566 METHOD VALIDATION scrutiny of any reviewer. The contents of this chapter concentrate on pharmaceutical methods, but the same principles can be applied to any HPLC method so as to ensure that it is suitable for its intended use REFERENCES 1. Guideline for Submitting Samples and Analytical Data for Methods Validation, USFDA-CDER (February 1987), http://www.fda.gov/cder/guidance/ameth.htm. 2. United States Pharmacopeia No. 31-NF 26, (2008), ch. 1225. 3. Analytical Procedures and Methods Validation, USFDA-CDER (Aug. 2000), http://www.fda.gov/cder/guidance/2396dft.htm. 4. Harmonized Tripartite Guideline, Validation of Analytical Procedures, Text and Methodology, Q2 (R1), International Conference on Harmonization, (Nov. 2005), http://www.ich.org/LOB/media/MEDIA417.pdf. 5. Guidance for Methods Development and Methods Validation for the Resource Con- servation and Recovery Act (RCRA) Program, US EPA, (1995), http://www.epa.gov/ epawaste/hazard/testmethods/pdfs/methdev.pdf. 6. ISO/IEC 17025, General Requirements for the Competence of Testing and Calibration Laboratories, (2005), http://www.iso.org/iso/iso catalogue/catalogue tc/ catalogue detail.htm?csnumber=39883. 7. Current Good Manufacturing Practice in Manufacturing, Processing, Packing, or Holding Of Drugs, 21 CFR Part 210, http://www.fda.gov/cder/dmpq/cgmpregs.htm. 8. Current Good Manufacturing Practice for Finished Pharmaceuticals, 21 CFR Part 211, http://www.fda.gov/cder/dmpq/cgmpregs.htm. 9. International Organization for Standardization, http://www.iso.org/iso/home.htm. 10. Reviewer Guidance, Validation of Chromatographic Methods, USFDA (November 1994) http://www.fda.gov/cder/guidance/cmc3.pdf. 11. L. R. Snyder, J. J. Kirkland, and J. L. Glajch, Practical HPLC Method Development, 2nd ed., Wiley-Interscience, New York, 1997. 12. United States Pharmacopeia No. 31-NF 26, (2008), ch. 621. 13. V. P. Shah, K. K. Midha, and S. V. Dighe, Pharm. Res., 9 (1992) 588. 14. V. P. Shah, K. K. Midha, J. W. A. Findlay, H. M. Hill, J. D. Hulse, I. J. McGilvaray, G. McKay, K. J. Miller, R. N. Patnaik, M. L. Powell, A. Tonelli, C. T. Viswanathan, and A. Yacobi, Pharm. Res., 17 (2000) 1551. 15. C. T. Viswanathan, S. Bansal, B. Booth, A. J. DeStafano, M. J. Rose, J. Sailstad, V. P. Shah, J. P. Skelly, P. G. Swann, and R. Weiner, AAPS J., 9(1), (2007) E30. See also: www.aapsj.org. 16. Guidance for Industry, Bioanalytical Method Validation, USFDA-CDER (May 2001), http://www.fda.gov/cder/guidance/4252fnl.pdf. 17. ISPE Good Practice Guide: Technology Transfer, ISPE, Tampa, FL (Mar. 2003), http://www.ispe.org/cs/ispe good practice guides section/ispe good practice guides. 18. PhRMA Analytical Research and Development Workshop, Wilmington DE, 20 Sept. 2000. 19. S. Scypinski, D. Roberts, M. Oates, and J. Etse, Pharm. Tech., (Mar. 2004) 84. 20. J. C. Miller and J. N. Miller, Statistics for Analytical Chemistry, Ellis Horwood, Chichester, UK, 1986. REFERENCES 567 21. NIST/SEMATECH e-Handbook of Statistical Methods, http://www.itl.nist.gov/div898/ handbook. 22. P. C. Meier and R. E. Zund, Statistical Methods in Analytical Chemistry, Wiley, New York, 1993. 23. M. Swartz and R. Plumb, unpublished results. 24. H. Pappa and M. Marques, presentation at USP Annual Scientific Meeting, Denver, 28 September 2006. See also: http://www.usp.org/USPNF/columns.html. 25. M. E. Swartz and I. S. Krull, LCGC, 23 (2005) 1100. 26. Pharmacopeial Forum, 31(2) (Mar.–Apr. 2005) 555. 27. FDA ORA Laboratory Procedure, ORA-LAB.5.4.5, USFDA (09/09/ 2005). See also: http://www.fda.gov/ora/science ref/lm/vol2/section/5 04 05.pdf. 28. W. B. Furman, J. G. Dorsey, and L. R. Snyder, Pharm. Technol., 22(6) (1998) 58. 29. Pharmacopeial Forum, 31(3) (May–Jun. 2005) 825. 30. Pharmacopeial Forum, 31(6) (Nov.–Dec. 2005) 1681. 31. M. E. Swartz and I. S. Krull, LCGC, 23 (2005) 46. 32. M. E. Swartz, unpublished data on the analysis of tricyclic amines at pH-7.2. 33. M. E. Swartz and I. S. Krull, LCGC, 24 (2006) 770. CHAPTER THIR TEEN BIOCHEMICAL AND SYNTHETIC POLYMER SEPARATIONS with Timothy Wehr, Carl Scandella, and Peter Schoenmakers 13.1 BIOMACROMOLECULES, 570 13.2 MOLECULAR STRUCTURE AND CONFORMATION, 571 13.2.1 Peptides and Proteins (Polypeptides), 571 13.2.2 Nucleic Acids, 574 13.2.3 Carbohydrates, 576 13.2.4 Viruses, 578 13.3 SPECIAL CONSIDERATIONS FOR BIOMOLECULE HPLC, 579 13.3.1 Column Characteristics, 579 13.3.2 Role of Protein Structure in Chromatographic Behavior, 583 13.4 SEPARATION OF PEPTIDES AND PROTEINS, 584 13.4.1 Reversed-Phase Chromatography (RPC), 584 13.4.2 Ion-Exchange Chromatography (IEC) and Related Techniques, 597 13.4.3 Hydrophobic Interaction Chromatography (HIC), 608 13.4.4 Hydrophilic Interaction Chromatography (HILIC), 613 13.4.5 Multidimensional Liquid Chromatography (MDLC) in Proteomics, 616 13.5 SEPARATION OF NUCLEIC ACIDS, 618 13.5.1 Anion-Exchange Chromatography, 619 13.5.2 Reversed-Phase Chromatography, 620 13.5.3 Hydrophobic Interaction Chromatography, 624 13.6 SEPARATION OF CARBOHYDRATES, 625 13.6.1 Hydrophilic Interaction Chromatography, 625 13.6.2 Ion-Moderated Partition Chromatography, 626 13.6.3 High-Performance Anion-Exchange Chromatography, 628 Introduction to Modern Liquid Chromatography, Third Edition, by Lloyd R. Snyder, Joseph J. Kirkland, and John W. Dolan Copyright © 2010 John Wiley & Sons, Inc. 569 570 BIOCHEMICAL AND SYNTHETIC POLYMER SEPARATIONS 13.7 SEPARATION OF VIRUSES, 630 13.8 SIZE-EXCLUSION CHROMATOGRAPHY (SEC), 631 13.8.1 SEC Retention Process, 632 13.8.2 Columns for Gel Filtration, 633 13.8.3 Mobile Phases for Gel Filtration, 636 13.8.4 Operational Considerations, 637 13.8.5 Advantages and Limitations of SEC, 638 13.8.6 Applications of SEC, 639 13.9 LARGE-SCALE PURIFICATION OF LARGE BIOMOLECULES, 641 13.9.1 Background, 641 13.9.2 Production-Scale Purification of rh-Insulin, 642 13.9.3 General Requirements for Prep-LC Separations of Proteins, 648 13.10 SYNTHETIC POLYMERS, 648 13.10.1 Background, 648 13.10.2 Techniques for Polymer Analysis, 651 13.10.3 Liquid-Chromatography Modes for Polymer Analysis, 653 13.10.4 Polymer Separations by Two-Dimensional Chromatography, 657 13.1 BIOMACROMOLECULES Since liquid chromatography was first developed, it has been an important tool for the isolation and characterization of biomolecules. However, the extension of HPLC to the successful separation of biopolymers such as polypeptides, nucleic acids, and carbohydrates required the development of column packings that were tailored for these molecules. This chapter will concentrate on the HPLC separation of these three most important classes of biomacromolecules, with an emphasis on analytical and semipreparative applications. We can assume that the general principles of HPLC separation for ‘‘small’’ molecules apply equally to the separation of biopolymers. However, the size and structure of a biomolecule lead to some important differences that will be examined in this chapter. As an introduction to the present chapter, the reader is encouraged to first review relevant earlier chapters, especially Chapter 2 on basic concepts and the control of separation, and Chapter 9 on gradient elution. The primary chromatographic modes for the low-pressure separation of biomacromolecules have been ion exchange, size exclusion, hydrophobic inter- action, metal chelate, and affinity chromatography; the HPLC versions of the first four techniques will be discussed here. For a detailed discussion of affinity chro- matography, see [1]. In addition reversed-phase HPLC (RPC) has been hugely 13.2 MOLECULAR STRUCTURE AND CONFORMATION 571 successful in the separation and characterization of peptides, and it serves as one of the major analytical tools for the development and characterization of protein-based biopharmaceuticals. The RPC separation of peptides and proteins will therefore be a major topic in this chapter. For more general guidelines for the preparative separation of all samples, see Chapter 15. 13.2 MOLECULAR STRUCTURE AND CONFORMATION Macromolecules found in living cells are polymers consisting of subunits of similar chemical properties, such as amino acids, nucleotides, and sugars. The amino-acid sequence of proteins and the nucleotide sequences of RNA and DNA are precisely specified by the genetic code. In contrast, the carbohydrate sequences in glycoprotein side chains are determined by the specificity of the biosynthetic enzyme systems and the availability of substrates, so they may be more variable with respect to structure and sites of attachment on the polypeptide backbone. The properties of the assembled polymer depend on the properties of the individual subunits, as well as how they are positioned within the molecule. These two aspects of biopolymer organization (sub- unit properties and three-dimensional structure) influence both biological function and chromatographic behavior. Although it was earlier thought that the chromatog- raphy of biopolymers depends on different principles than for small molecules, it has been shown that biopolymers interact chromatographically in the same manner as small molecules, albeit with complexities introduced by polymer size, folding state, and three-dimensional structure [2, 3]. These macromolecules, proteins in particular, show complex behavior in solution with respect to their structure, stability, and aggregation state. This behavior restricts the choice of chromatographic conditions. 13.2.1 Peptides and Proteins (Polypeptides) The fundamental subunits of polypeptides are amino acids, each of which consists of a carboxylic acid group, an amino group, and a side chain (Fig. 13.1). Amino acids differ in their side chains, which can be neutral and hydrophilic (e.g., serine, threonine), neutral and hydrophobic (e.g., leucine, phenylalanine), acidic (aspartic acid, glutamic acid), or basic (lysine, arginine, histidine). In polypeptide biosynthesis the carboxyl group of one amino acid (or residue) is linked to the amino group of the next amino acid with loss of water to form an amide or peptide bond (–CONH–). Of special interest is the amino acid cysteine, whose side-chain –SH group can be linked to that of another cysteine to form a disulfide bond (–SS–). Also noteworthy is the imidazole group of histidine, which can form coordination complexes with metal cations. The structures of the 20 common protein amino acids are shown in Figure 13.1, with their single- and three-letter codes, and the pKa values of the ionogenic side chains. 13.2.1.1 Primary Sequence This comprises the sequence of amino acids in the molecule (Fig. 13.2a). Peptides consist of 40 amino acids or less, with a mass of no more than about 5000 Da. Proteins are larger polypeptide chains that contain up to several hundred amino acids, with masses from 5000 to 250,000 Da or greater. Peptides with fewer than 15 572 BIOCHEMICAL AND SYNTHETIC POLYMER SEPARATIONS Acidic The Common Amino Acids 3.9 Aspartic acid (Asp, D) Lysine (Lys, K) Arginine (Arg, R) Glycine (Gly, G) Alanine (Ala, A) Valine (Val, V) Leucine (Leu, L) Asparagine (Asp, N) Glutamine (Gln, Q) Isoleucine (Ile, I) Histidine (His, H) Phenylalanine (Phe, F) Tryptophan (Trp, W) Tyrosine (Tyr, Y) Glutamic acid (Glu, E) Proline (Pro, P) Serine (Ser, S) Threonine (Thr, T) Cysteine (Cys, C) Methionine (Met, M) 4.3 pK a = pK a = 1.8-2.6 pK a = 8.8-10.8 10.8 12.5 6.0 pK a = Basic Aliphatic Imine Aliphatic alcohol Sulfur containing Amides Aromatic HO NH 2 O OH O NH 2 OH O HO NH 2 OH O H 2 N H 2 N NH 2 OH O NH HN NH NH 2 OH O N NH 2 OH O NH 2 OH O NH NH 2 OH O HO NH 2 R1 OH O α NH 2 OH O NH 2 OH O NH 2 OH O NH 2 OH O NH 2 OH O NH OH O HO NH 2 OH O NH 2 OH OHO HS NH 2 OH O NH 2 OH O S NH 2 NH 2 O OH O NH 2 OH O NH 2 O Figure 13.1 Structures of the amino acids commonly found in proteins. The amino acids are divided into groups according to the chemical properties of the side chains. The pK a values for the ionogenic side chains are shown for acidic and basic amino acids. Adapted from [7]. 13.2 MOLECULAR STRUCTURE AND CONFORMATION 573 Protein Structural Heirarchies (a) Primary Structure (b) Secondary Structure H 2 N-Asp-Glu-Phe-Arg-Asp-Ser Gly-Tyr-Glu-Val-His-Gln-Lys-Leu-COOH (c) Tertiary Structure (d) Quaternary Structure Figure 13.2 Polypeptide structures. (a) Linear arrangement of amino acids in a polypeptide determines the primary structure. (b) Arrangement of amino acids of a 14-residue alanine homo-oligomer as an α-helical secondary structure, showing representation as a stick figure, and with only the backbone shown, overlain with a ribbon representation of the helix. (c)Rib- bon diagram of the backbone of the hemoglobin β-subunit. (d ) Schematic representation of the multi-sub-unit enzyme catalase. Adapted from [7, 8]. amino acid residues exist in solution as random coils, and they behave substantially like small organic molecules in chromatography. As peptide length begins to exceed 15 residues, molecular folding introduces increasing structure, as described below. 13.2.1.2 Secondary Structure The spontaneous intramolecular interactions of a polypeptide during biosynthesis results in a secondary structure in which the three-dimensional shape of the final molecule is determined. Examples of the secondary structure (Fig. 13.2b) include the α-helix, which is stabilized by hydrogen bonds between residues located at intervals of about four amino acids along the primary sequence, and the β-sheet, which forms by hydrogen bonding between adjacent linear segments of primary sequence. 574 BIOCHEMICAL AND SYNTHETIC POLYMER SEPARATIONS 13.2.1.3 Tertiary and Quaternary Structure The final folded structure of a single polypeptide chain is the tertiary structure, which may consist of combinations of helices, β-sheets, turns, and random coil sections (Fig. 13.2c). Combinations of secondary-structure elements may exist as domains, the fundamental units of tertiary structure; each domain contains an individual hydrophobic core built from secondary structural units. The tertiary structure is stabilized by the summation of a great number of weak interactions, including hydrogen bonding, ionic bonds, and hydrophobic forces. In addition the tertiary structure may depend on disulfide bonds between cysteine residues, which can covalently join remote segments of the primary sequence. Quaternary structure represents the association of two or more folded protein chains to form a complex (13.2d) and depends on the same interactions involved in tertiary structure. The association of protein subunits (and conformational changes within the subunits) often plays a functional role in the regulation of protein activity. Similarly protein aggregation can be altered by the binding of substrates and small-molecule effectors. Denaturation refers to both a functional and a physical change in the state of the native (bioactive) protein. Functionally, denaturation results in a loss of biological activity. Physically, denaturation occurs when the folding state of protein is altered or abolished, resulting in loss of secondary and higher order structures. Denatured proteins in a random-coil state often form aggregates that precipitate from solution. The environment of the protein molecule (either dissolved in the mobile phase or bound to the stationary phase) is a common cause of denaturation. Denaturation with loss of secondary, tertiary, and quaternary structure commonly occurs during RPC, but is less likely in ion-exchange, hydrophobic interaction, or size-exclusion chromatography. 13.2.1.4 Post-translational Modifications A protein’s primary sequence, which is a direct reflection of the nucleotide sequence in its associated gene, largely determines folding. However, many proteins are modified after translation (the initial creation of the protein) by the addition of one or more groups, and these post-translational modifications (PTMs) are not inferable from the gene sequence. The same gene sequence may direct the synthesis of proteins with different PTMs when expressed in different cells. A huge variety of PTMs have been described, but the most frequent are addition of sugar groups to the side chains of serine, threonine, or asparagine residues (glycosylation) and phosphorylation of serine, threonine, or tyrosine groups. Some PTMs are important biologically because they are involved in the regulation of protein function, in signal transduction, and in receptor-ligand interactions, while others result from mistreatment of the protein during isolation and handling. From a separation standpoint, the presence of PTMs may alter the interaction of a protein with a chromatographic surface and its retention. 13.2.2 Nucleic Acids 13.2.2.1 Single-Stranded Nucleic Acids Single-stranded nucleic acids consist of a linear chain of nucleotides (Fig. 13.3), with each nucleotide consisting of a purine (adenine or guanine) or pyrimidine base 13.2 MOLECULAR STRUCTURE AND CONFORMATION 575 RNA DNA (a)(b)Oligonucleotide composition B2 B2 B2 B1 B1 B1 O O O O O SS S P P O O O O O O O O O O O O P H 3 C Common nucleobases Methylphosphonate Phosphorothioate Phosphorodithioate (c) Backbone-modified oligonucleotides B1 B2 O OH P P O O O O O O O O O NH NH 2 NH 2 NH 2 N N N N N N NH NH NH NH NH NH NH O O O O O O Adenine Guanine Thymine Cytosine Uracil Figure 13.3 Structure of nucleic acids. (a) Schematic composition of a single-stranded oligonucleotide; in RNA the 2 ribose position is hydroxylated (circled), whereas it is not in DNA. B1 and B2 represent the nucleobases, shown in (b). Adapted from [7]. (cytosine or thymine for DNA, cytosine or uracil for RNA) (Fig. 13.3b) linked to the C-1 carbon of ribose (RNA) or deoxyribose (DNA) (Fig. 13.3a). Nucleotide residues are linked through phosphodiester bonds between the 3 hydroxyl of one nucleotide and the 5 hydroxyl of the successive nucleotide. Oligonucleotides are short (usually single-stranded) nucleic acids, typically 13 to 25 bases in length, although lengths of 100 bases are sometimes referred to as oligonucleotides. Backbone-modified oligonucleotides (Fig. 13.3c) are synthetic derivatives used in ‘‘antisense’’ therapy, where the modified compound is able to combine with and deactivate the messenger RNA associated with a pathogen—because of the complementarity of the two molecular entities (as in following Section 13.2.2.2). 13.2.2.2 Double-Stranded Nucleic Acids These consist of two complementary polynucleotide chains in a helical structure, with both chains coiled around a common axis, and with the two chains oriented in opposite directions (Fig. 13.4). Bases attached to the external sugar-phosphate backbone are situated inside the helix and participate in specific, interchain hydro- gen bonds, with adenine (A) pairing with thymine (T) or uracil (U), and guanine (G) pairing with cytosine (C). As with native proteins, the molecular structure . Chromatography, 625 13.6.2 Ion-Moderated Partition Chromatography, 626 13.6.3 High-Performance Anion-Exchange Chromatography, 628 Introduction to Modern Liquid Chromatography, Third Edition, by Lloyd. Anion-Exchange Chromatography, 619 13.5.2 Reversed-Phase Chromatography, 620 13.5.3 Hydrophobic Interaction Chromatography, 624 13.6 SEPARATION OF CARBOHYDRATES, 625 13.6.1 Hydrophilic Interaction Chromatography,. Analysis, 651 13.10.3 Liquid- Chromatography Modes for Polymer Analysis, 653 13.10.4 Polymer Separations by Two-Dimensional Chromatography, 657 13.1 BIOMACROMOLECULES Since liquid chromatography was first