Proteins as Drugs: Analysis, Formulation and Delivery

  • C. R. Middaugh
  • R. Pearlman
Part of the Handbook of Experimental Pharmacology book series (HEP, volume 137)


The use of proteins as drugs is by no means new. Insulin, gamma-globulin and protein-containing vaccines have been routinely employed for decades. However, the advent of recombinant DNA technology has resulted in a dramatic expansion of interest in their pharmaceutical applications. It now appears that we can make virtually any desired protein in sufficient quantities for therapeutic use, although often with significant difficulty. It is considerably more problematic, however, to take the appropriate macromolecule and prepare it as a clinically acceptable drug substance. This problem arises from several sources. First, proteins are intrinsically less stable than their lower molecular weight (MW) pharmaceutical counterparts. Although this has turned out to be less of a problem than first anticipated, it remains a continuing challenge to formulate proteins that can be handled without damage throughout their entire lives; from their initial preparation, through distribution within the complex bio-medical system, into their ultimate clinical use in a hospital or doctor’s office or perhaps even in the home. Second, to make optimum use of a protein as a pharmaceutical agent, it is necessary to get it to the relevant in vivo site of action with maximum efficiency. A major potential power of proteins as therapeutic agents resides in their intrinsic compatibility with living systems. As critical components of virtually all biochemical processes, the presentation of a natural protein or one with specifically altered functional properties offers the opportunity to intervene in a pathological process with a high degree of specificity and minimal perturbation of normal processes.


Nuclear Magnetic Resonance Circular Dichroism Human Growth Hormone Protein Drug Luteinizing Hormone Release Hormone 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. Arakawa T, Kita Y, Carpenter JF (1991) Protein-solvent interactions in pharmaceuti¬cal formulations. Pharm Res 8: 285 – 291PubMedCrossRefGoogle Scholar
  2. Arakawa T, Prestrelski SJ, Kenney WC, Carpenter JF (1993) Factors affecting the short-term and long-term stabilities of proteins. Adv Drug Del Rev 10: 1 – 28CrossRefGoogle Scholar
  3. Amott D, Shabanowitz J, Hunt DF (1993) Mass spectrometry of proteins and peptides: sensitive and accurate mass measurement and sequence analysis. Clin Chem 39: 2005 – 2010Google Scholar
  4. Bax A (1991) Experimental NMR techniques for studies of biopolymers. Curr Opin Struct Biol 1: 1030 – 1035CrossRefGoogle Scholar
  5. Bechgaard E, J¿rgensen L, Larsen R, Gizurarson S, Carstensen J, Hvass (1993) Insulin and didecanoyl-L-a-phosphatidylcholine: in vitro study of the transport through rabbit nasal mucosal tissue. Int J Pharm 89: 147 – 154Google Scholar
  6. Becker GW, Tackitt PM, Bromer WW, Lefeber DS, Riggin RM (1988) Isolation and characterization of a sulfoxide and a desamido derivative of biosynthetic human growth hormone. Biotechnol Appi Biochem 10: 326 – 337Google Scholar
  7. Becker GW, Bowsher, RR, Mackellar WC, Poor ML, Tackitt PM, Riggin RM (1987) Chemical, physical, and biological characterization of a dimeric form of biosyn¬thetic human growth hormone. Biotechnol Appi Biochem 9: 478 – 487Google Scholar
  8. Bennett WF, Builder SE, Gatlin LA (1988) Stabilized human tissue plasminogen activator compositions. United States Patent Number 4,777, 043Google Scholar
  9. Billeci TM, Stults JT (1993) Tryptic mapping of recombinant proteins by matrix- assisted laser desorption/ionization mass spectrometry. Anal Chem 65: 1709 – 1716PubMedCrossRefGoogle Scholar
  10. Burke CJ, Steadman BL, Volkin DB, Tsai PK, Bruner MW, Middaugh CR (1992) The adsorption of proteins to container surfaces. Int J Pharm 86: 89 – 93CrossRefGoogle Scholar
  11. Burstein EA, Vedenkina NS, Ivkova MN (1973) Fluorescence and the location of tryptophan residues in protein molecules. Photochem Photobiol 18: 263 – 279PubMedCrossRefGoogle Scholar
  12. Carpenter JF, Prestrelski SJ, Arakawa T (1993) Separation of freezing- and drying- induced denaturation of Iyophilized proteins using stress-specific stabilization. I. Enzyme activity and calorimetric studies. Arch Biochem Biophys 303: 456 – 464PubMedCrossRefGoogle Scholar
  13. Carr SA, Hemling ME, Bean MF, Roberts GD (1991) Integration of mass spectrometry in analytical biochemistry. Anal Chem 63: 2802 – 2824PubMedCrossRefGoogle Scholar
  14. Chait BT, Kent SBH (1992) Weighing naked proteins: practical, high-accuracy mass measurement of peptides and proteins. Science 257: 1885 – 1894PubMedCrossRefGoogle Scholar
  15. Cipolla D, Gonda I, Shire S J (1994) Characterization of aerosols of human recombinant deoxyribonuclease I (rhDNase) generated by jet nebulizers. Pharm Res 11: 491 – 498PubMedCrossRefGoogle Scholar
  16. Claes P, Fowell S, Woollin C, Kenney A (1990) On-line molecular size Determination for protein chromatography. Am Lab 22: 58 – 62Google Scholar
  17. Cleary S, Mulkerrin M, Kelley R (1989) Purification and characterization of tissue plasminogen activator kringle-2 domain expressed in Escherichia coli. Biochemistry 28: 1844 – 1890CrossRefGoogle Scholar
  18. Cleland JL, Powell MF, Shire SJ (1993) The development of stable protein formulations: a close look at protein aggregation, deamidation, and oxidation. Crit Rev Therapeutic Drug Carrier Sys 10: 307 – 377Google Scholar
  19. Clore GM, Gronenborn AM (1994) Structures of larger proteins, protein-ligand and protein-DNA complexes by multidimensional heteronuclear NMR. Protein Sci 3: 372 – 390PubMedCrossRefGoogle Scholar
  20. Dabora JM, Sanyal G, Middaugh CR (1991) Effects of polyanions on the refolding of human acidic fibroblast growth factor. J Biol Chem 266: 23637 – 23640PubMedGoogle Scholar
  21. Davis SS (1990) Overcoming barriers to the oral administration of peptide drugs. Trends Pharmacol Sci 11: 353 – 355PubMedCrossRefGoogle Scholar
  22. The Diabetes Control and Complications Trial Research Group (1993) The effect of intensive treatment of diabetes on the development and progression of long-term complications in insulin-dependent diabetes mellitus. New Engl J Med 329: 977 – 986Google Scholar
  23. Dollinger G, Cunico B, Kunitani M, Johnson D, Jones R (1992) Practical on-line determination of biopolymer molecular weights by high-performance liquid chromatography with classical light-scattering detection. J Chromatogr 592: 215 – 228CrossRefGoogle Scholar
  24. Ealick SE, Walter RL (1993) Synchroton beamlines for macromolecular crystallography. Curr Opin Struct Biol 3: 725 – 736CrossRefGoogle Scholar
  25. Ebel JP (1990) A method for quantifying particle absorption from the small intestine. Pharm Res 7: 848 – 851PubMedCrossRefGoogle Scholar
  26. Edman P, Bjsrk (1992) Nasal delivery of peptide drugs. Adv Drug Delivery Rev 8: 165 – 178CrossRefGoogle Scholar
  27. Eppstein DA, Schryver BB (1991) Controlled release of macromolecular peptides. United States Patent Number 4,962, 091Google Scholar
  28. Faure P, Micu A, Perahia D, Doucet J, Smith JC, Benoit JP (1994) Correlated intramo-lecular motions and diffuse X-ray scattering in Iysozyme. Struct Biol 1: 124 – 128CrossRefGoogle Scholar
  29. Feng R, Konishi Y (1993) Stepwise refolding of acid-denatured myoglobin: Evidence from electrospray mass spectrometry. J Am Soc Mass Spectrom 4: 638 – 645CrossRefGoogle Scholar
  30. Fersht AR, Jackson SE, Serrano L (1993) Protein stability: experimental data from protein engineering. Philos Trans R Soc London, Ser A 345: 141 – 151Google Scholar
  31. Fontana A (1991) Analysis and modulation of protein stability. Curr Opin Biotechnol 2: 551 – 560PubMedCrossRefGoogle Scholar
  32. Franks F (1990) Freeze drying: from empiricism to predictability. Cryo-Letters 11: 93 – 110Google Scholar
  33. Frenz J, Shire SJ, Slikowski MB (1994) Purified forms of DNase. United States Patent Number 5,279, 823Google Scholar
  34. Gombotz WR, Healey MS, Brown LR (1991) Very low temperature casting of con-trolled release microspheres. United States Patent Number 5,019, 400Google Scholar
  35. Gombotz WR, Pankey SC, Phan D, Drager R, Donaldson K, Antonsen KP, Hoffman AS, Raff HV (1994) The stabilization of a human IgM monoclonal antibody with poly(vinylpyrrolidone). Pharm Res 11: 624 – 632PubMedCrossRefGoogle Scholar
  36. Granzow R, Reed R (1992) Interactions in the fourth dimension. Bio/Technology 10: 390 – 393PubMedCrossRefGoogle Scholar
  37. Guzzetta AW, Basa LJ, Hancock WS, Keyt, Bennett NNT (1993) Identification of carbohydrate structures in glycoprotein peptide maps by the use of LC/MS with selected ion extraction and special reference to tissue plasminogen activator and glycosylation variant produced by site directed mutagenesis. Anal Chem 65: 2953 – 2962PubMedCrossRefGoogle Scholar
  38. Harris RJ, van Halbeek H, Glushka J, Basa LJ, Ling VT, Smith KJ, Spellman MW (1993) Identification and structural analysis of the tetrasaccharide NeuAca(2→6)Galβ (1→4)GlcNacβ (1→3)Fucα1O-linked to Serine 61 of human factor IX. Biochemistry 32: 6539 – 6547PubMedCrossRefGoogle Scholar
  39. Hovgaard L, Mack EJ, Kim SW (1992) Insulin stabilization and GI absorption. J Controlled Release 19: 99 – 108CrossRefGoogle Scholar
  40. Hsu CC, Ward CA, Pearlman R, Nguyen HM, Yeung DA, Curley JA (1991) Determining the optimum residual moisture in lyophilized protein pharmaceuticals in: Developments in Biological Standardization: International Symposium on Biological Product Freeze- Drying and Formulation, Vol. 74, Karger, Basel, pp 255 – 271Google Scholar
  41. Hsu CC, Pearlman R, Bewley TA, Yeung DA, Koe GS, Brooks DA, Nguyen HM (1995) The effect of freezing rate during lyophilization on the stability of tissue type plasminogen activator. Pharm Res 12: 69 – 77PubMedCrossRefGoogle Scholar
  42. Jian L, Li Wan Po A (1993) Effects of insulin and nasal absorption enhancers on ciliary 23640 activity. Int J Pharm 95: 101 – 104CrossRefGoogle Scholar
  43. Johnson WC Jr (1990) Protein secondary structure and circular dichroism: a practical guide. Proteins: Struct, Fune, Genet 7: 205 – 214Google Scholar
  44. Jones AJS (1993) Analysis of polypeptides and proteins. Adv Drug Delivery Rev 10: 29 – 90CrossRefGoogle Scholar
  45. Jones AJS, Nguyen T, Cleland JL, Pearlman R (1994) New delivery systems for recombinant proteins - practical issues from proof of concept to clinic. In: Lee VHL, Hashida M, Mizushima M (eds) Trends and future perspectives in peptide and protein drug delivery. Drug targeting and delivery, Harwood Academic Publishers, Gmbh, Amsterdam, The NetherlandsGoogle Scholar
  46. Laube BL, Georgopoulos A, Adams GK (1993) Preliminary study of the efficacy of aerosolized insulin delivered by oral inhalation in diabetic patients. JAMA 269: 2106 – 2109PubMedCrossRefGoogle Scholar
  47. Lasky S J, Buttry DA (1990) Development of a real-time glucose biosensor by Enzyme immobilization on the quartz crystal microbalance. Am Biotech Lab 2: 8 – 16Google Scholar
  48. Lee VHL (ed) (1990) Peptide and protein drug delivery, Marcel Dekker, Inc., New YorkGoogle Scholar
  49. Mach H, Volkin DB, Middaugh CR (1995) Spectroscopic techniques to study protein folding: ultraviolet absorbance spectroscopy. In: Shirley BA (ed) Methods in Molecular Biology: Protein Stability and Folding Protocols 40: 91 – 114Google Scholar
  50. Mashida M, Sano K, Arakawa M, Hayashi M, Awazu S (1993) Absorption of recombinant human granulocyte colony-stimulating factor (rhG-CSF) from rat nasal mucosa. Pharm Res 10: 1372 – 1377CrossRefGoogle Scholar
  51. McConnell HM, Owicki JC, Parce JW, Miller DL, Baxter GT, Wada H G, Pitchford S (1992) The cytosensor microphysiometer: biological applications of silicon technology. Science 257: 1906 – 1912PubMedCrossRefGoogle Scholar
  52. Middaugh CR (1990) Biophysical approaches to the pharmaceutical development of proteins. Drug Develop Ind Pharm 16: 2635 – 2654CrossRefGoogle Scholar
  53. Mirza UA, Cohen SL, Chait BT (1993) Heat-induced conformational changes in proteins studied by electrospray ionization mass spectrometry. Anal Chem 65: 1 – 6PubMedCrossRefGoogle Scholar
  54. Mori A, Kennel S J, Huang L (1993) Immunotargeting of liposomes containing lipophilic antitumor prodrugs. Pharm Res 10: 507 – 514PubMedCrossRefGoogle Scholar
  55. Morishita M, Morishita I, Takayama K, Machida Y, Nagai T (1992a) Novel oral microspheres of insulin with protease inhibitor protecting from enzymatic degra¬dation. Int J Pharm 78: 1 – 8CrossRefGoogle Scholar
  56. Morishita I, Morishita M, Takayama K, Machida Y, Nagai T (1992b) Hypoglycemic effect of novel oral microspheres of insulin with protease inhibitor in normal and diabetic rats Int J Pharm 78: 9 – 16Google Scholar
  57. Morris W, Steinhoff MC, Russell PK (1994) Potential of Polymer Microencapsulation technology for vaccine innovation. Vaccine 12: 5 – 11PubMedCrossRefGoogle Scholar
  58. Nellans HN (1991) Mechanisms of peptide and protein absorption. Paracellular intestinal transport: modulation of absorption. Adv Drug Delivery Rev 7: 339 – 364CrossRefGoogle Scholar
  59. Niven RW, Lott FD, Cribbs JM (1993) Pulmonary absorption of recombinant methionyl human granulocyte colony stimulating factor r-hu-G-CSF after intratracheal instillation to the hamster. Pharm Res 10: 1604 – 1610PubMedCrossRefGoogle Scholar
  60. Nguyen TH, Ward C (1993) Stability characterization and formulation development of Alteplase, a recombinant tissue type plasminogen activator. In: Wang YJ, Pearlman R (eds) Stability and characterization of protein and peptide drugs: Case Histories, Plenum Press, New York, pp 91 – 134Google Scholar
  61. Okada H, Ogawa Y, Yashiki T (1987) Prolonged release microcapsule and its production. United States Patent Number 4,652, 441Google Scholar
  62. Okada H, Heya T, Igari Y, Yamamoto M, Ogawa Y, Toguchi H, Shimamato T (1989) One- month-release injectable microspheres of a superactive agonist of LHRH, leuprolide acetate. In: Therapeutic Peptides and Proteins. Formulation, Delivery and Targeting, Marshak D, Liu D (eds) Cold Spring Harbor Laboratory, NY, pp 107 – 112Google Scholar
  63. Okada H, Yamamoto M, Heya T, Inoue Y, Kamei S, Ogawa Y, Toguchi H (1994) Drug delivery using degradable microspheres. J Controlled Release 28: 121 – 130CrossRefGoogle Scholar
  64. Okumura K, Iwakawa S, Yoshida T, Seki T, Komada F (1992) Intratracheal delivery of insulin. Absorption from solution and aerosol by rat lung. Int J Pharm 88: 63 – 74CrossRefGoogle Scholar
  65. Patel DG, Ritschel WA, Chalasani P, Rao S (1991) Biological activity of insulin in microemulsion in mice. J Pharm Sci 80: 613 – 614PubMedCrossRefGoogle Scholar
  66. Patton JS, Platz RM (1992) Pulmonary delivery of peptides and proteins for Systemic action. Adv Drug Delivery Rev 8: 179 – 196CrossRefGoogle Scholar
  67. Pearlman R, Bewley TA (1993) Stability and characterization of human growth hormone. In: Wang YJ, Pearlman R (eds) Stability and characterization of protein and peptide drugs: case histories, Plenum Press, NY, pp 1 – 58Google Scholar
  68. Pearlman R, Nguyen T (1992) Pharmaceutics of protein drugs. J Pharm Pharmacol 44: 178 – 186PubMedGoogle Scholar
  69. Pearlman R, Oeswein JQ (1992) Human growth hormone formulation. United States Patent Number 5,096, 885Google Scholar
  70. Peppas NA, Langer R (1994) New challenges in biomaterials. Science 263:1715–1720Google Scholar
  71. Pflugrath JW (1992) Developments in X-ray detectors. Curr Opin Struct Biol 2: 811 – 815CrossRefGoogle Scholar
  72. Pikal MJ, Dellerman KM, Roy ML, Riggin RM (1991) The effects of formulation variables on the stability of freeze-dried human growth hormone. Pharm Res 8: 427 – 436PubMedCrossRefGoogle Scholar
  73. Porter CJH, Moghimi SM, Davies MC, Davis SS, Ilum L (1992) Differences in the molecular weight profile of poloxamer 407 affect its ability to redirect intra¬venously administered colloids. Int J Pharm 83: 273 – 276CrossRefGoogle Scholar
  74. Prestrelski S J, Arakawa T, Carpenter JF (1993) Separation of freezing- and drying- induced denaturation of lyophilized proteins using stress specific stabilization. II. Structural studies using infrared spectroscopy. Arch Biochem Biophys 303: 465 – 473PubMedCrossRefGoogle Scholar
  75. Pusztai A (1989) Transport of proteins through the membranes of the adult gastro-intestinal tract - a potential for drug delivery? Adv Drug Delivery Rev 3: 215 – 228CrossRefGoogle Scholar
  76. Saltzman WM (1993) Antibodies for treating and preventing disease: the potential role of polymeric controlled release. Crit Rev Therapeutic Drug Carrier Syst 10: 142Google Scholar
  77. Saudek CD (1993) Future developments in insulin delivery systems. Diabetes Care 16 (Suppl 3): 122 – 132PubMedGoogle Scholar
  78. Smith PL, Wall DA, Gochoco CH, Wilson G (1992) Oral absorption of peptides and proteins. Adv Drug Delivery Rev 8: 253 – 290CrossRefGoogle Scholar
  79. Smith PAG, Dewdney JM, Fears R, Poste G (1993) Chemical derivatization of thera¬peutic proteins. Trends Biotech 11: 397 – 403CrossRefGoogle Scholar
  80. Spellman M, Basa L, Leonard C, Chakel J, O’Connor J (1989) Carbohydrate structures of human tissue plasminogen activator expressed in Chinese hamster ovary cells. J Biol Chem 264: 14100 – 14111PubMedGoogle Scholar
  81. Surewicz WK, Mantsch HH, Chapman D (1993) Determination of protein secondary structure by Fourier transform infrared spectroscopy: a critical assessment. Biochemistry 32: 389 – 394PubMedCrossRefGoogle Scholar
  82. Tabata Y, Gutta S, Langer R (1993) Controlled delivery systems for proteins using polyanhydride microspheres. Pharm Res 10: 487 – 496PubMedCrossRefGoogle Scholar
  83. Talmadge JE (1993) The pharmaceutics and delivery of therapeutic polypeptides and proteins. Adv Drug Delivery Rev 10: 247 – 300CrossRefGoogle Scholar
  84. Thurow H, Geisen (1984) Stabilisation of dissolved proteins against denaturation at hydrophobic surfaces. Diabetologia 27: 212 – 218PubMedGoogle Scholar
  85. Tsai PK, Volkin DB, Dabora, JM, Thompson, KC, Bruner MW, Gress JO, Matuszewska B, Keogan M, Bondi JV, Middaugh CR (1993) Formulation design of acidic fibroblast growth factor. Pharm Res 10: 649 – 659PubMedCrossRefGoogle Scholar
  86. Volkin DB, Middaugh CR (1992) The effect of temperature on protein structure. In: Ahern TJ, Manning MC (eds) Stability of protein pharmaceuticals, Part A: Chemical and physical pathways of protein degradation, Plenum Press, NY, pp 215 – 247Google Scholar
  87. Volkin DB, Tsai PK, Dabora JM, Gress JO, Burke CJ, Linhardt RJ, Middaugh CR (1993) The stabilization of acidic fibroblast growth factor by poly anions. Arch Biochem Biophys 300: 30 – 41PubMedCrossRefGoogle Scholar
  88. Volkin DB, Burke C, Marfia K, Middaugh R, Oswald B, Hennessey J, Orella C, Hagan A, Sitrin R, Oliver C (1997) Biophysical characterization of hepatitis A virus (HAV). J Pharm Sci 86: 666 – 673PubMedCrossRefGoogle Scholar
  89. Wyatt PJ, Papazian LA (1993) The interdetector volume in modern light scattering and high performance size-exclusion chromatography. LC-GC 11: 862 – 872Google Scholar
  90. Wang Y-CJ, Hanson MA (1988) Parenteral formulations of proteins and peptides: stability and stabilizers. J Parent, Sci Tech 42 Suppl: 53 – 526Google Scholar
  91. Wang YJ, Pearlman R (eds) (1993) Stability and characterization of protein and peptide drugs: Case histories, Plenum Press, NYGoogle Scholar
  92. Wearley L (1991) Recent progress in protein and peptide delivery by non invasive routes. Crit Rev Therapeutic Drug Carrier Systems 8: 331 – 394Google Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 1999

Authors and Affiliations

  • C. R. Middaugh
  • R. Pearlman

There are no affiliations available

Personalised recommendations