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 s
Trang 1566 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
Trang 2REFERENCES 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.
Trang 4CHAPTER THIRTEEN
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
Trang 5570 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
Trang 613.2MOLECULAR 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
Trang 7572 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 NH2
O
OH O
NH2 OH
O HO
NH2 OH
O
H2N
H2N
NH2 OH O NH
HN
NH
NH2 OH O
N
NH2 OH O
NH2 OH O
NH
NH2 OH O
HO
NH2
R1
OH
O
α
NH2 OH O
NH2 OH O
NH2 OH O
NH2 OH O
NH2 OH O
NH OH O
HO
NH2 OH O
NH2 OH
O HO
HS
NH2 OH O
NH2 OH
O S
NH2 NH2
O
OH O
NH2 OH
O
NH2 O
Figure13.1 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 pKavalues for the ionogenic side chains are shown for acidic and basic amino acids Adapted from [7]
Trang 813.2MOLECULAR STRUCTURE AND CONFORMATION 573 Protein Structural Heirarchies
(a) Primary Structure (b) Secondary Structure
H2N-Asp-Glu-Phe-Arg-Asp-Ser
Gly-Tyr-Glu-Val-His-Gln-Lys-Leu-COOH
(c) Tertiary Structure (d ) Quaternary Structure
Figure13.2 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 anα-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 backRib-bone of the hemoglobinβ-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 theβ-sheet, which forms
by hydrogen bonding between adjacent linear segments of primary sequence
Trang 9574 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
Trang 1013.2MOLECULAR STRUCTURE AND CONFORMATION 575
RNA
DNA
(a)Oligonucleotide (b)
composition
O
O
O O
O S P S P S
O
O O
O
O O
O O
O O
P
H3C
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
NH2
NH2
NH2
N
N
N
N N
N
NH
NH
NH NH
NH
NH NH
O
O O
O
Figure13.3 Structure of nucleic acids (a) Schematic composition of a single-stranded
oligonucleotide; in RNA the 2ribose 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 3hydroxyl of one nucleotide and the 5hydroxyl 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