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Absolute Molecular Weight Distribution of Low-Molecular-Weight Heparins by Size-Exclusion Chromatography with Multiangle Laser Light Scattering Detection
ANALYTICAL BIOCHEMISTRY ARTICLE NO.
245, 231–241 (1997)
AB969984
Absolute Molecular Weight Distribution of Low-MolecularWeight Heparins by Size-Exclusion Chromatography with Multiangle Laser Light Scattering Detection James E. Knobloch1 and Patrick N. Shaklee Scientific Protein Laboratories, Subsidiary of American Home Products Corporation, 700 East Main Street, Waunakee, Wisconsin 53597
Received October 29, 1996
The absolute molecular weight (Mr) distribution of seven low-molecular-weight (LMW) heparin products was determined by size-exclusion chromatography (SEC) coupled with multiangle laser light scattering (MALLS) detection. The SEC/MALLS technique does not rely on relative Mr standards for column calibration and yields absolute Mr estimates directly from the angular dependence of scattered light intensity as a function of concentration, as formulated by light scattering theory. The SEC/MALLS method we describe is rapid, precise, and accurate. In 1 h it yields results from triplicate injections that agree well with the manufacturers’ own independent analyses and that exhibit coefficients of variation of Ç1%. By eliminating the requirement for finite quantities of highly purified, wellcharacterized Mr standards derived from heparin, the present procedure represents a clear improvement over relative methods of Mr determination. Thus, it is concluded that the SEC/MALLS method is ideally suited to routine quality control of commercial LMWheparin products. q 1997 Academic Press
Heparin, the well-known anticoagulant and antithrombotic agent, is a linear polysaccharide isolated from animal tissues. It is composed primarily of alternating residues of a(1 r 4)-linked L-iduronic acid and D-glucosamine (1). Selected hydroxyl groups in the heparin molecule are derivatized as their sulfate esters, and amino groups can be either sulfated or acetylated (1, 2). As a polyelectrolyte, heparin is usually supplied as the sodium salt (i.e., heparin sodium, United States Pharmacopeia). Heparin is not a homogeneous sub1 To whom correspondence should be addressed. Fax: (608) 8494053.
stance but rather a polydisperse mixture of molecules, ranging in molecular weight (Mr)2 from about 5000 to 30,000 and averaging 12,000–15,000 (3, 4). The heterogeneity of heparin can be ascribed to differences in chain length, as well as overall sulfation pattern (5). Low-molecular-weight (LMW) heparins are prepared by the controlled chemical or enzymatic depolymerization of unfractionated (UF) heparin (6) and are approximately one-third its size. Depending on the depolymerization approach, each manufacturing process yields a chemically distinct product in terms of end-group saccharides, partial desulfation, and Mr distribution (7). As with the heparin starting material, LMW-heparins also represent a heterogeneous mixture of polymers and not a single, discrete molecular entity. Although LMW-heparins enjoy wide acceptance in Europe and Japan, these drugs have received close scrutiny from the U.S. Food and Drug Administration (FDA) due in part to their heterogeneity (8). Each LMW-heparin product is considered to be a new drug entity, distinct from UF heparin (6). Lack of a universally recognized method for determining the Mr distribution of LMW-heparin has complicated the regulatory approval process. To characterize their product’s chemical composition, manufacturers have prepared Mr standards derived from heparin for use in calibrating size-exclusion chromatography columns (9–11). The accuracy of such relative methods of Mr determination 2 Abbreviations used: SEC, size-exclusion chromatography; MALLS, multiangle laser light scattering; LALLS, low-angle laser light scattering; LS, light scattering; RI, refractive index; UF heparin, unfractionated heparin; LMW-heparin, low-molecular-weight heparin; FDA, U.S. Food and Drug Administration; Mr , molecular weight; Mw , weight-average molecular weight; Mn , number-average molecular weight; Mp , peak molecular weight; dn/dc, refractive index increment; EGT, end-group titration; BSA, bovine serum albumin; CV, coefficient of variation; DSP, digital signal processing; EP, European Pharmacopeia.
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0003-2697/97 $25.00 Copyright q 1997 by Academic Press All rights of reproduction in any form reserved.
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KNOBLOCH AND SHAKLEE TABLE 1
LMW-Heparins Included in SEC/MALLS Study dn/dca
Trade name(s)
Approved name
Manufacturer(s)
Method of production
Fluxum Fragmin Fraxiparin Logiparin Lovenox, Clexane Normiflo
Parnaparin sodium Dalteparin sodium Nadroparin sodium Tinzaparin sodium Enoxaparin sodium Ardeparin sodium
Peroxidolysis Deaminative cleavage Deaminative cleavage Enzymatic b-elimination Chemical b-elimination Peroxidolysis
0.133 0.129 0.132 0.132 0.134 0.132
Sandoparin
Certiparin sodium
Opocrin S.p.A., Alfa Wasserman Pharmacia & Upjohn Sanofi Recherche Novo Nordisk A/S Rhoˆne-Poulenc Rorer Wyeth-Ayerst Research, Pharmacia Hepar, Inc. Sandoz AG
Deaminative cleavage
0.132
a The refractive index increment (ml/g) was measured at 257C in SEC eluant (0.1 M ammonium acetate) with an Optilab DSP interferometric refractometer (Wyatt Technology Corp.), operating at the same wavelength (690 nm) as the MALLS detector.
depends on the quality of the calibration standards themselves. Intrinsic viscometry (3), equilibrium ultracentrifugation (5, 12), and low-angle laser light scattering (LALLS) (11, 13) are typically employed for characterizing each standard. Considering the expense and effort involved in their preparation, it is understandable that authentic heparin Mr standards are not widely available. We sought a method for determining heparin’s Mr distribution that would avoid the problems associated with existing techniques. Lacking a source of relative heparin Mr standards, we developed and validated an absolute chromatographic method for UF heparin that employs multiangle laser light scattering (MALLS) technology in conjunction with high-performance sizeexclusion chromatography (SEC) (unpublished results). We then turned our attention to the LMW-heparins, to determine whether these products could also be analyzed by a similar procedure. The data presented in this paper clearly demonstrate the utility of the SEC/ MALLS methodology for determining the Mr distribution of a variety of commercial LMW-heparin products. Furthermore, the data suggest that the method may be generally useful for Mr analysis of other biopolymers. MATERIALS AND EQUIPMENT
LMW-Heparin Products The pharmaceutical companies that agreed to participate in the LMW-heparin study are listed in Table 1. Each manufacturer generously provided a sample of their LMW-heparin product (as the bulk drug substance, not the final formulated product), together with results from their own independent Mr analysis. All products were supplied as the sodium salt.
degassed, NANOpure water (Barstead-Thermolyne, Dubuque, IA) and vacuum-filtered through a 0.2-mm Anotop 47 inorganic membrane filter (Whatman, Fairfield, NJ) on the day of intended use. Description of the SEC/MALLS System The liquid chromatograph (Fig. 1) consisted of a single Waters 510 pump (Waters Corp., Milford, MA), with flow rate control provided by a Waters 680 gradient controller. To prevent dust contamination, the eluant reservoir was sealed with a protective cap (Lazar, Los Angeles, CA), which incorporated an integral 1-mm air intake filter. Before reaching the pump, the eluant was continuously degassed upon passage through both channels of an in-line degasser (Shodex KT-27 Degas, Showa Denko, Tokyo, Japan). After leaving the pump, the eluant passed sequentially through a pulse damper (Shodex DP-1), a 25-mm stainless steel in-line filter (No. XX4502500, Millipore Corp., Bedford, MA) fitted with a 0.1-mm cellulose ester membrane, a 500-psi Upchurch backpressure regulator (Chrom Tech, Apple Valley, MN), and a Waters 717 Plus autosampler. A Shodex OHpak SB-803 HQ column (8.0 1 300 mm) equipped with an OHpak SB-G guard column was employed for chromatographic analysis of LMW-heparins. Column effluent was monitored sequentially with a miniDAWN light scattering detector (Wyatt Technology Corp., Santa Barbara, CA) and a Waters R401 differential refractometer. The design of the miniDAWN flow cell incorporates a fixed photodiode detector array, capable of measuring scattered light intensity simultaneously at three angles (45, 90, and 1357). METHOD
Eluant Preparation
MALLS Detector Calibration and Normalization
SEC eluant (0.1 M ammonium acetate, pH 7, containing 0.05% sodium azide) was prepared in vacuum-
Each day before use the miniDAWN’s 907 detector was calibrated with toluene (Rayleigh factor of 9.9461
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FIG. 1. Configuration of the SEC/MALLS system. Solid arrows show the eluant flow path. Dashed lines indicate electrical connections between system components. BP Regulator denotes the location of the backpressure regulator.
1 1006 cm01 at 690 nm) according to the manufacturer’s recommended procedure. Calibration enabled photodiode output voltages to be converted to excess Rayleigh ratios, a measure of scattered light intensity above background. A syringe pump (Model 100, KD Scientific, Boston, MA) fitted with a 10-ml glass syringe (Becton–Dickinson, Franklin Lakes, NJ) was operated at 0.2 ml/min when backflushing the miniDAWN’s flow cell with toluene during calibration (99.8%, Aldrich, Milwaukee, WI), with methanol when switching to aqueous conditions (Burdick & Jackson, Muskegon, MI), with 2 N nitric acid for routine in situ cell cleaning, or with water for salt removal. To prevent particulate contamination, Anotop 25 syringe filters (Whatman) with 0.02-mm inorganic membranes were employed whenever backflushing the miniDAWN’s flow cell with the syringe pump. After calibration, the miniDAWN was connected to the SEC system preequilibrated at operating pressure. A 0.2-mg injection of bovine serum albumin (BSA) monomer (Ç98%, Sigma, St. Louis, MO) was employed for normalizing the 45 and 1357 light scattering (LS) detectors relative to the 907 detector. Normalization was required to correct for slight differences in light beam collimation, photodiode sensitivity, and refractive index effects among the three LS detectors. Use of
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a monodisperse material like BSA monomer also enabled the volume delay (0.077 ml) between the miniDAWN and refractometer to be determined, permitting proper alignment of the LS and refractive index (RI) signals as required for calculation of the weight-average molecular weight (Mw) for each data slice. The RI detector’s 100-mV output signal was amplified to 1 V and digitized by the miniDAWN’s digital signal processing (DSP) circuitry. The RI detector was calibrated at 321 attenuation over a linear concentration range (0.1–1.9 mg/ml) with seven evenly spaced sodium chloride standards prepared by direct dilution from a 5 mg/ml stock solution. Sample Preparation Solutions of each LMW-heparin (10 mg/ml) were prepared in ammonium acetate eluant the day before each analysis and stirred 4 h. The extended sample dissolution time was necessary to reduce the size of a high Mr aggregate peak detected by light scattering in the SEC column’s exclusion volume (data not shown). Aliquots were then centrifuged in Microfilterfuge tubes (Rainin, Woburn, MA; 0.2-mm cellulose acetate membrane) prior to injection. Two hundred microliters was injected, representing Ç2 mg of LMW-heparin on column.
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Dextran Control A 10 mg/ml solution of a 5000 Mr dextran standard (Pharmacosmos, Viby Sj., Denmark; distributed through American Polymer Standards Corp., Mentor, OH) was prepared in ammonium acetate eluant. It was syringe-filtered through a 0.2-mm Anotop 25 membrane and stored under refrigeration for use as a system suitability control throughout the LMW-heparin study. Each analysis incorporated five 200-ml control injections, interspersed at the beginning and end of the injection sequence as well as between each subset of LMW-heparin replicates. The manufacturer determined the Mw of this dextran standard by the LALLS technique. These control injections provided independent confirmation of the accuracy of the Mw estimates generated by the SEC/MALLS method. The dextran standard served as a control and was not utilized for calibration of the miniDAWN detector or SEC column. Refractive Index Increment Determination The differential refractive index increment (dn/dc) defines how a solution’s refractive index (n) changes with solute concentration (c). This parameter was required for converting RI voltages to solute concentrations at each data slice across a chromatographic peak and was determined individually for each LMW-heparin product (see Table 1). Measurements were conducted in 0.1 M ammonium acetate at 257C, using a calibrated Optilab DSP interferometric refractometer (Wyatt) operating at 690 nm (the same wavelength as the miniDAWN’s 20-mW GaAs semiconductor laser). Before calculating dn/dc, the mass concentration at four to six test dilutions was corrected for the moisture content of each LMW-heparin. For BSA and dextran, dn/dc values of 0.180 and 0.148 ml/g were employed, respectively. Chromatography The Shodex OHpak SB-803 HQ column was equilibrated overnight at operating pressure in the same batch of ammonium acetate eluant used for sample preparation. A gradient controller allowed the flow rate to be changed gradually (linear gradient between 0.20 and 0.80 ml/min over 30 min), thereby protecting the column from sudden pressure surges and prolonging its suitability for light scattering applications. Throughout the study a noise bandwidth of Ç5 mV was observed at the 457 LS detector, the one most sensitive to particulate shedding from the column. Each analysis was conducted at 0.80 ml/min and ambient temperature (20–227C). Data Collection Three analog signals from the LS detector array and one RI signal were digitized by the miniDAWN’s DSP
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circuitry before acquisition by a Pentium-based computer running Astra for Windows software (Wyatt). After an initial 5-min (4-ml) collection delay, data acquisition proceeded at a rate of 2 samples/s for 12.5 min (10 ml), yielding 1500 data slices per chromatogram. Each raw data slice thus incorporated three LS signals and one RI signal and corresponded to an elution volume of 6.7 ml. Study Design To assess the SEC/MALLS method’s reproducibility, accuracy, and precision, the seven LMW-heparin products were subjected to three independent analyses conducted at 1-week intervals. Each day of analysis single injections of all seven LMW-heparins were made, and the entire injection sequence was repeated three times. In this manner triplicate injections from each sample solution occurred 2.7 h apart, thereby testing the stability of the entire chromatographic system. Each week the injection order of the LMW-heparins was randomized to remove potential sequence bias. RESULTS
Details of the SEC/MALLS Analysis for Fragmin We chose the Fragmin data to illustrate the analytical procedure for two reasons. First, the manufacturer provided estimates for all moments of the Mr distribution (compiled in Table 4). Second, Fragmin is one of only two LMW-heparin products currently approved for clinical use in the United States by the FDA (the other being Lovenox). A three-dimensional plot of the RI and three LS detector signals recorded during a single analysis of Fragmin is presented in Fig. 2. After setting baselines and defining peak boundaries, the software converted the RI voltages from 286 volume slices across the chromatographic peak to solute concentrations (g/ml) by multiplying each voltage by the RI calibration constant (2.60 1 1004) and dividing by the product’s dn/dc value (0.129 ml/g). A trace of high Mr aggregate can be seen at 5.5 ml in the 907 LS profile (Fig. 3), marking the exclusion volume of the column. The prominent RI peak at 11 ml marks the column’s inclusion volume and apparently results from counterion exchange between the LMWheparin polyelectrolyte (as the sodium salt) and the ammonium acetate eluant. The intensity of this sodium peak is dramatically reduced in chromatograms of the dextran control, a nonionic polysaccharide. This phenomenon may be related to the occurrence of two counterion forms in sodium heparin (‘‘condensed and delocalized’’ vs ‘‘active and dissociated’’), as postulated by Manning’s polyelectrolyte theory (14). Although the 907 LS and RI chromatograms appear
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FIG. 2. Three-dimensional plot of detector signals acquired during a single SEC/MALLS analysis of Fragmin. Chromatograms (from left to right) correspond to the RI, 138.57 LS, 907 LS, and 41.57 LS detectors and are oriented with the injection on the left.
FIG. 3. Overlay of the RI (light) and 907 LS (dark) chromatograms for a single SEC/MALLS analysis of Fragmin. The Mr distribution is calculated for the elution volume between the vertical markers.
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to be offset in Fig. 3, this actually indicates that the LMW-heparin sample is polydisperse, i.e., composed of a mixture of molecules of varying Mr . Because the LS detector responds to differences in Mr , the light scattering signal is strongest on the leading edge of the SEC peak where the larger molecules elute. Data Processing and Calculation of Results To describe our LMW-heparin data further, we must first review a few general light scattering concepts discussed in greater detail by Wyatt (15). In the Rayleigh– Gans–Debye approximation (Eq. [1]) K*c 1 Å / 2A2c, R(u) MrP(u)
[1]
the quantities on the left are measurable (as described below), whereas most on the right must be derived. Here M is the weight-average molecular weight and P(u) is the theoretical form factor, describing the angular and size dependence of scattered light intensity. For small molecules such as LMW-heparins (rms radius ! l/20), the intensity of scattered light does not show strong angular dependence and P(u) approaches unity. The second virial coefficient, A2 , corrects for solute/solvent interaction and in a first approximation is usually small enough to be ignored at the concentrations encountered in chromatographic analyses. In practice, Eq. [1] is solved by linear regression analysis (weighted for the observed background noise at each LS detector) of K*c/R(u) versus sin2(u/2) in a Debye plot (Fig. 4), which provides an estimate of 1/M for each volume slice directly from the y-intercept. Here, (i) K* is an optical constant incorporating the solvent’s refractive index, the solute’s dn/dc, the wavelength of incident light in vacuum (690 nm), and Avogadro’s number; (ii) c is the solute concentration (g/ml) in each volume slice, as measured by the on-line RI detector; and (iii) R(u) is the excess Rayleigh scattering (i.e., scattered light intensity above background) at angle u. Table 2 presents the Mw for each daily replicate injection of Fragmin, calculated as ( (cirMi)/( ci over 286 volume slices in the chromatographic peak. When graphed together, these slice Mw values form the Mr vs volume plot shown in Fig. 5, the familiar SEC column calibration curve. These data may also be used to construct a cumulative distribution plot (Fig. 6), from which the proportion of sample below a given Mr can be tabulated through the software. Similarly, the numberaverage molecular weight values (Mn) reported in Table 2 for each daily replicate of Fragmin were calculated according to the formula ( ci/( (ci/Mi). Peak molecular weight values (Mp) were obtained through the software in the Debye plot window. Mp corresponds to the Mr
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FIG. 4. Debye plot of K*c/R(u) versus sin2(u/2) from a single SEC/ MALLS analysis of Fragmin. Plot shows information from one data slice within the designated peak boundaries. Plotted points correspond to the three LS detectors. The y-intercept of the weighted regression line provides an estimate of 1/Mw for this particular data slice. The LS detector angles employed in the calculations are corrected for the refractive index of water (1.330) and correspond to observed angles (uobs) of 41.5, 90.0, and 138.57.
at the apex of the RI peak, i.e., the Mr of the sample component present in highest concentration. Comparison of Results from Seven LMW-Heparins Each LMW-heparin product included in the SEC/ MALLS study was analyzed in triplicate in three independent analyses performed at 1-week intervals. Tables 3A and 3B summarize the results for each LMWheparin by day and provide their overall means and coefficients of variation [CV Å standard deviation/ mean]. Approximately 80% of the daily CVs were less than 1%, whereas 98% were less than 2% and none exceeded 3%. These data clearly demonstrate the excellent intraassay precision (i.e., same day) of the SEC/ MALLS method for the analysis of LMW-heparins. Interassay precision (i.e., different days) was generally somewhat lower (i.e., exhibited higher CVs). Of 28 overall CVs (n Å 9) reported for the LMW-heparins in Tables 3A and 3B, 7 were õ1%, 11 were õ2%, 23 were õ3%, 26 were õ4%, and none exceeded 5%. This level of interassay precision is quite acceptable for routine quality control purposes, being comparable to relative SEC methods employing calibration standards. Table 4 compares our overall (n Å 9) SEC/MALLS results (mean { 95% confidence interval) with the values reported by the LMW-heparin manufacturers. Most of the manufacturers’ own estimates for Mw agreed remarkably well with our results, being within 175 of the reported value for Fluxum, Fragmin, Lovenox, and Sandoparin. Generally, the Mn , Mp , and polydispersity figures were also in reasonably good agreement. Taking experi-
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SEC/MALLS ANALYSIS OF LMW-HEPARINS TABLE 2
SEC/MALLS Analysis of Fragmin Day
Injection
Mpa
Mwb
Mnc
Mw /Mnd
1
A B C Mean CV (%) A B C Mean CV (%) A B C Mean CV (%) Mean CV (%)
4797 { 55e 4802 { 90 4779 { 20 4793 0.25 4513 { 80 4586 { 60 4532 { 57 4544 0.83 4648 { 45 4649 { 50 4638 { 65 4645 0.13 4660 2.37
6157 { 200 6066 { 200 6123 { 200 6115 0.75 6018 { 300 6074 { 200 6041 { 200 6044 0.47 6146 { 100 6179 { 200 6141 { 100 6155 0.34 6105 0.93
5562 { 200 5499 { 200 5532 { 200 5531 0.57 5370 { 300 5420 { 300 5368 { 300 5386 0.55 5493 { 100 5530 { 200 5475 { 100 5499 0.51 5472 1.29
1.107 { 0.042 1.103 { 0.053 1.107 { 0.048 1.106 0.20 1.121 { 0.088 1.121 { 0.081 1.125 { 0.071 1.122 0.24 1.119 { 0.032 1.117 { 0.042 1.122 { 0.036 1.119 0.19 1.116 0.71
2
3
Overall (n Å 9)
a Mp (peak molecular weight) was measured at the apex of the refractive index peak and corresponded to the molecular weight of the sample component present in highest concentration. b Mw is the weight-average molecular weight. c Mn is to the number-average molecular weight. d Mw /Mn represents the polydispersity of the sample. e Statistical uncertainty reported by the MALLS software. Based on the background noise at each detector, these uncertainties are intended to express the statistical consistency of the data and not to set absolute limits on the accuracy of results.
mental error into consideration, the SEC/MALLS method yielded results consistent with the manufacturers’ own independent analyses.
FIG. 5. Molecular weight versus elution volume curve from a single SEC/MALLS analysis of Fragmin. The corresponding RI chromatogram is overlaid for reference. The Mr distribution across the peak is linear until a compact, dense sample fraction emerges along the trailing edge of the peak. This fraction remains undetected by RI.
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Dextran Control Results Results from the SEC/MALLS analysis of the dextran control are also summarized in Table 3B. This Mr standard was routinely included in each analysis to verify system performance. It was not employed for column calibration purposes. Intraassay precision for Mp and Mw were consistently high, with CVs £0.8% for five replicate injections occurring 2.7 h apart. For Mn and polydispersity intraassay precision was slightly lower, with CVs ranging from 1.2 to 2.2%. Interassay precision, as measured by overall CVs based on a total of 15 determinations, was comparable to the LMWheparin figures: õ0.5% for Mw , õ2% for Mp , and õ3% for both Mn and polydispersity. Our overall (n Å 15) Mw of 5445 { 15 (mean { 95% confidence interval) for the dextran control compared reasonably well (within 4.5%) with the LALLS value of 5700 reported by the manufacturer (Table 4). Their Mn estimate of 3326 was determined by reducing endgroup titration (EGT) and was substantially lower than the present MALLS estimate of 4299 { 70. Polydispersity (Mw/Mn) varied accordingly: 1.268 { 0.020 (found) vs 1.71 (reported, as the LALLS/EGT ratio). The manufacturer also provided an Mp estimate of 4440 by gelpermeation chromatography with relative standardization, which compared very well (within 2.5%) with our estimate of 4552 { 47. Considering the different analytical techniques em-
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FIG. 6. Cumulative distribution plot from a single SEC/MALLS analysis of Fragmin. The utility of this plot is suggested by the dashed lines, which indicate that 50% of the sample mass is below (and conversely, above) 5350 in molecular weight.
ployed, the SEC/MALLS method yielded reasonable results for the dextran standard. Its use as a control (not as a column calibrant) in the present SEC/MALLS procedure served to increase operator confidence in the accuracy, consistency, and general applicability of the LMW-heparin results. DISCUSSION
Before use in Mr analysis, a new SEC column is usually calibrated with a set of relative standards. The
validity of an existing calibration curve can then be verified by including high and low Mr standards in each sample set. Kristensen et al. (11) markedly improved the reliability of their relative SEC method by adding an internal standard (sucrose) to all standard and sample solutions and tracking its elution volume over time. They demonstrated that a 2% error in flow rate can result in a 24% error in Mp and a 40% error in Mn and Mw . This large potential error results from the fact that molecular weights are plotted logarithmically against
TABLE 3A
SEC/MALLS Analyses of LMW-Heparins
Product
Day
Fluxum
Overall Fragmin
(n
Overall Fraxiparin
(n
Overall Logiparin
(n
Overall
(n
1 2 3 Å 1 2 3 Å 1 2 3 Å 1 2 3 Å
9)
9)
9)
9)
Mp mean (CV %)a 4003 3795 3865 3888 4793 4544 4645 4660 3300 3107 3167 3191 4596 4368 4488 4484
(0.59) (0.19) (0.46) (2.39) (0.25) (0.83) (0.13) (2.37) (0.62) (0.97) (0.92) (2.78) (0.23) (0.81) (0.39) (2.25)
Mw mean (CV %) 5173 5078 5107 5119 6115 6044 6155 6105 4340 4291 4337 4323 6256 6243 6183 6227
a
(0.73) (0.43) (0.24) (0.93) (0.75) (0.47) (0.34) (0.93) (0.41) (0.62) (1.08) (0.86) (0.30) (0.52) (0.74) (0.72)
Mn mean (CV %) 4346 4155 4100 4200 5531 5386 5499 5472 3714 3590 3565 3623 5000 4973 4702 4892
(1.08) (0.56) (0.06) (2.74) (0.57) (0.55) (0.51) (1.29) (0.93) (0.82) (1.72) (2.18) (0.52) (0.78) (0.89) (2.98)
Mw /Mn mean (CV %) 1.190 1.222 1.246 1.219 1.106 1.122 1.119 1.116 1.169 1.195 1.217 1.194 1.251 1.256 1.315 1.274
(0.37) (0.16) (0.27) (1.99) (0.20) (0.24) (0.19) (0.71) (0.55) (0.27) (0.81) (1.82) (0.24) (0.57) (0.32) (2.44)
Unless otherwise indicated, the daily means correspond to triplicate injections from the same LMW-heparin solution that were spaced 2.7 h apart. For the dextran control, daily means are from five injections spaced 2.7 h apart.
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SEC/MALLS ANALYSIS OF LMW-HEPARINS TABLE 3B
SEC/MALLS Analyses of LMW-Heparins
Product
Mp mean (CV %)a
Day
Lovenox
Overall Normiflo
(n
Overall Sandoparin
(n
Overall Dextran
(n
Overall
(n
1 2 3 Å 9) 1 2 3 Å 9) 1 2 3 Å 9) 1 2 3 Å 15)
2935 2748 2821 2835 5537 5225 5318 5360 3952 3761 3863 3859 4633 4448 4574 4552
Mw mean (CV %)
(0.59) (1.54) (0.92) (3.02) (0.59) (0.38) (0.52) (2.63) (1.28) (0.94) (0.39) (2.30) (0.80) (0.46) (0.57) (1.85)
4011 3923 3957 3964 6845 6772 6736 6784 5050 5008 4995 5018 5440 5453 5443 5445
Mn mean (CV %)
(0.61) (0.90) (0.95) (1.20) (0.88) (0.37) (0.43) (0.88) (1.31) (0.75) (0.63) (0.96) (0.42) (0.73) (0.32) (0.49)
3292 3139 3096 3176 5416 5371 5069 5286 3704 3681 3373 3586 4361 4384 4152 4299
(0.84) (1.31) (1.43) (3.00) (0.85) (1.49) (1.41) (3.28) (1.47) (2.95) (1.30) (4.81) (1.46) (2.22) (1.16) (2.96)
Mw /Mn mean (CV %) 1.218 1.250 1.278 1.249 1.264 1.261 1.329 1.285 1.363 1.361 1.481 1.402 1.248 1.244 1.311 1.268
(1.34) (0.56) (0.48) (2.21) (1.11) (1.13) (0.99) (2.76) (0.97) (2.17) (1.05) (4.42) (1.54) (1.54) (1.18) (2.83)
a Unless otherwise indicated, the daily means correspond to triplicate injections from the same LMW-heparin solution that were spaced 2.7 h apart. For the dextran control, daily means are from five injections spaced 2.7 h apart.
elution volume when constructing column calibration curves. Recently, the European Pharmacopeia (EP) Commission proposed an official method for determining the
Mr distribution of LMW-heparins by SEC. Based on the work of van Dedem and Nielsen (17, 18), the EP calibrant was prepared by partial enzymatic depolymerization of UF heparin with heparinase. When run
TABLE 4
Comparison of SEC/MALLS Results for LMW-Heparins Product (lot no.)
Mp (reported)
Mw (reported)
Mn (reported)
Mw /Mn (reported)
Fluxum (90248) Fragmin (62158-51) Fraxiparin (4096XH) Logiparin (LMW9405) Lovenox (PRS122) Normiflo (RD13390) Sandoparin (76465002) Dextran (096001)
3888 { 71a (4200) 4664 { 85 (5100) 3191 { 68 (4162) 4484 { 78 (4500) 2835 { 66 (n/a) 5360 { 108 (n/a) 3859 { 68 (n/a) 4552 { 47 (4440) d
5119 { 37 (5240) 6105 { 44 (6250) 4323 { 28 (4991) 6227 { 35 (n/a)b 3964 { 37 (4139) c 6784 { 46 (5953) 5018 { 37 (5020) c 5445 { 15 (5700) e
4200 { 88 (4190) 5472 { 54 (5100) 3623 { 61 (4074) 4892 { 112 (n/a) 3176 { 73 (n/a) 5286 { 133 (5235) 3586 { 132 (n/a) 4299 { 70 (3326) f
1.219 { 0.019 (1.251) 1.116 { 0.006 (1.226) 1.194 { 0.017 (1.225) 1.274 { 0.024 (n/a) 1.249 { 0.021 (n/a) 1.285 { 0.027 (1.137) 1.402 { 0.048 (n/a) 1.268 { 0.020 (1.71) g
a
Mean { 95% confidence interval (n Å 9 for LMW-heparins, n Å 15 for the dextran control). Not available (information not reported by the manufacturer). c Reported simply as the ‘‘average’’ molecular weight. d Measured by SEC using relative standardization. e Determined by LALLS. f Determined by reducing end group titration (EGT). g LALLS/EGT ratio. b
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on a specific brand of column under defined conditions, the EP standard yields a jagged elution profile with the lower oligomers of heparin (di- through tetradecasaccharides) visible as regularly spaced peaks. Equidistant time points are then extrapolated into the high Mr region of the chromatogram, where the column does not resolve higher oligomers of heparin into distinct peaks. A calibration curve can then be constructed for calculating the molecular weight of LMW-heparins analyzed under the same conditions. Because the EP standard was prepared from UF heparin depolymerized enzymatically by b-elimination, the fragments contain a D4,5-unsaturated uronic acid residue at the nonreducing terminus, permitting their detection at 235 nm. Assuming the molar extinction coefficient of such fragments to be constant, RI and ultraviolet (uv) absorbance detectors operated in series provide a relative measure of Mr as the ratio of the RI/ uv peak heights (i.e., grml01/molrml01 Å g/mol). One tentatively assigns the identity of the octasaccharide peak and then normalizes the peak height ratios of the remaining peaks against it. The principle of the EP method maintains that these normalized peak height ratios should be close to integer values, if the octasaccharide peak was correctly assigned. The Mr of all the peaks are then calculated, based on the assumption that the average Mr of the repeating disaccharide unit of heparin is 600. Simplifying assumptions such as these are commonly made when preparing relative heparin Mr standards. The EP method was validated by analyzing heparin Mr standards (characterized by LALLS) on an SEC column calibrated with the EP standard and obtaining comparable results. Jeske et al. (19) reported that the Mr range obtained with the EP standard is too narrow for most LMWheparins and that it also overestimates the proportion of components above Mr 7500. They proposed an improved SEC calibrant prepared by alkaline hydrolysis of heparin benzyl ester (20, 21), which also results in depolymerization by b-elimination and introduction of a uv chromophore. Unfortunately, neither the EP calibrant nor this improved version is suitable for determining the Mr distribution of UF heparin. Compared to relative calibration techniques, the absolute SEC/MALLS approach we describe is applicable to both unfractionated and LMW-heparins. It makes no simplifying assumptions about the Mr of the basic heparin disaccharide unit. Furthermore, the SEC technique avoids the critical dependency of relative chromatographic methods on constant flow rate. By contrast, absolute Mr estimates derived from light scattering measurements are independent of flow rate: reanalysis of the same LMW-heparin solutions at 0.4 ml/min (one-half the normal flow rate) yielded identical results within experimental error of measurement (data not shown). Moreover, the present SEC/MALLS method delivers precise
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and accurate results within a remarkably short time. Whereas the dual-column EP method requires about an hour to complete a single analysis at a flow rate of 0.5 ml/min, our method performs triplicate analyses in the same amount of time by employing a single SEC column and a higher flow rate. System suitability and method accuracy can be verified by including a dextran control in each sample set. It should be noted that our results for Normiflo agree with an independent assessment of this same lot (No. RD 13390) reported by Atha et al. (16) of the National Institute of Standards and Technology (Gaithersburg, MD). By SEC analysis using relative standardization according to the EP method described above, they obtained values of 6850 for Mw , 5300 for Mn , and 1.3 for polydispersity, compared to our estimates of 6784, 5286, and 1.285, respectively. Low-angle laser light scattering detectors have successfully been employed for the Mr determination of UF heparin and heparin fractions (22, 23). LALLS instruments, however, are inherently more sensitive to noise interference from extraneous dust and column shedding than multiangle detectors, which extrapolate to zero angle rather than directly measuring scattered light intensity near it (i.e., at 3 to 77). The MALLS technique has previously been applied to heparin analysis (15), using the research-grade DAWN-F instrument (Wyatt) that measured scattering intensity simultaneously at 15 angles. The recent introduction of the less expensive, triple-angle miniDAWN detector employed here provides LMW-heparin manufacturers with a practical tool for routine quality control. With the above-described method, absolute Mr determinations can now be performed on LMW-heparins directly, without reference to relative heparin Mr standards that ultimately must be characterized by absolute methods. The commercial availability of this modern instrumentation, together with versatile Windows-based software for data acquisition and analysis, should now make SEC/MALLS technology accessible to a broader range of polymer chemists. ACKNOWLEDGMENTS The authors sincerely thank the following individuals and their corporate sponsors for participating in this study and graciously providing the LMW-heparin samples and independent Mr analyses: Dr. Carl Magnus Svahn (Pharmacia & Upjohn), Dr. Giuseppe Mascellani (Opocrin S.p.A.), Dr. Theodore Spiro (Rhoˆne-Poulenc Rorer), Dr. D. Scott Holloway (Wyeth-Ayerst Research), Dr. Helmut Wolf (Sandoz AG), Hanne Kristensen (Novo Nordisk A/S), and Drs. Maurice Petitou and Jean-Marc Herbert (Sanofi Recherche). The authors also express their appreciation to Edward Mancilla and Dr. Paul Weiss of Scientific Protein Labs for their support and encouragement during development of the analytical method. We are grateful to Dr. Philip Wyatt, Dr. David Shortt, and Lena Nilsson of Wyatt Technology Corp. for their expert advice and acknowledge the technical assistance of Dirk Sarcinelli and Robert Paulson in performing dn/dc measurements.
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REFERENCES 1. Rode´n, L. (1989) in Heparin: Chemical and Biological Properties, Clinical Applications (Lane, D. A., and Lindahl, U., Eds.), pp. 1– 23, Edward Arnold, London. 2. Lindahl, U., Ba¨ckstro¨m, G., Thunberg, L., and Leder, I. G. (1980) Proc. Natl. Acad. Sci. USA 77, 6551. 3. Johnson, E. A., and Mulloy, B. (1976) Carbohydr. Res. 51, 119– 127. 4. Olson, S. T., and Bjo¨rk, I. (1993) Perspect. Drug Discovery Design 1, 479–501. 5. Lasker, S. E. (1977) Fed. Proc. 36, 92–97.
12. 13. 14.
15. 16. 17.
6. Mardiguian, J. (1992) in Low Molecular Weight Heparins in Clinical Practice (Doutremepuich, C., Ed.), pp. 7–12, Dekker, New York.
18. 19.
7. Wolf, H. (1994) Common Bleeding Clotting Disorders 78, 733– 743.
20.
8. Linhardt, R. J., and Hileman, R. E. (1995) Gen. Pharmacol. 26, 443–451.
21.
9. Rodriguez, H. J. (1976) Anal. Lett. 9, 497–506.
22.
10. Harenberg, J., and De Vries, J. X. (1983) J. Chromatogr. 261, 287–292. 11. Kristensen, H. I., Tromborg, E. M., Nielsen, J. R., Nielsen, J. I.,
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23.
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Johansen, K. B., and Østergaard, P. B. (1991) Thromb. Res. 64, 131–141. Barlow, G. H. (1985) Semin. Thromb. Hemostasis 11, 26–28. Miklautz, H., Riemann, J., and Vidic, H. J. (1986) J. Liq. Chromatogr. 9, 2073–2093. Nieduszynski, I. (1989) in Heparin: Chemical and Biological Properties, Clinical Applications (Lane, D. A., and Lindahl, U., Eds.), pp. 56–59, CRC Press, Boca Raton, FL. Wyatt, P. J. (1993) Anal. Chim. Acta 272, 1–40. Atha, D. A., Coxon, B., Reipa, V., and Gaigalas, A. K. (1995) J. Pharm. Sci. 84, 360–364. Van Dedem, G., and Nielsen, J. I. (1991) Pharmeuropa 3, 202– 218. Nielsen, J. I. (1992) Thromb. Haemostasis 68, 478–480. Jeske, W., Ahsan, A., and Fareed, J. (1993) Thromb. Res. 70, 39–50. Ahsan, A., Jeske, W., and Fareed, J. (1993) Semin. Thromb. Hemostasis 19, 63–68. Ahsan, A., Jeske, W., Mardiguian, J., and Fareed, J. (1994) J. Pharm. Sci. 83, 197–201. Hennink, W. E., van den Berg, J. W. A., and Feijen, J. (1987) Thromb. Res. 45, 463–475. Komatsu, H., Yoshii, K., Ishimitsu, S., Okada, S., and Takahata, T. (1993) J. Chromatogr. 644, 17–24.
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AB969984
Absolute Molecular Weight Distribution of Low-MolecularWeight Heparins by Size-Exclusion Chromatography with Multiangle Laser Light Scattering Detection James E. Knobloch1 and Patrick N. Shaklee Scientific Protein Laboratories, Subsidiary of American Home Products Corporation, 700 East Main Street, Waunakee, Wisconsin 53597
Received October 29, 1996
The absolute molecular weight (Mr) distribution of seven low-molecular-weight (LMW) heparin products was determined by size-exclusion chromatography (SEC) coupled with multiangle laser light scattering (MALLS) detection. The SEC/MALLS technique does not rely on relative Mr standards for column calibration and yields absolute Mr estimates directly from the angular dependence of scattered light intensity as a function of concentration, as formulated by light scattering theory. The SEC/MALLS method we describe is rapid, precise, and accurate. In 1 h it yields results from triplicate injections that agree well with the manufacturers’ own independent analyses and that exhibit coefficients of variation of Ç1%. By eliminating the requirement for finite quantities of highly purified, wellcharacterized Mr standards derived from heparin, the present procedure represents a clear improvement over relative methods of Mr determination. Thus, it is concluded that the SEC/MALLS method is ideally suited to routine quality control of commercial LMWheparin products. q 1997 Academic Press
Heparin, the well-known anticoagulant and antithrombotic agent, is a linear polysaccharide isolated from animal tissues. It is composed primarily of alternating residues of a(1 r 4)-linked L-iduronic acid and D-glucosamine (1). Selected hydroxyl groups in the heparin molecule are derivatized as their sulfate esters, and amino groups can be either sulfated or acetylated (1, 2). As a polyelectrolyte, heparin is usually supplied as the sodium salt (i.e., heparin sodium, United States Pharmacopeia). Heparin is not a homogeneous sub1 To whom correspondence should be addressed. Fax: (608) 8494053.
stance but rather a polydisperse mixture of molecules, ranging in molecular weight (Mr)2 from about 5000 to 30,000 and averaging 12,000–15,000 (3, 4). The heterogeneity of heparin can be ascribed to differences in chain length, as well as overall sulfation pattern (5). Low-molecular-weight (LMW) heparins are prepared by the controlled chemical or enzymatic depolymerization of unfractionated (UF) heparin (6) and are approximately one-third its size. Depending on the depolymerization approach, each manufacturing process yields a chemically distinct product in terms of end-group saccharides, partial desulfation, and Mr distribution (7). As with the heparin starting material, LMW-heparins also represent a heterogeneous mixture of polymers and not a single, discrete molecular entity. Although LMW-heparins enjoy wide acceptance in Europe and Japan, these drugs have received close scrutiny from the U.S. Food and Drug Administration (FDA) due in part to their heterogeneity (8). Each LMW-heparin product is considered to be a new drug entity, distinct from UF heparin (6). Lack of a universally recognized method for determining the Mr distribution of LMW-heparin has complicated the regulatory approval process. To characterize their product’s chemical composition, manufacturers have prepared Mr standards derived from heparin for use in calibrating size-exclusion chromatography columns (9–11). The accuracy of such relative methods of Mr determination 2 Abbreviations used: SEC, size-exclusion chromatography; MALLS, multiangle laser light scattering; LALLS, low-angle laser light scattering; LS, light scattering; RI, refractive index; UF heparin, unfractionated heparin; LMW-heparin, low-molecular-weight heparin; FDA, U.S. Food and Drug Administration; Mr , molecular weight; Mw , weight-average molecular weight; Mn , number-average molecular weight; Mp , peak molecular weight; dn/dc, refractive index increment; EGT, end-group titration; BSA, bovine serum albumin; CV, coefficient of variation; DSP, digital signal processing; EP, European Pharmacopeia.
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0003-2697/97 $25.00 Copyright q 1997 by Academic Press All rights of reproduction in any form reserved.
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LMW-Heparins Included in SEC/MALLS Study dn/dca
Trade name(s)
Approved name
Manufacturer(s)
Method of production
Fluxum Fragmin Fraxiparin Logiparin Lovenox, Clexane Normiflo
Parnaparin sodium Dalteparin sodium Nadroparin sodium Tinzaparin sodium Enoxaparin sodium Ardeparin sodium
Peroxidolysis Deaminative cleavage Deaminative cleavage Enzymatic b-elimination Chemical b-elimination Peroxidolysis
0.133 0.129 0.132 0.132 0.134 0.132
Sandoparin
Certiparin sodium
Opocrin S.p.A., Alfa Wasserman Pharmacia & Upjohn Sanofi Recherche Novo Nordisk A/S Rhoˆne-Poulenc Rorer Wyeth-Ayerst Research, Pharmacia Hepar, Inc. Sandoz AG
Deaminative cleavage
0.132
a The refractive index increment (ml/g) was measured at 257C in SEC eluant (0.1 M ammonium acetate) with an Optilab DSP interferometric refractometer (Wyatt Technology Corp.), operating at the same wavelength (690 nm) as the MALLS detector.
depends on the quality of the calibration standards themselves. Intrinsic viscometry (3), equilibrium ultracentrifugation (5, 12), and low-angle laser light scattering (LALLS) (11, 13) are typically employed for characterizing each standard. Considering the expense and effort involved in their preparation, it is understandable that authentic heparin Mr standards are not widely available. We sought a method for determining heparin’s Mr distribution that would avoid the problems associated with existing techniques. Lacking a source of relative heparin Mr standards, we developed and validated an absolute chromatographic method for UF heparin that employs multiangle laser light scattering (MALLS) technology in conjunction with high-performance sizeexclusion chromatography (SEC) (unpublished results). We then turned our attention to the LMW-heparins, to determine whether these products could also be analyzed by a similar procedure. The data presented in this paper clearly demonstrate the utility of the SEC/ MALLS methodology for determining the Mr distribution of a variety of commercial LMW-heparin products. Furthermore, the data suggest that the method may be generally useful for Mr analysis of other biopolymers. MATERIALS AND EQUIPMENT
LMW-Heparin Products The pharmaceutical companies that agreed to participate in the LMW-heparin study are listed in Table 1. Each manufacturer generously provided a sample of their LMW-heparin product (as the bulk drug substance, not the final formulated product), together with results from their own independent Mr analysis. All products were supplied as the sodium salt.
degassed, NANOpure water (Barstead-Thermolyne, Dubuque, IA) and vacuum-filtered through a 0.2-mm Anotop 47 inorganic membrane filter (Whatman, Fairfield, NJ) on the day of intended use. Description of the SEC/MALLS System The liquid chromatograph (Fig. 1) consisted of a single Waters 510 pump (Waters Corp., Milford, MA), with flow rate control provided by a Waters 680 gradient controller. To prevent dust contamination, the eluant reservoir was sealed with a protective cap (Lazar, Los Angeles, CA), which incorporated an integral 1-mm air intake filter. Before reaching the pump, the eluant was continuously degassed upon passage through both channels of an in-line degasser (Shodex KT-27 Degas, Showa Denko, Tokyo, Japan). After leaving the pump, the eluant passed sequentially through a pulse damper (Shodex DP-1), a 25-mm stainless steel in-line filter (No. XX4502500, Millipore Corp., Bedford, MA) fitted with a 0.1-mm cellulose ester membrane, a 500-psi Upchurch backpressure regulator (Chrom Tech, Apple Valley, MN), and a Waters 717 Plus autosampler. A Shodex OHpak SB-803 HQ column (8.0 1 300 mm) equipped with an OHpak SB-G guard column was employed for chromatographic analysis of LMW-heparins. Column effluent was monitored sequentially with a miniDAWN light scattering detector (Wyatt Technology Corp., Santa Barbara, CA) and a Waters R401 differential refractometer. The design of the miniDAWN flow cell incorporates a fixed photodiode detector array, capable of measuring scattered light intensity simultaneously at three angles (45, 90, and 1357). METHOD
Eluant Preparation
MALLS Detector Calibration and Normalization
SEC eluant (0.1 M ammonium acetate, pH 7, containing 0.05% sodium azide) was prepared in vacuum-
Each day before use the miniDAWN’s 907 detector was calibrated with toluene (Rayleigh factor of 9.9461
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FIG. 1. Configuration of the SEC/MALLS system. Solid arrows show the eluant flow path. Dashed lines indicate electrical connections between system components. BP Regulator denotes the location of the backpressure regulator.
1 1006 cm01 at 690 nm) according to the manufacturer’s recommended procedure. Calibration enabled photodiode output voltages to be converted to excess Rayleigh ratios, a measure of scattered light intensity above background. A syringe pump (Model 100, KD Scientific, Boston, MA) fitted with a 10-ml glass syringe (Becton–Dickinson, Franklin Lakes, NJ) was operated at 0.2 ml/min when backflushing the miniDAWN’s flow cell with toluene during calibration (99.8%, Aldrich, Milwaukee, WI), with methanol when switching to aqueous conditions (Burdick & Jackson, Muskegon, MI), with 2 N nitric acid for routine in situ cell cleaning, or with water for salt removal. To prevent particulate contamination, Anotop 25 syringe filters (Whatman) with 0.02-mm inorganic membranes were employed whenever backflushing the miniDAWN’s flow cell with the syringe pump. After calibration, the miniDAWN was connected to the SEC system preequilibrated at operating pressure. A 0.2-mg injection of bovine serum albumin (BSA) monomer (Ç98%, Sigma, St. Louis, MO) was employed for normalizing the 45 and 1357 light scattering (LS) detectors relative to the 907 detector. Normalization was required to correct for slight differences in light beam collimation, photodiode sensitivity, and refractive index effects among the three LS detectors. Use of
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a monodisperse material like BSA monomer also enabled the volume delay (0.077 ml) between the miniDAWN and refractometer to be determined, permitting proper alignment of the LS and refractive index (RI) signals as required for calculation of the weight-average molecular weight (Mw) for each data slice. The RI detector’s 100-mV output signal was amplified to 1 V and digitized by the miniDAWN’s digital signal processing (DSP) circuitry. The RI detector was calibrated at 321 attenuation over a linear concentration range (0.1–1.9 mg/ml) with seven evenly spaced sodium chloride standards prepared by direct dilution from a 5 mg/ml stock solution. Sample Preparation Solutions of each LMW-heparin (10 mg/ml) were prepared in ammonium acetate eluant the day before each analysis and stirred 4 h. The extended sample dissolution time was necessary to reduce the size of a high Mr aggregate peak detected by light scattering in the SEC column’s exclusion volume (data not shown). Aliquots were then centrifuged in Microfilterfuge tubes (Rainin, Woburn, MA; 0.2-mm cellulose acetate membrane) prior to injection. Two hundred microliters was injected, representing Ç2 mg of LMW-heparin on column.
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Dextran Control A 10 mg/ml solution of a 5000 Mr dextran standard (Pharmacosmos, Viby Sj., Denmark; distributed through American Polymer Standards Corp., Mentor, OH) was prepared in ammonium acetate eluant. It was syringe-filtered through a 0.2-mm Anotop 25 membrane and stored under refrigeration for use as a system suitability control throughout the LMW-heparin study. Each analysis incorporated five 200-ml control injections, interspersed at the beginning and end of the injection sequence as well as between each subset of LMW-heparin replicates. The manufacturer determined the Mw of this dextran standard by the LALLS technique. These control injections provided independent confirmation of the accuracy of the Mw estimates generated by the SEC/MALLS method. The dextran standard served as a control and was not utilized for calibration of the miniDAWN detector or SEC column. Refractive Index Increment Determination The differential refractive index increment (dn/dc) defines how a solution’s refractive index (n) changes with solute concentration (c). This parameter was required for converting RI voltages to solute concentrations at each data slice across a chromatographic peak and was determined individually for each LMW-heparin product (see Table 1). Measurements were conducted in 0.1 M ammonium acetate at 257C, using a calibrated Optilab DSP interferometric refractometer (Wyatt) operating at 690 nm (the same wavelength as the miniDAWN’s 20-mW GaAs semiconductor laser). Before calculating dn/dc, the mass concentration at four to six test dilutions was corrected for the moisture content of each LMW-heparin. For BSA and dextran, dn/dc values of 0.180 and 0.148 ml/g were employed, respectively. Chromatography The Shodex OHpak SB-803 HQ column was equilibrated overnight at operating pressure in the same batch of ammonium acetate eluant used for sample preparation. A gradient controller allowed the flow rate to be changed gradually (linear gradient between 0.20 and 0.80 ml/min over 30 min), thereby protecting the column from sudden pressure surges and prolonging its suitability for light scattering applications. Throughout the study a noise bandwidth of Ç5 mV was observed at the 457 LS detector, the one most sensitive to particulate shedding from the column. Each analysis was conducted at 0.80 ml/min and ambient temperature (20–227C). Data Collection Three analog signals from the LS detector array and one RI signal were digitized by the miniDAWN’s DSP
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circuitry before acquisition by a Pentium-based computer running Astra for Windows software (Wyatt). After an initial 5-min (4-ml) collection delay, data acquisition proceeded at a rate of 2 samples/s for 12.5 min (10 ml), yielding 1500 data slices per chromatogram. Each raw data slice thus incorporated three LS signals and one RI signal and corresponded to an elution volume of 6.7 ml. Study Design To assess the SEC/MALLS method’s reproducibility, accuracy, and precision, the seven LMW-heparin products were subjected to three independent analyses conducted at 1-week intervals. Each day of analysis single injections of all seven LMW-heparins were made, and the entire injection sequence was repeated three times. In this manner triplicate injections from each sample solution occurred 2.7 h apart, thereby testing the stability of the entire chromatographic system. Each week the injection order of the LMW-heparins was randomized to remove potential sequence bias. RESULTS
Details of the SEC/MALLS Analysis for Fragmin We chose the Fragmin data to illustrate the analytical procedure for two reasons. First, the manufacturer provided estimates for all moments of the Mr distribution (compiled in Table 4). Second, Fragmin is one of only two LMW-heparin products currently approved for clinical use in the United States by the FDA (the other being Lovenox). A three-dimensional plot of the RI and three LS detector signals recorded during a single analysis of Fragmin is presented in Fig. 2. After setting baselines and defining peak boundaries, the software converted the RI voltages from 286 volume slices across the chromatographic peak to solute concentrations (g/ml) by multiplying each voltage by the RI calibration constant (2.60 1 1004) and dividing by the product’s dn/dc value (0.129 ml/g). A trace of high Mr aggregate can be seen at 5.5 ml in the 907 LS profile (Fig. 3), marking the exclusion volume of the column. The prominent RI peak at 11 ml marks the column’s inclusion volume and apparently results from counterion exchange between the LMWheparin polyelectrolyte (as the sodium salt) and the ammonium acetate eluant. The intensity of this sodium peak is dramatically reduced in chromatograms of the dextran control, a nonionic polysaccharide. This phenomenon may be related to the occurrence of two counterion forms in sodium heparin (‘‘condensed and delocalized’’ vs ‘‘active and dissociated’’), as postulated by Manning’s polyelectrolyte theory (14). Although the 907 LS and RI chromatograms appear
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FIG. 2. Three-dimensional plot of detector signals acquired during a single SEC/MALLS analysis of Fragmin. Chromatograms (from left to right) correspond to the RI, 138.57 LS, 907 LS, and 41.57 LS detectors and are oriented with the injection on the left.
FIG. 3. Overlay of the RI (light) and 907 LS (dark) chromatograms for a single SEC/MALLS analysis of Fragmin. The Mr distribution is calculated for the elution volume between the vertical markers.
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to be offset in Fig. 3, this actually indicates that the LMW-heparin sample is polydisperse, i.e., composed of a mixture of molecules of varying Mr . Because the LS detector responds to differences in Mr , the light scattering signal is strongest on the leading edge of the SEC peak where the larger molecules elute. Data Processing and Calculation of Results To describe our LMW-heparin data further, we must first review a few general light scattering concepts discussed in greater detail by Wyatt (15). In the Rayleigh– Gans–Debye approximation (Eq. [1]) K*c 1 Å / 2A2c, R(u) MrP(u)
[1]
the quantities on the left are measurable (as described below), whereas most on the right must be derived. Here M is the weight-average molecular weight and P(u) is the theoretical form factor, describing the angular and size dependence of scattered light intensity. For small molecules such as LMW-heparins (rms radius ! l/20), the intensity of scattered light does not show strong angular dependence and P(u) approaches unity. The second virial coefficient, A2 , corrects for solute/solvent interaction and in a first approximation is usually small enough to be ignored at the concentrations encountered in chromatographic analyses. In practice, Eq. [1] is solved by linear regression analysis (weighted for the observed background noise at each LS detector) of K*c/R(u) versus sin2(u/2) in a Debye plot (Fig. 4), which provides an estimate of 1/M for each volume slice directly from the y-intercept. Here, (i) K* is an optical constant incorporating the solvent’s refractive index, the solute’s dn/dc, the wavelength of incident light in vacuum (690 nm), and Avogadro’s number; (ii) c is the solute concentration (g/ml) in each volume slice, as measured by the on-line RI detector; and (iii) R(u) is the excess Rayleigh scattering (i.e., scattered light intensity above background) at angle u. Table 2 presents the Mw for each daily replicate injection of Fragmin, calculated as ( (cirMi)/( ci over 286 volume slices in the chromatographic peak. When graphed together, these slice Mw values form the Mr vs volume plot shown in Fig. 5, the familiar SEC column calibration curve. These data may also be used to construct a cumulative distribution plot (Fig. 6), from which the proportion of sample below a given Mr can be tabulated through the software. Similarly, the numberaverage molecular weight values (Mn) reported in Table 2 for each daily replicate of Fragmin were calculated according to the formula ( ci/( (ci/Mi). Peak molecular weight values (Mp) were obtained through the software in the Debye plot window. Mp corresponds to the Mr
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FIG. 4. Debye plot of K*c/R(u) versus sin2(u/2) from a single SEC/ MALLS analysis of Fragmin. Plot shows information from one data slice within the designated peak boundaries. Plotted points correspond to the three LS detectors. The y-intercept of the weighted regression line provides an estimate of 1/Mw for this particular data slice. The LS detector angles employed in the calculations are corrected for the refractive index of water (1.330) and correspond to observed angles (uobs) of 41.5, 90.0, and 138.57.
at the apex of the RI peak, i.e., the Mr of the sample component present in highest concentration. Comparison of Results from Seven LMW-Heparins Each LMW-heparin product included in the SEC/ MALLS study was analyzed in triplicate in three independent analyses performed at 1-week intervals. Tables 3A and 3B summarize the results for each LMWheparin by day and provide their overall means and coefficients of variation [CV Å standard deviation/ mean]. Approximately 80% of the daily CVs were less than 1%, whereas 98% were less than 2% and none exceeded 3%. These data clearly demonstrate the excellent intraassay precision (i.e., same day) of the SEC/ MALLS method for the analysis of LMW-heparins. Interassay precision (i.e., different days) was generally somewhat lower (i.e., exhibited higher CVs). Of 28 overall CVs (n Å 9) reported for the LMW-heparins in Tables 3A and 3B, 7 were õ1%, 11 were õ2%, 23 were õ3%, 26 were õ4%, and none exceeded 5%. This level of interassay precision is quite acceptable for routine quality control purposes, being comparable to relative SEC methods employing calibration standards. Table 4 compares our overall (n Å 9) SEC/MALLS results (mean { 95% confidence interval) with the values reported by the LMW-heparin manufacturers. Most of the manufacturers’ own estimates for Mw agreed remarkably well with our results, being within 175 of the reported value for Fluxum, Fragmin, Lovenox, and Sandoparin. Generally, the Mn , Mp , and polydispersity figures were also in reasonably good agreement. Taking experi-
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SEC/MALLS ANALYSIS OF LMW-HEPARINS TABLE 2
SEC/MALLS Analysis of Fragmin Day
Injection
Mpa
Mwb
Mnc
Mw /Mnd
1
A B C Mean CV (%) A B C Mean CV (%) A B C Mean CV (%) Mean CV (%)
4797 { 55e 4802 { 90 4779 { 20 4793 0.25 4513 { 80 4586 { 60 4532 { 57 4544 0.83 4648 { 45 4649 { 50 4638 { 65 4645 0.13 4660 2.37
6157 { 200 6066 { 200 6123 { 200 6115 0.75 6018 { 300 6074 { 200 6041 { 200 6044 0.47 6146 { 100 6179 { 200 6141 { 100 6155 0.34 6105 0.93
5562 { 200 5499 { 200 5532 { 200 5531 0.57 5370 { 300 5420 { 300 5368 { 300 5386 0.55 5493 { 100 5530 { 200 5475 { 100 5499 0.51 5472 1.29
1.107 { 0.042 1.103 { 0.053 1.107 { 0.048 1.106 0.20 1.121 { 0.088 1.121 { 0.081 1.125 { 0.071 1.122 0.24 1.119 { 0.032 1.117 { 0.042 1.122 { 0.036 1.119 0.19 1.116 0.71
2
3
Overall (n Å 9)
a Mp (peak molecular weight) was measured at the apex of the refractive index peak and corresponded to the molecular weight of the sample component present in highest concentration. b Mw is the weight-average molecular weight. c Mn is to the number-average molecular weight. d Mw /Mn represents the polydispersity of the sample. e Statistical uncertainty reported by the MALLS software. Based on the background noise at each detector, these uncertainties are intended to express the statistical consistency of the data and not to set absolute limits on the accuracy of results.
mental error into consideration, the SEC/MALLS method yielded results consistent with the manufacturers’ own independent analyses.
FIG. 5. Molecular weight versus elution volume curve from a single SEC/MALLS analysis of Fragmin. The corresponding RI chromatogram is overlaid for reference. The Mr distribution across the peak is linear until a compact, dense sample fraction emerges along the trailing edge of the peak. This fraction remains undetected by RI.
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Dextran Control Results Results from the SEC/MALLS analysis of the dextran control are also summarized in Table 3B. This Mr standard was routinely included in each analysis to verify system performance. It was not employed for column calibration purposes. Intraassay precision for Mp and Mw were consistently high, with CVs £0.8% for five replicate injections occurring 2.7 h apart. For Mn and polydispersity intraassay precision was slightly lower, with CVs ranging from 1.2 to 2.2%. Interassay precision, as measured by overall CVs based on a total of 15 determinations, was comparable to the LMWheparin figures: õ0.5% for Mw , õ2% for Mp , and õ3% for both Mn and polydispersity. Our overall (n Å 15) Mw of 5445 { 15 (mean { 95% confidence interval) for the dextran control compared reasonably well (within 4.5%) with the LALLS value of 5700 reported by the manufacturer (Table 4). Their Mn estimate of 3326 was determined by reducing endgroup titration (EGT) and was substantially lower than the present MALLS estimate of 4299 { 70. Polydispersity (Mw/Mn) varied accordingly: 1.268 { 0.020 (found) vs 1.71 (reported, as the LALLS/EGT ratio). The manufacturer also provided an Mp estimate of 4440 by gelpermeation chromatography with relative standardization, which compared very well (within 2.5%) with our estimate of 4552 { 47. Considering the different analytical techniques em-
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FIG. 6. Cumulative distribution plot from a single SEC/MALLS analysis of Fragmin. The utility of this plot is suggested by the dashed lines, which indicate that 50% of the sample mass is below (and conversely, above) 5350 in molecular weight.
ployed, the SEC/MALLS method yielded reasonable results for the dextran standard. Its use as a control (not as a column calibrant) in the present SEC/MALLS procedure served to increase operator confidence in the accuracy, consistency, and general applicability of the LMW-heparin results. DISCUSSION
Before use in Mr analysis, a new SEC column is usually calibrated with a set of relative standards. The
validity of an existing calibration curve can then be verified by including high and low Mr standards in each sample set. Kristensen et al. (11) markedly improved the reliability of their relative SEC method by adding an internal standard (sucrose) to all standard and sample solutions and tracking its elution volume over time. They demonstrated that a 2% error in flow rate can result in a 24% error in Mp and a 40% error in Mn and Mw . This large potential error results from the fact that molecular weights are plotted logarithmically against
TABLE 3A
SEC/MALLS Analyses of LMW-Heparins
Product
Day
Fluxum
Overall Fragmin
(n
Overall Fraxiparin
(n
Overall Logiparin
(n
Overall
(n
1 2 3 Å 1 2 3 Å 1 2 3 Å 1 2 3 Å
9)
9)
9)
9)
Mp mean (CV %)a 4003 3795 3865 3888 4793 4544 4645 4660 3300 3107 3167 3191 4596 4368 4488 4484
(0.59) (0.19) (0.46) (2.39) (0.25) (0.83) (0.13) (2.37) (0.62) (0.97) (0.92) (2.78) (0.23) (0.81) (0.39) (2.25)
Mw mean (CV %) 5173 5078 5107 5119 6115 6044 6155 6105 4340 4291 4337 4323 6256 6243 6183 6227
a
(0.73) (0.43) (0.24) (0.93) (0.75) (0.47) (0.34) (0.93) (0.41) (0.62) (1.08) (0.86) (0.30) (0.52) (0.74) (0.72)
Mn mean (CV %) 4346 4155 4100 4200 5531 5386 5499 5472 3714 3590 3565 3623 5000 4973 4702 4892
(1.08) (0.56) (0.06) (2.74) (0.57) (0.55) (0.51) (1.29) (0.93) (0.82) (1.72) (2.18) (0.52) (0.78) (0.89) (2.98)
Mw /Mn mean (CV %) 1.190 1.222 1.246 1.219 1.106 1.122 1.119 1.116 1.169 1.195 1.217 1.194 1.251 1.256 1.315 1.274
(0.37) (0.16) (0.27) (1.99) (0.20) (0.24) (0.19) (0.71) (0.55) (0.27) (0.81) (1.82) (0.24) (0.57) (0.32) (2.44)
Unless otherwise indicated, the daily means correspond to triplicate injections from the same LMW-heparin solution that were spaced 2.7 h apart. For the dextran control, daily means are from five injections spaced 2.7 h apart.
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SEC/MALLS ANALYSIS OF LMW-HEPARINS TABLE 3B
SEC/MALLS Analyses of LMW-Heparins
Product
Mp mean (CV %)a
Day
Lovenox
Overall Normiflo
(n
Overall Sandoparin
(n
Overall Dextran
(n
Overall
(n
1 2 3 Å 9) 1 2 3 Å 9) 1 2 3 Å 9) 1 2 3 Å 15)
2935 2748 2821 2835 5537 5225 5318 5360 3952 3761 3863 3859 4633 4448 4574 4552
Mw mean (CV %)
(0.59) (1.54) (0.92) (3.02) (0.59) (0.38) (0.52) (2.63) (1.28) (0.94) (0.39) (2.30) (0.80) (0.46) (0.57) (1.85)
4011 3923 3957 3964 6845 6772 6736 6784 5050 5008 4995 5018 5440 5453 5443 5445
Mn mean (CV %)
(0.61) (0.90) (0.95) (1.20) (0.88) (0.37) (0.43) (0.88) (1.31) (0.75) (0.63) (0.96) (0.42) (0.73) (0.32) (0.49)
3292 3139 3096 3176 5416 5371 5069 5286 3704 3681 3373 3586 4361 4384 4152 4299
(0.84) (1.31) (1.43) (3.00) (0.85) (1.49) (1.41) (3.28) (1.47) (2.95) (1.30) (4.81) (1.46) (2.22) (1.16) (2.96)
Mw /Mn mean (CV %) 1.218 1.250 1.278 1.249 1.264 1.261 1.329 1.285 1.363 1.361 1.481 1.402 1.248 1.244 1.311 1.268
(1.34) (0.56) (0.48) (2.21) (1.11) (1.13) (0.99) (2.76) (0.97) (2.17) (1.05) (4.42) (1.54) (1.54) (1.18) (2.83)
a Unless otherwise indicated, the daily means correspond to triplicate injections from the same LMW-heparin solution that were spaced 2.7 h apart. For the dextran control, daily means are from five injections spaced 2.7 h apart.
elution volume when constructing column calibration curves. Recently, the European Pharmacopeia (EP) Commission proposed an official method for determining the
Mr distribution of LMW-heparins by SEC. Based on the work of van Dedem and Nielsen (17, 18), the EP calibrant was prepared by partial enzymatic depolymerization of UF heparin with heparinase. When run
TABLE 4
Comparison of SEC/MALLS Results for LMW-Heparins Product (lot no.)
Mp (reported)
Mw (reported)
Mn (reported)
Mw /Mn (reported)
Fluxum (90248) Fragmin (62158-51) Fraxiparin (4096XH) Logiparin (LMW9405) Lovenox (PRS122) Normiflo (RD13390) Sandoparin (76465002) Dextran (096001)
3888 { 71a (4200) 4664 { 85 (5100) 3191 { 68 (4162) 4484 { 78 (4500) 2835 { 66 (n/a) 5360 { 108 (n/a) 3859 { 68 (n/a) 4552 { 47 (4440) d
5119 { 37 (5240) 6105 { 44 (6250) 4323 { 28 (4991) 6227 { 35 (n/a)b 3964 { 37 (4139) c 6784 { 46 (5953) 5018 { 37 (5020) c 5445 { 15 (5700) e
4200 { 88 (4190) 5472 { 54 (5100) 3623 { 61 (4074) 4892 { 112 (n/a) 3176 { 73 (n/a) 5286 { 133 (5235) 3586 { 132 (n/a) 4299 { 70 (3326) f
1.219 { 0.019 (1.251) 1.116 { 0.006 (1.226) 1.194 { 0.017 (1.225) 1.274 { 0.024 (n/a) 1.249 { 0.021 (n/a) 1.285 { 0.027 (1.137) 1.402 { 0.048 (n/a) 1.268 { 0.020 (1.71) g
a
Mean { 95% confidence interval (n Å 9 for LMW-heparins, n Å 15 for the dextran control). Not available (information not reported by the manufacturer). c Reported simply as the ‘‘average’’ molecular weight. d Measured by SEC using relative standardization. e Determined by LALLS. f Determined by reducing end group titration (EGT). g LALLS/EGT ratio. b
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on a specific brand of column under defined conditions, the EP standard yields a jagged elution profile with the lower oligomers of heparin (di- through tetradecasaccharides) visible as regularly spaced peaks. Equidistant time points are then extrapolated into the high Mr region of the chromatogram, where the column does not resolve higher oligomers of heparin into distinct peaks. A calibration curve can then be constructed for calculating the molecular weight of LMW-heparins analyzed under the same conditions. Because the EP standard was prepared from UF heparin depolymerized enzymatically by b-elimination, the fragments contain a D4,5-unsaturated uronic acid residue at the nonreducing terminus, permitting their detection at 235 nm. Assuming the molar extinction coefficient of such fragments to be constant, RI and ultraviolet (uv) absorbance detectors operated in series provide a relative measure of Mr as the ratio of the RI/ uv peak heights (i.e., grml01/molrml01 Å g/mol). One tentatively assigns the identity of the octasaccharide peak and then normalizes the peak height ratios of the remaining peaks against it. The principle of the EP method maintains that these normalized peak height ratios should be close to integer values, if the octasaccharide peak was correctly assigned. The Mr of all the peaks are then calculated, based on the assumption that the average Mr of the repeating disaccharide unit of heparin is 600. Simplifying assumptions such as these are commonly made when preparing relative heparin Mr standards. The EP method was validated by analyzing heparin Mr standards (characterized by LALLS) on an SEC column calibrated with the EP standard and obtaining comparable results. Jeske et al. (19) reported that the Mr range obtained with the EP standard is too narrow for most LMWheparins and that it also overestimates the proportion of components above Mr 7500. They proposed an improved SEC calibrant prepared by alkaline hydrolysis of heparin benzyl ester (20, 21), which also results in depolymerization by b-elimination and introduction of a uv chromophore. Unfortunately, neither the EP calibrant nor this improved version is suitable for determining the Mr distribution of UF heparin. Compared to relative calibration techniques, the absolute SEC/MALLS approach we describe is applicable to both unfractionated and LMW-heparins. It makes no simplifying assumptions about the Mr of the basic heparin disaccharide unit. Furthermore, the SEC technique avoids the critical dependency of relative chromatographic methods on constant flow rate. By contrast, absolute Mr estimates derived from light scattering measurements are independent of flow rate: reanalysis of the same LMW-heparin solutions at 0.4 ml/min (one-half the normal flow rate) yielded identical results within experimental error of measurement (data not shown). Moreover, the present SEC/MALLS method delivers precise
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and accurate results within a remarkably short time. Whereas the dual-column EP method requires about an hour to complete a single analysis at a flow rate of 0.5 ml/min, our method performs triplicate analyses in the same amount of time by employing a single SEC column and a higher flow rate. System suitability and method accuracy can be verified by including a dextran control in each sample set. It should be noted that our results for Normiflo agree with an independent assessment of this same lot (No. RD 13390) reported by Atha et al. (16) of the National Institute of Standards and Technology (Gaithersburg, MD). By SEC analysis using relative standardization according to the EP method described above, they obtained values of 6850 for Mw , 5300 for Mn , and 1.3 for polydispersity, compared to our estimates of 6784, 5286, and 1.285, respectively. Low-angle laser light scattering detectors have successfully been employed for the Mr determination of UF heparin and heparin fractions (22, 23). LALLS instruments, however, are inherently more sensitive to noise interference from extraneous dust and column shedding than multiangle detectors, which extrapolate to zero angle rather than directly measuring scattered light intensity near it (i.e., at 3 to 77). The MALLS technique has previously been applied to heparin analysis (15), using the research-grade DAWN-F instrument (Wyatt) that measured scattering intensity simultaneously at 15 angles. The recent introduction of the less expensive, triple-angle miniDAWN detector employed here provides LMW-heparin manufacturers with a practical tool for routine quality control. With the above-described method, absolute Mr determinations can now be performed on LMW-heparins directly, without reference to relative heparin Mr standards that ultimately must be characterized by absolute methods. The commercial availability of this modern instrumentation, together with versatile Windows-based software for data acquisition and analysis, should now make SEC/MALLS technology accessible to a broader range of polymer chemists. ACKNOWLEDGMENTS The authors sincerely thank the following individuals and their corporate sponsors for participating in this study and graciously providing the LMW-heparin samples and independent Mr analyses: Dr. Carl Magnus Svahn (Pharmacia & Upjohn), Dr. Giuseppe Mascellani (Opocrin S.p.A.), Dr. Theodore Spiro (Rhoˆne-Poulenc Rorer), Dr. D. Scott Holloway (Wyeth-Ayerst Research), Dr. Helmut Wolf (Sandoz AG), Hanne Kristensen (Novo Nordisk A/S), and Drs. Maurice Petitou and Jean-Marc Herbert (Sanofi Recherche). The authors also express their appreciation to Edward Mancilla and Dr. Paul Weiss of Scientific Protein Labs for their support and encouragement during development of the analytical method. We are grateful to Dr. Philip Wyatt, Dr. David Shortt, and Lena Nilsson of Wyatt Technology Corp. for their expert advice and acknowledge the technical assistance of Dirk Sarcinelli and Robert Paulson in performing dn/dc measurements.
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