, 2000. Characterization and downstream processing of HIV-1 core and virus-like-particles produced in serum free medium. Enzyme Microb Technol, 26:61-70.