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Inhibition of Taq polymerase as a method for screening heparin for oversulfated contaminants
Biomaterials 29 (2008) 4808–4814
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Biomaterials journal homepage: www.elsevier.com/locate/biomaterials
Inhibition of Taq polymerase as a method for screening heparin for oversulfated contaminants Cecilia Tami a, Montserrat Puig a, John C. Reepmeyer b, Hongping Ye b, D. Andre D’Avignon c, Lucinda Buhse b, Daniela Verthelyi a, * a
Laboratory of Immunology, Division of Therapeutic Proteins, Office of Biotechnology Products, Center for Drugs Evaluation and Research, Food and Drug Administration, Rockville Pike, Bethesda, MD 20892, United States Division of Pharmaceutical Analysis, Center for Drugs Evaluation and Research, Food and Drug Administration, United States c Department of Chemistry, Washington University, St. Louis, MO, United States b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 23 June 2008 Accepted 20 August 2008 Available online 17 September 2008
Heparin and low molecular heparins are extensively used in the treatment of a wide range of diseases in addition to their classic anticoagulant activity and can be found coating medical devices such as catheters, stents and filters. Early in 2008, a sharp increase in heparin-associated severe adverse events, including over 80 deaths, was linked to the presence of a contaminant identified as hypersulfated chondroitin sulfate (OS-CS). OS-CS is one of several oversulfated glycosaminoglycans (GAGs) of different origins that can potentially cause similar clinical problems underscoring the need to develop robust screening methods for contaminants in existing and future lots of heparin. This study demonstrates that oversulfated GAGs block the activity of Taq polymerase used for real time PCR. Based on this finding we developed a simple, rapid, sensitive and high throughput screening method to detect and quantify oversulfated chondroitin sulfate (OS-CS) and other potential oversulfated contaminants in commercial lots of heparin. This method requires less than 100 miliUnits (mU) of heparin as starting material, therefore avoiding the need to lyophilize and concentrate samples, and has a limit of detection of <1 ng for all oversulfated GAGs tested. Published by Elsevier Ltd.
Keywords: Heparin Oversulfated chondroitin sulfate Glycosaminoglycan Taq polymerase PCR Safety
1. Introduction Heparin and low molecular heparins are widely used in the treatment of a wide range of diseases in addition to their classic anticoagulant activity [1–3]. Indeed, millions of doses of heparin are dispensed every month. The most significant adverse event linked to heparin have traditionally been increased bleeding and heparin-induce thrombocytopenia [4]. Between January 1, 2007 and April 13, 2008, the FDA received over 700 reports of adverse events in patients receiving heparin as part of their dialysis treatment or surgical procedures [5]. Adverse events included severe hypotension, vasodilation, facial swelling, tachycardia, urticaria, nausea, vomiting, diarrhea and abdominal pain, and resulted in over 80 deaths. Researchers at the Centers for Disease Control realized that the adverse events were associated with the receipt of
* Corresponding author. Laboratory of Immunology, Division of Therapeutic Products, Office of Biotechnology Products, Center for Drugs Evaluation and Research, Food and Drug Administration, Building 29A, Room 3B19, 8800 Rockville Pike, Bethesda, MD 20892, United States. Tel.: þ1 301 827 1702; fax: þ1 301 480 3256. E-mail address: [email protected] (D. Verthelyi). 0142-9612/$ – see front matter Published by Elsevier Ltd. doi:10.1016/j.biomaterials.2008.08.024
heparin sodium for injection (1000 U/ml, in 10 ml and 30 ml multidose vials), manufactured by Baxter Healthcare [6]. As a result, Baxter Healthcare issued recalls for all remaining lots and doses of its multidose and single-dose vials of heparin sodium for injection and HEP-LOCK heparin flush products in January and February 2008 [6–8]. This was followed by recalls for a number of medical devices that contain or are coated with heparin [5]. In March 2008, a similar recall was issued by Rotexmedica GmbH Arzneimittelwerk in Trittau, Germany and since then suspect lots have been identified in 11 other countries [9,10]. This indicated an extensive problem with heparin manufacture that was unlikely to be restricted to a single source. Using multidimensional nuclear magnetic resonance (NMR), enzymatic digestion followed by high-performance liquid chromatography, and liquid chromatography with mass spectrometry, Guerrini et al. identified an unusual oversulfated form of chondroitin sulfate (OS-CS) as a contaminant present in suspect lots of heparin [11]. The OS-CS contained a tetrasulfated disaccharide unit consisting of glucuronic acid linked to N-acetyl-D-galactosamine that was not evident in lots of heparin that were not linked to adverse events [11]. Kishimoto et al. [12] then were able to partially reproduce the clinical syndrome in a porcine model by
C. Tami et al. / Biomaterials 29 (2008) 4808–4814
inoculating a large dose of the pure contaminant (5 mg/kg i.v. in bolus) suggesting that the presence of OS-CS was linked to or possibly responsible for the adverse events. Proton nuclear magnetic resonance (H-NMR spectroscopy) and capillary electrophoresis (CE) tests were identified by the FDA as tests available to assess for the presence of OS-CS contaminant in products containing heparin sodium [8]. However, OS-CS is only one of numerous oversulfated compounds of animal, vegetable, insect or completely synthetic origin that could potentially be designed to co-purify and co-elute with heparin [12–16]. Most of these synthetic compounds have anticoagulant activity by current US pharmacopeia (clotting-based) tests and could be designed to give similar spectra by H-NMR as heparin, avoiding the identification by the tests currently in place. Such compounds would require methods such as high field spectra NMR for identification [15]. Further, for finished dosage forms, traditional tests such as NMR or CE cannot determine the presence of contaminant without lyophilizing and concentrating each sample, and may not be suitable for testing finished medical devises. 2. Materials and methods 2.1. Samples and reagents Heparin samples: active pharmaceutical ingredient (API) and final drug product (FDP) for samples B1–B3 and C1–C3 were obtained by the FDA from Baxter Healthcare (1000 U/ml or 5000 U/ml in 10 ml and 30 ml vials). Eight blinded samples (Blind #1 to 8) were obtained during FDA’s inspections. Chondroitin sulfate E was obtained from Seikagaku (Japan) and characterized by the HNMR method stated below. Heparinase I was obtained from Sigma (St Louis, MO, USA). RNA was extracted from MDA-MB-231 human breast cancer cell line grown in DMEM/F12 (50/50) media. 2.2. Heparinase treatment C
in Heparin was treated with heparinase I (1:1 unit:unit reaction) for 2 h at 25 a buffer containing 4 mM Tris–HCl pH 7.5, 0.8 mM CaCl, 10 mM NaCl and 20 U of RNase inhibitor (Applied Biosystems, Foster City, CA, USA) (ABI). After treatment, the heparin was diluted to a final volume of 50 ml and serial dilutions were prepared in DEPC water as specified. Where indicated, other GAGs and oversulfated GAGs (dermatan sulfate, chondroitin sulfate, heparan sulfate) were treated with heparinase under the same conditions as described above. 2.3. Screening assay cDNA was generated using total RNA from MDA-MB-231 human breast cancer cell line extracted using Trizol reagent (Invitrogen, Carlsbad, CA, USA) as per manufacturer’s instructions. For each cDNA reaction 1 mg total RNA was reverse transcribed using High Capacity cDNA Reverse Transcription Kit (ABI) in a volume of 20 ml and further diluted in DEPC water to 100 ml final volume. The concentration of cDNA was determined using Quant-iT OliGreen ssDNA Reagent (Invitrogen). For each Taqman reaction, 25 ng of cDNA in a 2.5 ml volume were mixed with 2.5 ml of each dilution of – heparinase-treated– heparin or hypersulfated GAGs and then subjected to real time PCR using Taqman Gene Expression assay for human 18S rRNA (20) and 2X Universal PCR master mix (ABI) in a 25 ml reaction. Amplification levels of 18S are expressed as CT values. CT values represent the cycle at which amplification of a target gene is first detected. The amplification was analyzed using manual settings with a threshold value of 0.1 and the SDS2.3 software from ABI. CT values < 16 cycles indicate there was no significant inhibition of the Taq polymerase activity while CT values of >35 indicate complete inhibition of the assay. Quantification of the oversulfated GAGs was performed by establishing a cutoff at the minimum concentration of synthetic OS-CS that completely blocks Taq polymerase (500 pg) and tittering all the samples until the activity of Taq polymerase was restored. The maximal dilution that completely blocked the enzyme was then assumed to have at least 500 pg of contaminant. 2.4. H-NMR analysis All samples were analyzed using a Varian500 MHzInova instrument. Samples were prepared by dissolving approximately 10 mg of sample in 0.6 ml of deutered water spiked with a reference compound TSP (tri-methyl-silyl propionate, sodium salt). The reference TSP signal is set to 0.00 ppm, which is referenced at 0.00 ppm. Samples were run at 25 C. Spectral parameters include no less than 16 transient, 90 pulse width, acquisition time of at least 1 s, time between transients of 20 s and a spectral window of 8000 Hz.
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2.5. Capillary electrophoresis CE was conducted on a Hewlett Packard 3D-CE instrument equipped with a diode array detector set at a wavelength of 200 nM (band width 10 nM). Separations were performed in a bare fused silica capillary, internal diameter 50 mM, 64.5 cm-total length, 56 cm-effective length with a column temperature of 25 C. The polarity was negative with a voltage of 30 kV. Samples were dissolved in Milli-Q water at a concentration of approximately 10 mg/ml and filtered through 0.2 mM cellulose acetate membrane filters (Micro-Spin filter tubes, Alltech Associates, Deerfield, IL, USA). The sample solutions were injected using hydrodynamic pressure at 50 mbar for 10 s. The electrolyte solution was 36 mM phosphate buffer (pH 3.5) filtered with a 0.2 mM cellulose acetate syringe filter (Grace, Deerfield, IL, USA). The capillary column was preconditioned at the beginning of each day by flushing with 1 M NaOH, 0.1 M NaOH, and water, each for 2 min, and prior to running each sample by flushing with water for 2 min and electrolyte solution for 2 min. Monobasic sodium phosphate, monohydrate, ACS grade, and phosphoric acid 85%, N.F. Food Grade, were obtained from Mallinckrodt Baker, Inc (Phillipsburg, NJ, USA). 1 M and 0.1 M Sodium hydroxide solutions for High-Performance Capillary Electrophoresis (HPCE) were from Hewlett Packard (Waldbronn, Germany). 2.6. Chemical sulfonation of chondroitin sulfate Fully sulfated chondroitin sulfate was prepared from chondroitin sulfate as described [14]. Thus, 139 mg chondroitin sulfate tributylamine salt and 1.2 g sulfur trioxide pyridine complex were dissolved in 2 ml dry N,N-dimethylformamide and heated for 1 h at 40 C. The reaction solution was adjusted to pH 9 with 1 M NaOH and diluted with 3 volumes of ethanol saturated with sodium sulfate, generating a precipitate. The mixture was cooled in a refrigerator, centrifuged, and the solid material was purified by dialysis and lyophilization. Heparan sulfate, dermatan sulfate, chondroitin sulfate A, and E as well as oversulfated heparin (OS-HS) and dermatan sulfate (OS-DS) were synthesized in house as described [15,17] and characterized by H-NMR (Supplementary Fig. 1), CE and elemental analysis for sulfur content (performed at Galbraith Laboratories).
3. Results 3.1. Effect of heparin contaminants on gene amplification by Taqman PCR Real time PCR (Taqman PCR) is a highly sensitive method for detecting changes in gene expression. Gene amplification depends on a thermostable DNA polymerase from Thermus aquaticus (Taq pol). Previous studies have shown that heparin competitively inhibits several different cellular DNA polymerases including Taq pol [18–21], but this inhibition can be overcome by the use of heparinase [20,22]. Shown in Fig. 1 are the levels of 18S amplification from 25 ng cDNA derived from MDA-MB-231 cells as determined by Taqman PCR. Addition of progressively higher amounts of heparin induces a corresponding reduction in Taq pol activity that can be monitored by assessing 18S cDNA amplification. Cycle thresholds (CT) < 16 indicate no inhibition of the Taq pol activity whereas CT values > 35 denote complete inhibition of the assay. Treatment of heparin with 1 U of heparinase for 2 h at 25 C overcomes the blocking activity of up to 50 mU of heparin (Fig. 1a). To determine whether the presence of contaminants modulates the blocking effect of heparin on Taq pol, we designed an assay in which heparin from lots that were associated with adverse clinical effects (B1, B2 and B3) as well as from control lots with no visible clinical effects (C1, C2, C3) were treated with heparinase (or left untreated). Sequential dilutions of heparin were then added onto 25 ng of cDNA and 18S was amplified by Taqman PCR. As shown in Fig. 1b, while 18S amplification was maximal in control heparinasetreated lots (C1–C3), heparinase treatment did not restore 18S amplification of cDNA exposed to lots of heparin associated with adverse events (B1–B3). This suggested that a contaminant in the heparin inhibits the Taq pol enzyme. Of note, addition of as little as 6.25 mU of heparinase-treated heparin to the Taqman reaction was sufficient to clearly differentiate between contaminated and uncontaminated lots regardless of whether the starting material was finished formulated drug product or the corresponding
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a
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Heparinase treatment restores amplification by RT-PCR
Lot
Hepa- Hepari 100 rin nase
50
25
12.5
C1
+
-
N/T
N/T
N/T
N/T
C1
+
+
16.8
14.8
14.1
13.9
b
6.3
13.6
3.1
13.7
1.6
0.8
0.4
0.2
0.1
0.1
0.0
25.1
19.2
15.6
14.4
13.8
13.9
14.1
13.8
N/T
N/T
N/T
N/T
13.9
1.6
0.8
0.4
0.2
0.1
0.1
0.0
25.1
19.2
15.6
14.4
13.8
13.9
13.8
N/T
N/T
N/T
N/T
24.7
17.5
16.3
14.0
14.1
N/T
N/T
N/T
N/T
30.5
18.6
15.2
14.6
13.8
13.7
N/T
N/T
N/T
N/T
20.3
19.2
14.3
13.8
14.1
13.5
21.8
16.2
14.2
Heparinase does not restore amplification in contaminated lots 100
C1
FDP
C2
FDP
FDP
FDP
12.5
6.3
3.1
16.8
14.8
14.1
13.9
13.6
13.7
14.1
N/T 14.8
14.2
14.0
14.0
14.1
13.8
13.9
+
B1
25
+
C3
50
+
14.8
14.1
14.0
14.0
13.9
14.0
14.0
+
B2
FDP
B3
FDP
22.3
15.5
+
17.6
14.4
14.0
13.9
-
26.0
18.3
14.9
14.0
+
c
mUnits
mUnits
14.37 13.89 13.76 13.97 13.93
Oversulfated chondroitin sulfate blocks 18S amplification 1.25
1
0.75
0.5
0.25
0.08
0
No amplification (Ct >35)
ng
OS-CS
-
-
17.3
14.14 14.2
Reduced amplification (Ct 16-35)
OS-CS
C1
+
15.0
14.2
Maximal amplification (Ct<16)
d
14.1
Effect of natural and oversulfated glycosaminoglycans on 18S amplification by RT-PCR 125
62.5
25
8.3
2.7
0.9
0.3
0
ng
% sulfur content
DS
C1
+
14.9
14.2
13.9
13.7
13.7
N/T
N/T
13.6
6.3
HS
C1
+
13.9
13.8
13.7
13.5
13.5
N/T
N/T
13.6
5.0
CS-A
C1
+
13.6
13.6
13.7
13.6
13.6
N/T
N/T
13.6
6.4
CS-E
C1
+
19.7
15.6
14.2
13.7
13.6
N/T
N/T
13.6
10.6
OS-DS
C1
+
32.7
15.6
13.6
16.0
OS-DS
C1
+
27.0
15.38 13.6
13.1
OS-HS
C1
+
32.9
14.6
13.6
16.4
OS-CS
C1
+
16.1
13.6
19.2
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non-formulated active pharmaceutical ingredient (API) diluted in water (Fig. 2a). 3.2. Use of Taq polymerase inhibition to screen for the presence of OS-CS in heparin As mentioned above, OS-CS was identified as a contaminant present in lots of heparin that have been linked to adverse events [11]. Previous studies have established that persulphated chondroitin sulfate is not susceptible to heparinase I or II (or chondroitinase) degradation. To determine whether OS-CS directly inhibits Taq pol activity, decreasing concentrations of OS-CS were added to cDNA and amplified by Taqman PCR. As shown in Fig. 1c, addition of 500 pg of OS-CS completely blocked Taq pol mediated 18S cDNA amplification. In order to determine the minimum % (w/w) of contaminant that could be detected in heparin, we used a representative potency of heparin (6.25 mg/U). Since 500 pg of OS-CS completely block 18S amplification, the lowest titer of heparin that completely blocked amplification was assumed to contain at least 500 pg of OS-CS. From this, the limit of detection was calculated as 0.16% (w/w), which is below the level of detection for both proton NMR and CE. Importantly, the presence of heparin and heparinase did not modify the sensitivity of the assay (Fig. 1c). These results indicate that failure to amplify 18S cDNA can be used as a rapid and sensitive diagnostic test to screen for the presence of OS-CS in heparin. Lastly, similar gene amplification inhibition levels were evident whether the suspect heparin or the OS-CS were added to the cDNA or to the RNA used to generate the cDNA, indicating that any contaminant present likely co-purifies with nucleic acids (Fig. 2b). This raises the possibility that the presence of oversulfated contaminants in heparin may have interfered with PCR-based assays that are currently used to test heparin lots for contaminants from material from other natural sources such as bovine heparin. 3.3. Comparison of the Taq pol inhibition method with current methods used to screen and quantify OS-CS To verify the method as a screening assay for the presence of oversulfated contaminants, 8 lots of heparin API were tested in a blinded manner. Each was treated with heparinase I, and titered into a Taqman PCR reaction. Addition of 6.25 mU of heparin resulted in complete inhibition of amplification for two samples (blind #7 and 8) (Fig. 3a). Those samples were later identified as having 15 and 27% OS-CS contamination by CE, and showing significant peaks at 2.16 ppm by NMR (Fig. 3b and not shown). For blind samples #1 and 2, there was reduced amplification of 18S at 6.25 mU and complete inhibition when 25 or more mU of heparin were added to the PCR reaction. Subsequent unblinding of the NMR and CE profiles for these particular samples showed the presence of a weak signal at 2.16 ppm by H-NMR but no visible peak by CE, a profile that suggests marginal OS-CS contamination (Fig. 3b). Samples 3 and 4 showed a reduced 18S amplification at heparin concentrations of 100 mU and 25 mU of heparin but did not completely block it suggesting that trace levels of contaminants could be present in these samples. This indicated that Taq pol inhibition is more sensitive to OS-CS contamination than CE or H-NMR. The remaining samples (Bl #5 and 6) did not inhibit 18S
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amplification. Lastly, comparison of the OS-CS concentration, as determined by inhibition of 18S amplification, to the percent OS-CS as determined by CE shows a high level of correlation (r2 ¼ 0.9, p < 0.001) confirming that the Taqman-inhibition based method is an effective semi-quantitative screening assay, likely to identify very low levels of oversulfated contaminants in heparin. 3.4. Susceptibility of Taq polymerase to other potential oversulfated GAGs Heparin is commonly extracted from porcine intestinal mucosa or bovine lung, and preparations may contain small amounts of other glycosaminoglycans. The presence of naturally occurring dermatan sulfate (DS), heparan sulfate (HS) or chondroitin sulfates A (CS-A), when spiked into heparin and treated with heparinase did not inhibit Taq pol activity at any of the concentrations tested (0–125 ng) (Fig. 1c). Chondriotin sulfate E (CS-E), which is more sulfated that chondroitin sulfate A (CS-A) or DS, did show a trace of inhibitory effect at the highest concentration tested (125 ng). In contrast, under the same conditions, all oversulfated glycosaminoglycans tested, including oversulfated heparan sulfate (OS-HS), two different forms of oversulfated dermatan sulfate (OS-DS) and OS-CS significantly reduced 18S amplification. Further, the magnitude of the inhibitory effect on Taq pol correlated with the degree of sulfation of each compound as determined by elemental analysis (r2 ¼ 0.93, p < 0.001). This indicates that Taq pol inhibition is a useful tool to screen for other potential oversulfated contaminants that could contaminate heparin causing adverse effects in the future. 4. Discussion Heparin is an essential drug for many patients. Licensed since the early 20th century, it is widely used as an anticoagulant in multiple settings including invasive surgical and dialysis procedures, and deep venous thrombosis treatment. Although usually well tolerated, in recent months hundreds of reports of serious adverse responses were followed by the discovery of contaminating OS-CS in heparin lots around the world. While the adverse events were linked to lots that have >5% OS-CS, at this time, we do not have the necessary data to establish the pathogenic impact of the presence of smaller amounts (<2%) of the OS-CS, nor understand the precise mechanism by which OS-CS may cause severe adverse events in a small but significant percentage of patients. Indeed, although the activation of the bradikinin–kallikrein system has been identified as potentially mediating some of the adverse events that have been associated with tainted heparins, other potential mechanisms are under investigation and the long term effects of these contaminants will need to be carefully considered. Importantly, future investigations will have to include an assessment of the potential for OS-CS and other oversulfated compounds to form more stable complexes with chemokine PF4, which could increment the immunogenicity of heparin and the risk of heparininduced thrombocytopenia (HIT) [23]. Together, these events have underscored the need to develop robust characterization methods to ensure the integrity of the global supply of heparin. This study demonstrates that the inhibition of Taq polymerase can be used as a highly sensitive assay to test
Fig. 1. Effect of glycosaminoglycans on 18S amplification. The effect of heparin on Taq polymerase activity was evaluated by quantitative real time PCR. Heparin lots were treated with heparinase I (1:1 U/U) for 2 h at 25 C or left untreated. Gene amplification was evaluated by real time PCR using the Taqman Gene Expression assay for human 18S rRNA (20) and 2X Universal PCR master mix (ABI) in a 25 ml reaction in the presence of increasing concentrations (0.5–100 mU) of heparin (heparinase-treated or untreated). Shown in: a) are the effects of heparin and heparinase-treated heparin on 18S amplification. b) Comparison of 3 final drug product lots of heparin characterized as contaminated by NMR and CE (B1–B3) with 3 lots of control heparin lots (C1–C3). c) Effect of synthetic OS-CS on 18S amplification in the presence and the absence of heparinase digested heparin. d) Effect of natural and oversulfated synthetic glycosaminoglycans on 18S amplification by PCR, DS: dermatan sulfate, HS: heparan sulfate, CS-A and E: chondroitin sulfate type A and E respectively, OS-HS: oversulfated heparan sulfate, OS-DS: oversulfated dermatan sulfate; OS-CS: oversulfated chondroitin sulfate.
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a
Taq polymerase inhibition by heparinase-treated API or FDP heparin Heparinase
Lot
C1 API
+
C1 FDP
+
B1 API
+
B1 FDP
+
B2 API
+
B2 FDP
+
mUnits 100
50
25
13
6.3
3.1
1.6
0.8
0.4
0.2
0.1
0.05
15.0
14.1
13.8
13.7
13.6
13.6
14.2
13.3
N/T
N/T
N/T
N/T
16.8
14.8
14.1
13.9
13.6
13.7
14.1
13.8
N/T
N/T
N/T
N/T
27.2
31.6
20.2
15.1
14.0
22.3
15.5
13.8
14.1
13.5
15.7
14.1
14.0
13.9
17.6
14.4
14.0
13.9
0.0
13.9
No amplification (Ct >35) Reduced amplification (Ct 16-35) Maximal amplification (Ct<16)
b
OS-CS co-purifies with RNA and blocks gene amplification
-
-
-
-
-
B
-
+
-
-
-
C
+
-
-
-
-
D
-
-
-
+
-
E
-
-
-
+
+
F
-
-
+
-
-
G
-
-
+
-
+
Gene amplification
Heparinase on
cDNA
1U Heparin on
cDNA
1U Heparin on RNA
OSCS (30ng) on
total RNA
OSCS (30ng) on
cDNA
cDNA Sample A
No amplification (Ct >35) Maximal amplification (Ct<16) Fig. 2. Sensitivity of the Taq pol assay. a) Active pharmaceutical ingredient or final drug product similarly inhibit Taq polymerase activity. Three lots of API and their corresponding FDP were screened by Taq pol inhibition. API was diluted in RNAse free water. As shown, similar degrees of amplification were observed indicating that both API and FDP can be used as the starting material for screening heparin. b) Addition of OS-CS (B and C; 30 ng) or heparin (D and F; 25 mU of uncontaminated lot C1) to cDNA or to the RNA used to generate the cDNA resulted in similar Taq pol inhibition. Treatment of heparin – but not OS-CS – with heparinase for 2 h at room temperature restores gene amplification (E and G). Controls include cDNA treated with heparinase buffer (A).
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Titer(mU)
%(w/w)
Blind #1
6.25
1.28
Blind #2
6.25
1.28
Blind #3
25
0.32
Blind #4
50
0.16
Blind #5
>100
<0.08
Blind #6
>100
<0.08
0.4
20.48
0.4
20.48
100
<0.08
0.78
10.24
6.25 mU
a
100 mU
25 mU
Blind #7 Blind #8 C1 B1 0 10 20 30 40 50 CT value 18S 10 8 6 4 2 0
mAU
30
c
Bl #5
25
0
2
4 6 min
8
10
18 16 14 12 10 8 6 4 2 0
2.3 2.2 2.1 2.0 1.9 1.8 ppm
r2= 0.9
20 15 10 5 0
Bl #8
5 0 10 15 20 25 % OS-CS (taq pol inhibition)
0
mAU
0 10 20 30 40 50 CT value 18S
% OS-CS (CE)
mAU
b
0 10 20 30 40 50 CT value 18S
2
4 6 min
8
2.3 2.2 2.1 2.0 1.9 1.8 ppm
10
8 7 6 5 4 3 2 1 0
Bl #2
0
2
4 6 min
8
10
2.3 2.2 2.1 2.0 1.9 1.8 ppm
Fig. 3. Screening of heparin samples for oversulfated contaminants. a) Eight blinded lots of heparin (Bl #1–8) were treated with heparinase and tested for gene amplification by Taqman PCR at 100 mU, 25 mU or 6.25 mU. b) Examples of CE and N-acetyl region of NMR profiles for selected samples. Note that for the N-acetyl region of NMR the heparin signals at 2.04 ppm, dermatan sulfate at 2.08 ppm and OS-CS at 2.16 as previously described [11]. For sample 5, CE shows a sharp peak for the contaminant before heparin (2.08 ppm) corresponding to DS. For sample 8, contaminant is evident by CE as a sharp peak after the heparin and confirmed by NMR with a proton peak at 2.16 ppm (arrows) in the N-acetyl region. Note that for sample 2, CE shows no contaminant while NMR shows a small peak consistent with the acetyl proton shift of OS-CS. High field (500 MHz) NMR is more sensitive than CE for low contaminant levels. c) Correlation of estimated OS-CS content as determined by PCR inhibition and by capillary electrophoresis. The % OS-CS (w/w) was estimated based on the assumption that 500 pg of OS-CS completely block Taq pol (CT value of 40) and 1 mU of heparin has 6.25 ng of active pharmaceutical ingredient.
for the presence of oversulfated contaminants in heparin. As shown, this method can reveal the presence of OS-CS (limit of quantitation: 500 pg or 0.16% w/w) and other oversulfated glycosaminoglycans (limit of quantitation: 2.7 ng) in as little as 0.6 mg and/or 100 mU of heparinase-treated heparin, as opposed to the higher amounts of heparin required for CE and NMR
(approximately 2 mg and 7 mg, respectively) and the lower limits of detection for OS-CS (1% for CE and 0.3 to 0.5% for NMR). In addition, assessment of Taq pol inhibition allows for the amount of contaminant to be quantified by using known amounts of OS-CS as standard controls and tittering down the heparin concentration to determine the lowest concentration that blocks gene amplification.
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The results obtained using the Taq pol inhibition method showed a high level of correlation with existing methods such as NMR and CE. Of note, naturally occurring GAGs such as dermatan sulfate or heparan sulfate did not block Taq pol activity reducing the concerns for false positive results. Together, this suggests that after proper validation Taq pol inhibition can be a used as a sensitive screening method to detect a broad spectrum of oversulfated glycosaminoglycans while NMR and CE may be used to confirm the presence and identify the nature of any oversulfated impurities. An additional advantage of this method is that it allows for testing of samples with low heparin concentration. For example, currently testing of heparin flush-lock syringes (100 U/ml) by CE or NMR requires extensive sample preparation including pooling of about 20 syringes, evaporation, resuspension and desalting, which would be unnecessary with the Taq pol inhibition assay. Lastly, the low requirement for starting material could make Taq pol inhibition a viable method for testing heparin retrieved from heparin coated devices such as syringes and tubing. Currently there are no practical methods available for evaluating contaminants in coated devices, since the collection of heparin from those devices does not render enough material to be analyzed by CE or NMR methods without very extensive pooling and concentrating of samples. 5. Conclusions Once validated, this simple yet highly sensitive in vitro test may be an effective way of screening heparin lots not only for OS-CS, the contaminant associated with the current adverse events reported, but for other oversulfated glycosaminoglycans that could potentially endanger the world supply of heparin in the future. The low requirement of starting material by the Taq pol inhibition assay provides an advantage over CE and NMR, potentially allowing for testing of both low concentrated heparin samples and heparin coating devices without extensive sample pooling and concentration. Acknowledgements The authors thank Dr Amy Rosenberg, Dr Steve Kozlowski, and Dr Moheb Nasr for reviewing the manuscript. The assertions herein are the private ones from the authors and are not to be construed as official or reflecting the views of the Food and Drug Administration. Appendix. Supplementary material Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.biomaterials.2008.08. 024.
References [1] Dudas B, Rose M, Cornelli U, Pavlovich A, Hanin I. Neuroprotective properties of glycosaminoglycans: potential treatment for neurodegenerative disorders. Neurodegener Dis 2008;5:200–5. [2] Sjalander A, Jansson JH, Bergqvist D, Eriksson H, Carlberg B, Svensson P. Efficacy and safety of anticoagulant prophylaxis to prevent venous thromboembolism in acutely ill medical inpatients: a meta-analysis. J Intern Med 2008;263:52–60. [3] Falanga A, Marchetti M. Heparin in tumor progression and metastatic dissemination. Semin Thromb Hemost 2007:688–94. [4] Baglin TP. Heparin induced thrombocytopenia thrombosis (HIT/T) syndrome: diagnosis and treatment. J Clin Pathol 2001;54:272–4. [5] Available from:; 2008. [6] Available from: ; 2008. [7] Available from: ; 2008. [8] Available from: ; 2008. [9] Available from: ; 2008. [10] Jia H. Regulators scramble to tighten loopholes after heparin debacle. Nat Biotechnol 2008;26:477–8. [11] Guerrini M, Beccati D, Shriver Z, Naggi A, Viswanathan K, Bisio A, et al. Oversulfated chondroitin sulfate is a contaminant in heparin associated with adverse clinical events. Nat Biotechnol 2008 [advanced online publication]. [12] Kishimoto TK, Viswanathan K, Ganguly T, Elankumaran S, Smith S, Pelzer K, et al. Contaminated heparin associated with adverse clinical events and activation of the contact system. N Engl J Med 2008. NEJMoa0803200. [13] Lindahl U, Li J, Kusche-Gullberg M, Salmivirta M, Alaranta S, Veromaa T, et al. Generation of ‘‘Neoheparin’’ from E. coli K5 capsular polysaccharide. J Med Chem 2005;48:349–52. [14] Maruyama T, Toida T, Imanari T, Yu G, Linhardt RJ. Conformational changes and anticoagulant activity of chondroitin sulfate following its O-sulfonation. Carbohydr Res 1998;306:35–43. [15] Chen J, Avci FY, Munoz EM, McDowell LM, Chen M, Pedersen LC, et al. Enzymatic redesigning of biologically active heparan sulfate. J Biol Chem 2005;280:42817–25. [16] Linhardt R, Dordick J, Deangelis P, Liu J. Enzymatic synthesis of glycosaminoglycan heparin. Semin Thromb Hemost 2007:453–65. [17] Nadkarni VD, Toida T, Van Gorp CL, Schubert RL, Weiler JM, Hansen KP, et al. Preparation and biological activity of N-sulfonated chondroitin and dermatan sulfate derivatives. Carbohydr Res 1996;290:87–96. [18] Holodniy M, Kim S, Katzenstein D, Konrad M, Groves E, Merigan TC. Inhibition of human immunodeficiency virus gene amplification by heparin. J Clin Microbiol 1991;29:676–9. [19] Furukawa K, Bhavanandan VP. Influences of anionic polysaccharides on DNA synthesis in isolated nuclei and by DNA polymerases a: correlation of observed effects with properties of the polysaccharides. Biochim Biophys Acta 1983;740:466–75. [20] Izraeli S, Pfleinderer C, Lion T. Detection of gene expression by PCR amplification of RNA derived from frozen heparinized whole blood. Nucleic Acids Res 1991;19(21):6051. [21] Yakota M, Tatsumi, Nathalang O, Yamada T, Tsuda I. Effects of heparin on polymerase chain reaction for blood white cells. J Clin Lab Anal 1999;13:133–40. [22] Johnson ML, Navanukraw C, Grazul-Bilska AT, Reynolds LP, Redmer DA. Heparinase treatment of RNA before quantitative real-time RT-PCR. Biotechniques 2003;35(6):1140–4. [23] Greinacher A, Michels I, Schafer MK, Kiefel V, Muller-Exkhardt C. Heparinassociated thrombocytopenia in a patient treated with polysulphated chondroitin sulphate: evidence for immunological crossreactivity between heparin and polysulphated glycosaminoglycan. Br J Haematol 1992;81: 252–4.
Contents lists available at ScienceDirect
Biomaterials journal homepage: www.elsevier.com/locate/biomaterials
Inhibition of Taq polymerase as a method for screening heparin for oversulfated contaminants Cecilia Tami a, Montserrat Puig a, John C. Reepmeyer b, Hongping Ye b, D. Andre D’Avignon c, Lucinda Buhse b, Daniela Verthelyi a, * a
Laboratory of Immunology, Division of Therapeutic Proteins, Office of Biotechnology Products, Center for Drugs Evaluation and Research, Food and Drug Administration, Rockville Pike, Bethesda, MD 20892, United States Division of Pharmaceutical Analysis, Center for Drugs Evaluation and Research, Food and Drug Administration, United States c Department of Chemistry, Washington University, St. Louis, MO, United States b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 23 June 2008 Accepted 20 August 2008 Available online 17 September 2008
Heparin and low molecular heparins are extensively used in the treatment of a wide range of diseases in addition to their classic anticoagulant activity and can be found coating medical devices such as catheters, stents and filters. Early in 2008, a sharp increase in heparin-associated severe adverse events, including over 80 deaths, was linked to the presence of a contaminant identified as hypersulfated chondroitin sulfate (OS-CS). OS-CS is one of several oversulfated glycosaminoglycans (GAGs) of different origins that can potentially cause similar clinical problems underscoring the need to develop robust screening methods for contaminants in existing and future lots of heparin. This study demonstrates that oversulfated GAGs block the activity of Taq polymerase used for real time PCR. Based on this finding we developed a simple, rapid, sensitive and high throughput screening method to detect and quantify oversulfated chondroitin sulfate (OS-CS) and other potential oversulfated contaminants in commercial lots of heparin. This method requires less than 100 miliUnits (mU) of heparin as starting material, therefore avoiding the need to lyophilize and concentrate samples, and has a limit of detection of <1 ng for all oversulfated GAGs tested. Published by Elsevier Ltd.
Keywords: Heparin Oversulfated chondroitin sulfate Glycosaminoglycan Taq polymerase PCR Safety
1. Introduction Heparin and low molecular heparins are widely used in the treatment of a wide range of diseases in addition to their classic anticoagulant activity [1–3]. Indeed, millions of doses of heparin are dispensed every month. The most significant adverse event linked to heparin have traditionally been increased bleeding and heparin-induce thrombocytopenia [4]. Between January 1, 2007 and April 13, 2008, the FDA received over 700 reports of adverse events in patients receiving heparin as part of their dialysis treatment or surgical procedures [5]. Adverse events included severe hypotension, vasodilation, facial swelling, tachycardia, urticaria, nausea, vomiting, diarrhea and abdominal pain, and resulted in over 80 deaths. Researchers at the Centers for Disease Control realized that the adverse events were associated with the receipt of
* Corresponding author. Laboratory of Immunology, Division of Therapeutic Products, Office of Biotechnology Products, Center for Drugs Evaluation and Research, Food and Drug Administration, Building 29A, Room 3B19, 8800 Rockville Pike, Bethesda, MD 20892, United States. Tel.: þ1 301 827 1702; fax: þ1 301 480 3256. E-mail address: [email protected] (D. Verthelyi). 0142-9612/$ – see front matter Published by Elsevier Ltd. doi:10.1016/j.biomaterials.2008.08.024
heparin sodium for injection (1000 U/ml, in 10 ml and 30 ml multidose vials), manufactured by Baxter Healthcare [6]. As a result, Baxter Healthcare issued recalls for all remaining lots and doses of its multidose and single-dose vials of heparin sodium for injection and HEP-LOCK heparin flush products in January and February 2008 [6–8]. This was followed by recalls for a number of medical devices that contain or are coated with heparin [5]. In March 2008, a similar recall was issued by Rotexmedica GmbH Arzneimittelwerk in Trittau, Germany and since then suspect lots have been identified in 11 other countries [9,10]. This indicated an extensive problem with heparin manufacture that was unlikely to be restricted to a single source. Using multidimensional nuclear magnetic resonance (NMR), enzymatic digestion followed by high-performance liquid chromatography, and liquid chromatography with mass spectrometry, Guerrini et al. identified an unusual oversulfated form of chondroitin sulfate (OS-CS) as a contaminant present in suspect lots of heparin [11]. The OS-CS contained a tetrasulfated disaccharide unit consisting of glucuronic acid linked to N-acetyl-D-galactosamine that was not evident in lots of heparin that were not linked to adverse events [11]. Kishimoto et al. [12] then were able to partially reproduce the clinical syndrome in a porcine model by
C. Tami et al. / Biomaterials 29 (2008) 4808–4814
inoculating a large dose of the pure contaminant (5 mg/kg i.v. in bolus) suggesting that the presence of OS-CS was linked to or possibly responsible for the adverse events. Proton nuclear magnetic resonance (H-NMR spectroscopy) and capillary electrophoresis (CE) tests were identified by the FDA as tests available to assess for the presence of OS-CS contaminant in products containing heparin sodium [8]. However, OS-CS is only one of numerous oversulfated compounds of animal, vegetable, insect or completely synthetic origin that could potentially be designed to co-purify and co-elute with heparin [12–16]. Most of these synthetic compounds have anticoagulant activity by current US pharmacopeia (clotting-based) tests and could be designed to give similar spectra by H-NMR as heparin, avoiding the identification by the tests currently in place. Such compounds would require methods such as high field spectra NMR for identification [15]. Further, for finished dosage forms, traditional tests such as NMR or CE cannot determine the presence of contaminant without lyophilizing and concentrating each sample, and may not be suitable for testing finished medical devises. 2. Materials and methods 2.1. Samples and reagents Heparin samples: active pharmaceutical ingredient (API) and final drug product (FDP) for samples B1–B3 and C1–C3 were obtained by the FDA from Baxter Healthcare (1000 U/ml or 5000 U/ml in 10 ml and 30 ml vials). Eight blinded samples (Blind #1 to 8) were obtained during FDA’s inspections. Chondroitin sulfate E was obtained from Seikagaku (Japan) and characterized by the HNMR method stated below. Heparinase I was obtained from Sigma (St Louis, MO, USA). RNA was extracted from MDA-MB-231 human breast cancer cell line grown in DMEM/F12 (50/50) media. 2.2. Heparinase treatment C
in Heparin was treated with heparinase I (1:1 unit:unit reaction) for 2 h at 25 a buffer containing 4 mM Tris–HCl pH 7.5, 0.8 mM CaCl, 10 mM NaCl and 20 U of RNase inhibitor (Applied Biosystems, Foster City, CA, USA) (ABI). After treatment, the heparin was diluted to a final volume of 50 ml and serial dilutions were prepared in DEPC water as specified. Where indicated, other GAGs and oversulfated GAGs (dermatan sulfate, chondroitin sulfate, heparan sulfate) were treated with heparinase under the same conditions as described above. 2.3. Screening assay cDNA was generated using total RNA from MDA-MB-231 human breast cancer cell line extracted using Trizol reagent (Invitrogen, Carlsbad, CA, USA) as per manufacturer’s instructions. For each cDNA reaction 1 mg total RNA was reverse transcribed using High Capacity cDNA Reverse Transcription Kit (ABI) in a volume of 20 ml and further diluted in DEPC water to 100 ml final volume. The concentration of cDNA was determined using Quant-iT OliGreen ssDNA Reagent (Invitrogen). For each Taqman reaction, 25 ng of cDNA in a 2.5 ml volume were mixed with 2.5 ml of each dilution of – heparinase-treated– heparin or hypersulfated GAGs and then subjected to real time PCR using Taqman Gene Expression assay for human 18S rRNA (20) and 2X Universal PCR master mix (ABI) in a 25 ml reaction. Amplification levels of 18S are expressed as CT values. CT values represent the cycle at which amplification of a target gene is first detected. The amplification was analyzed using manual settings with a threshold value of 0.1 and the SDS2.3 software from ABI. CT values < 16 cycles indicate there was no significant inhibition of the Taq polymerase activity while CT values of >35 indicate complete inhibition of the assay. Quantification of the oversulfated GAGs was performed by establishing a cutoff at the minimum concentration of synthetic OS-CS that completely blocks Taq polymerase (500 pg) and tittering all the samples until the activity of Taq polymerase was restored. The maximal dilution that completely blocked the enzyme was then assumed to have at least 500 pg of contaminant. 2.4. H-NMR analysis All samples were analyzed using a Varian500 MHzInova instrument. Samples were prepared by dissolving approximately 10 mg of sample in 0.6 ml of deutered water spiked with a reference compound TSP (tri-methyl-silyl propionate, sodium salt). The reference TSP signal is set to 0.00 ppm, which is referenced at 0.00 ppm. Samples were run at 25 C. Spectral parameters include no less than 16 transient, 90 pulse width, acquisition time of at least 1 s, time between transients of 20 s and a spectral window of 8000 Hz.
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2.5. Capillary electrophoresis CE was conducted on a Hewlett Packard 3D-CE instrument equipped with a diode array detector set at a wavelength of 200 nM (band width 10 nM). Separations were performed in a bare fused silica capillary, internal diameter 50 mM, 64.5 cm-total length, 56 cm-effective length with a column temperature of 25 C. The polarity was negative with a voltage of 30 kV. Samples were dissolved in Milli-Q water at a concentration of approximately 10 mg/ml and filtered through 0.2 mM cellulose acetate membrane filters (Micro-Spin filter tubes, Alltech Associates, Deerfield, IL, USA). The sample solutions were injected using hydrodynamic pressure at 50 mbar for 10 s. The electrolyte solution was 36 mM phosphate buffer (pH 3.5) filtered with a 0.2 mM cellulose acetate syringe filter (Grace, Deerfield, IL, USA). The capillary column was preconditioned at the beginning of each day by flushing with 1 M NaOH, 0.1 M NaOH, and water, each for 2 min, and prior to running each sample by flushing with water for 2 min and electrolyte solution for 2 min. Monobasic sodium phosphate, monohydrate, ACS grade, and phosphoric acid 85%, N.F. Food Grade, were obtained from Mallinckrodt Baker, Inc (Phillipsburg, NJ, USA). 1 M and 0.1 M Sodium hydroxide solutions for High-Performance Capillary Electrophoresis (HPCE) were from Hewlett Packard (Waldbronn, Germany). 2.6. Chemical sulfonation of chondroitin sulfate Fully sulfated chondroitin sulfate was prepared from chondroitin sulfate as described [14]. Thus, 139 mg chondroitin sulfate tributylamine salt and 1.2 g sulfur trioxide pyridine complex were dissolved in 2 ml dry N,N-dimethylformamide and heated for 1 h at 40 C. The reaction solution was adjusted to pH 9 with 1 M NaOH and diluted with 3 volumes of ethanol saturated with sodium sulfate, generating a precipitate. The mixture was cooled in a refrigerator, centrifuged, and the solid material was purified by dialysis and lyophilization. Heparan sulfate, dermatan sulfate, chondroitin sulfate A, and E as well as oversulfated heparin (OS-HS) and dermatan sulfate (OS-DS) were synthesized in house as described [15,17] and characterized by H-NMR (Supplementary Fig. 1), CE and elemental analysis for sulfur content (performed at Galbraith Laboratories).
3. Results 3.1. Effect of heparin contaminants on gene amplification by Taqman PCR Real time PCR (Taqman PCR) is a highly sensitive method for detecting changes in gene expression. Gene amplification depends on a thermostable DNA polymerase from Thermus aquaticus (Taq pol). Previous studies have shown that heparin competitively inhibits several different cellular DNA polymerases including Taq pol [18–21], but this inhibition can be overcome by the use of heparinase [20,22]. Shown in Fig. 1 are the levels of 18S amplification from 25 ng cDNA derived from MDA-MB-231 cells as determined by Taqman PCR. Addition of progressively higher amounts of heparin induces a corresponding reduction in Taq pol activity that can be monitored by assessing 18S cDNA amplification. Cycle thresholds (CT) < 16 indicate no inhibition of the Taq pol activity whereas CT values > 35 denote complete inhibition of the assay. Treatment of heparin with 1 U of heparinase for 2 h at 25 C overcomes the blocking activity of up to 50 mU of heparin (Fig. 1a). To determine whether the presence of contaminants modulates the blocking effect of heparin on Taq pol, we designed an assay in which heparin from lots that were associated with adverse clinical effects (B1, B2 and B3) as well as from control lots with no visible clinical effects (C1, C2, C3) were treated with heparinase (or left untreated). Sequential dilutions of heparin were then added onto 25 ng of cDNA and 18S was amplified by Taqman PCR. As shown in Fig. 1b, while 18S amplification was maximal in control heparinasetreated lots (C1–C3), heparinase treatment did not restore 18S amplification of cDNA exposed to lots of heparin associated with adverse events (B1–B3). This suggested that a contaminant in the heparin inhibits the Taq pol enzyme. Of note, addition of as little as 6.25 mU of heparinase-treated heparin to the Taqman reaction was sufficient to clearly differentiate between contaminated and uncontaminated lots regardless of whether the starting material was finished formulated drug product or the corresponding
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a
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Heparinase treatment restores amplification by RT-PCR
Lot
Hepa- Hepari 100 rin nase
50
25
12.5
C1
+
-
N/T
N/T
N/T
N/T
C1
+
+
16.8
14.8
14.1
13.9
b
6.3
13.6
3.1
13.7
1.6
0.8
0.4
0.2
0.1
0.1
0.0
25.1
19.2
15.6
14.4
13.8
13.9
14.1
13.8
N/T
N/T
N/T
N/T
13.9
1.6
0.8
0.4
0.2
0.1
0.1
0.0
25.1
19.2
15.6
14.4
13.8
13.9
13.8
N/T
N/T
N/T
N/T
24.7
17.5
16.3
14.0
14.1
N/T
N/T
N/T
N/T
30.5
18.6
15.2
14.6
13.8
13.7
N/T
N/T
N/T
N/T
20.3
19.2
14.3
13.8
14.1
13.5
21.8
16.2
14.2
Heparinase does not restore amplification in contaminated lots 100
C1
FDP
C2
FDP
FDP
FDP
12.5
6.3
3.1
16.8
14.8
14.1
13.9
13.6
13.7
14.1
N/T 14.8
14.2
14.0
14.0
14.1
13.8
13.9
+
B1
25
+
C3
50
+
14.8
14.1
14.0
14.0
13.9
14.0
14.0
+
B2
FDP
B3
FDP
22.3
15.5
+
17.6
14.4
14.0
13.9
-
26.0
18.3
14.9
14.0
+
c
mUnits
mUnits
14.37 13.89 13.76 13.97 13.93
Oversulfated chondroitin sulfate blocks 18S amplification 1.25
1
0.75
0.5
0.25
0.08
0
No amplification (Ct >35)
ng
OS-CS
-
-
17.3
14.14 14.2
Reduced amplification (Ct 16-35)
OS-CS
C1
+
15.0
14.2
Maximal amplification (Ct<16)
d
14.1
Effect of natural and oversulfated glycosaminoglycans on 18S amplification by RT-PCR 125
62.5
25
8.3
2.7
0.9
0.3
0
ng
% sulfur content
DS
C1
+
14.9
14.2
13.9
13.7
13.7
N/T
N/T
13.6
6.3
HS
C1
+
13.9
13.8
13.7
13.5
13.5
N/T
N/T
13.6
5.0
CS-A
C1
+
13.6
13.6
13.7
13.6
13.6
N/T
N/T
13.6
6.4
CS-E
C1
+
19.7
15.6
14.2
13.7
13.6
N/T
N/T
13.6
10.6
OS-DS
C1
+
32.7
15.6
13.6
16.0
OS-DS
C1
+
27.0
15.38 13.6
13.1
OS-HS
C1
+
32.9
14.6
13.6
16.4
OS-CS
C1
+
16.1
13.6
19.2
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non-formulated active pharmaceutical ingredient (API) diluted in water (Fig. 2a). 3.2. Use of Taq polymerase inhibition to screen for the presence of OS-CS in heparin As mentioned above, OS-CS was identified as a contaminant present in lots of heparin that have been linked to adverse events [11]. Previous studies have established that persulphated chondroitin sulfate is not susceptible to heparinase I or II (or chondroitinase) degradation. To determine whether OS-CS directly inhibits Taq pol activity, decreasing concentrations of OS-CS were added to cDNA and amplified by Taqman PCR. As shown in Fig. 1c, addition of 500 pg of OS-CS completely blocked Taq pol mediated 18S cDNA amplification. In order to determine the minimum % (w/w) of contaminant that could be detected in heparin, we used a representative potency of heparin (6.25 mg/U). Since 500 pg of OS-CS completely block 18S amplification, the lowest titer of heparin that completely blocked amplification was assumed to contain at least 500 pg of OS-CS. From this, the limit of detection was calculated as 0.16% (w/w), which is below the level of detection for both proton NMR and CE. Importantly, the presence of heparin and heparinase did not modify the sensitivity of the assay (Fig. 1c). These results indicate that failure to amplify 18S cDNA can be used as a rapid and sensitive diagnostic test to screen for the presence of OS-CS in heparin. Lastly, similar gene amplification inhibition levels were evident whether the suspect heparin or the OS-CS were added to the cDNA or to the RNA used to generate the cDNA, indicating that any contaminant present likely co-purifies with nucleic acids (Fig. 2b). This raises the possibility that the presence of oversulfated contaminants in heparin may have interfered with PCR-based assays that are currently used to test heparin lots for contaminants from material from other natural sources such as bovine heparin. 3.3. Comparison of the Taq pol inhibition method with current methods used to screen and quantify OS-CS To verify the method as a screening assay for the presence of oversulfated contaminants, 8 lots of heparin API were tested in a blinded manner. Each was treated with heparinase I, and titered into a Taqman PCR reaction. Addition of 6.25 mU of heparin resulted in complete inhibition of amplification for two samples (blind #7 and 8) (Fig. 3a). Those samples were later identified as having 15 and 27% OS-CS contamination by CE, and showing significant peaks at 2.16 ppm by NMR (Fig. 3b and not shown). For blind samples #1 and 2, there was reduced amplification of 18S at 6.25 mU and complete inhibition when 25 or more mU of heparin were added to the PCR reaction. Subsequent unblinding of the NMR and CE profiles for these particular samples showed the presence of a weak signal at 2.16 ppm by H-NMR but no visible peak by CE, a profile that suggests marginal OS-CS contamination (Fig. 3b). Samples 3 and 4 showed a reduced 18S amplification at heparin concentrations of 100 mU and 25 mU of heparin but did not completely block it suggesting that trace levels of contaminants could be present in these samples. This indicated that Taq pol inhibition is more sensitive to OS-CS contamination than CE or H-NMR. The remaining samples (Bl #5 and 6) did not inhibit 18S
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amplification. Lastly, comparison of the OS-CS concentration, as determined by inhibition of 18S amplification, to the percent OS-CS as determined by CE shows a high level of correlation (r2 ¼ 0.9, p < 0.001) confirming that the Taqman-inhibition based method is an effective semi-quantitative screening assay, likely to identify very low levels of oversulfated contaminants in heparin. 3.4. Susceptibility of Taq polymerase to other potential oversulfated GAGs Heparin is commonly extracted from porcine intestinal mucosa or bovine lung, and preparations may contain small amounts of other glycosaminoglycans. The presence of naturally occurring dermatan sulfate (DS), heparan sulfate (HS) or chondroitin sulfates A (CS-A), when spiked into heparin and treated with heparinase did not inhibit Taq pol activity at any of the concentrations tested (0–125 ng) (Fig. 1c). Chondriotin sulfate E (CS-E), which is more sulfated that chondroitin sulfate A (CS-A) or DS, did show a trace of inhibitory effect at the highest concentration tested (125 ng). In contrast, under the same conditions, all oversulfated glycosaminoglycans tested, including oversulfated heparan sulfate (OS-HS), two different forms of oversulfated dermatan sulfate (OS-DS) and OS-CS significantly reduced 18S amplification. Further, the magnitude of the inhibitory effect on Taq pol correlated with the degree of sulfation of each compound as determined by elemental analysis (r2 ¼ 0.93, p < 0.001). This indicates that Taq pol inhibition is a useful tool to screen for other potential oversulfated contaminants that could contaminate heparin causing adverse effects in the future. 4. Discussion Heparin is an essential drug for many patients. Licensed since the early 20th century, it is widely used as an anticoagulant in multiple settings including invasive surgical and dialysis procedures, and deep venous thrombosis treatment. Although usually well tolerated, in recent months hundreds of reports of serious adverse responses were followed by the discovery of contaminating OS-CS in heparin lots around the world. While the adverse events were linked to lots that have >5% OS-CS, at this time, we do not have the necessary data to establish the pathogenic impact of the presence of smaller amounts (<2%) of the OS-CS, nor understand the precise mechanism by which OS-CS may cause severe adverse events in a small but significant percentage of patients. Indeed, although the activation of the bradikinin–kallikrein system has been identified as potentially mediating some of the adverse events that have been associated with tainted heparins, other potential mechanisms are under investigation and the long term effects of these contaminants will need to be carefully considered. Importantly, future investigations will have to include an assessment of the potential for OS-CS and other oversulfated compounds to form more stable complexes with chemokine PF4, which could increment the immunogenicity of heparin and the risk of heparininduced thrombocytopenia (HIT) [23]. Together, these events have underscored the need to develop robust characterization methods to ensure the integrity of the global supply of heparin. This study demonstrates that the inhibition of Taq polymerase can be used as a highly sensitive assay to test
Fig. 1. Effect of glycosaminoglycans on 18S amplification. The effect of heparin on Taq polymerase activity was evaluated by quantitative real time PCR. Heparin lots were treated with heparinase I (1:1 U/U) for 2 h at 25 C or left untreated. Gene amplification was evaluated by real time PCR using the Taqman Gene Expression assay for human 18S rRNA (20) and 2X Universal PCR master mix (ABI) in a 25 ml reaction in the presence of increasing concentrations (0.5–100 mU) of heparin (heparinase-treated or untreated). Shown in: a) are the effects of heparin and heparinase-treated heparin on 18S amplification. b) Comparison of 3 final drug product lots of heparin characterized as contaminated by NMR and CE (B1–B3) with 3 lots of control heparin lots (C1–C3). c) Effect of synthetic OS-CS on 18S amplification in the presence and the absence of heparinase digested heparin. d) Effect of natural and oversulfated synthetic glycosaminoglycans on 18S amplification by PCR, DS: dermatan sulfate, HS: heparan sulfate, CS-A and E: chondroitin sulfate type A and E respectively, OS-HS: oversulfated heparan sulfate, OS-DS: oversulfated dermatan sulfate; OS-CS: oversulfated chondroitin sulfate.
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a
Taq polymerase inhibition by heparinase-treated API or FDP heparin Heparinase
Lot
C1 API
+
C1 FDP
+
B1 API
+
B1 FDP
+
B2 API
+
B2 FDP
+
mUnits 100
50
25
13
6.3
3.1
1.6
0.8
0.4
0.2
0.1
0.05
15.0
14.1
13.8
13.7
13.6
13.6
14.2
13.3
N/T
N/T
N/T
N/T
16.8
14.8
14.1
13.9
13.6
13.7
14.1
13.8
N/T
N/T
N/T
N/T
27.2
31.6
20.2
15.1
14.0
22.3
15.5
13.8
14.1
13.5
15.7
14.1
14.0
13.9
17.6
14.4
14.0
13.9
0.0
13.9
No amplification (Ct >35) Reduced amplification (Ct 16-35) Maximal amplification (Ct<16)
b
OS-CS co-purifies with RNA and blocks gene amplification
-
-
-
-
-
B
-
+
-
-
-
C
+
-
-
-
-
D
-
-
-
+
-
E
-
-
-
+
+
F
-
-
+
-
-
G
-
-
+
-
+
Gene amplification
Heparinase on
cDNA
1U Heparin on
cDNA
1U Heparin on RNA
OSCS (30ng) on
total RNA
OSCS (30ng) on
cDNA
cDNA Sample A
No amplification (Ct >35) Maximal amplification (Ct<16) Fig. 2. Sensitivity of the Taq pol assay. a) Active pharmaceutical ingredient or final drug product similarly inhibit Taq polymerase activity. Three lots of API and their corresponding FDP were screened by Taq pol inhibition. API was diluted in RNAse free water. As shown, similar degrees of amplification were observed indicating that both API and FDP can be used as the starting material for screening heparin. b) Addition of OS-CS (B and C; 30 ng) or heparin (D and F; 25 mU of uncontaminated lot C1) to cDNA or to the RNA used to generate the cDNA resulted in similar Taq pol inhibition. Treatment of heparin – but not OS-CS – with heparinase for 2 h at room temperature restores gene amplification (E and G). Controls include cDNA treated with heparinase buffer (A).
C. Tami et al. / Biomaterials 29 (2008) 4808–4814
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Titer(mU)
%(w/w)
Blind #1
6.25
1.28
Blind #2
6.25
1.28
Blind #3
25
0.32
Blind #4
50
0.16
Blind #5
>100
<0.08
Blind #6
>100
<0.08
0.4
20.48
0.4
20.48
100
<0.08
0.78
10.24
6.25 mU
a
100 mU
25 mU
Blind #7 Blind #8 C1 B1 0 10 20 30 40 50 CT value 18S 10 8 6 4 2 0
mAU
30
c
Bl #5
25
0
2
4 6 min
8
10
18 16 14 12 10 8 6 4 2 0
2.3 2.2 2.1 2.0 1.9 1.8 ppm
r2= 0.9
20 15 10 5 0
Bl #8
5 0 10 15 20 25 % OS-CS (taq pol inhibition)
0
mAU
0 10 20 30 40 50 CT value 18S
% OS-CS (CE)
mAU
b
0 10 20 30 40 50 CT value 18S
2
4 6 min
8
2.3 2.2 2.1 2.0 1.9 1.8 ppm
10
8 7 6 5 4 3 2 1 0
Bl #2
0
2
4 6 min
8
10
2.3 2.2 2.1 2.0 1.9 1.8 ppm
Fig. 3. Screening of heparin samples for oversulfated contaminants. a) Eight blinded lots of heparin (Bl #1–8) were treated with heparinase and tested for gene amplification by Taqman PCR at 100 mU, 25 mU or 6.25 mU. b) Examples of CE and N-acetyl region of NMR profiles for selected samples. Note that for the N-acetyl region of NMR the heparin signals at 2.04 ppm, dermatan sulfate at 2.08 ppm and OS-CS at 2.16 as previously described [11]. For sample 5, CE shows a sharp peak for the contaminant before heparin (2.08 ppm) corresponding to DS. For sample 8, contaminant is evident by CE as a sharp peak after the heparin and confirmed by NMR with a proton peak at 2.16 ppm (arrows) in the N-acetyl region. Note that for sample 2, CE shows no contaminant while NMR shows a small peak consistent with the acetyl proton shift of OS-CS. High field (500 MHz) NMR is more sensitive than CE for low contaminant levels. c) Correlation of estimated OS-CS content as determined by PCR inhibition and by capillary electrophoresis. The % OS-CS (w/w) was estimated based on the assumption that 500 pg of OS-CS completely block Taq pol (CT value of 40) and 1 mU of heparin has 6.25 ng of active pharmaceutical ingredient.
for the presence of oversulfated contaminants in heparin. As shown, this method can reveal the presence of OS-CS (limit of quantitation: 500 pg or 0.16% w/w) and other oversulfated glycosaminoglycans (limit of quantitation: 2.7 ng) in as little as 0.6 mg and/or 100 mU of heparinase-treated heparin, as opposed to the higher amounts of heparin required for CE and NMR
(approximately 2 mg and 7 mg, respectively) and the lower limits of detection for OS-CS (1% for CE and 0.3 to 0.5% for NMR). In addition, assessment of Taq pol inhibition allows for the amount of contaminant to be quantified by using known amounts of OS-CS as standard controls and tittering down the heparin concentration to determine the lowest concentration that blocks gene amplification.
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The results obtained using the Taq pol inhibition method showed a high level of correlation with existing methods such as NMR and CE. Of note, naturally occurring GAGs such as dermatan sulfate or heparan sulfate did not block Taq pol activity reducing the concerns for false positive results. Together, this suggests that after proper validation Taq pol inhibition can be a used as a sensitive screening method to detect a broad spectrum of oversulfated glycosaminoglycans while NMR and CE may be used to confirm the presence and identify the nature of any oversulfated impurities. An additional advantage of this method is that it allows for testing of samples with low heparin concentration. For example, currently testing of heparin flush-lock syringes (100 U/ml) by CE or NMR requires extensive sample preparation including pooling of about 20 syringes, evaporation, resuspension and desalting, which would be unnecessary with the Taq pol inhibition assay. Lastly, the low requirement for starting material could make Taq pol inhibition a viable method for testing heparin retrieved from heparin coated devices such as syringes and tubing. Currently there are no practical methods available for evaluating contaminants in coated devices, since the collection of heparin from those devices does not render enough material to be analyzed by CE or NMR methods without very extensive pooling and concentrating of samples. 5. Conclusions Once validated, this simple yet highly sensitive in vitro test may be an effective way of screening heparin lots not only for OS-CS, the contaminant associated with the current adverse events reported, but for other oversulfated glycosaminoglycans that could potentially endanger the world supply of heparin in the future. The low requirement of starting material by the Taq pol inhibition assay provides an advantage over CE and NMR, potentially allowing for testing of both low concentrated heparin samples and heparin coating devices without extensive sample pooling and concentration. Acknowledgements The authors thank Dr Amy Rosenberg, Dr Steve Kozlowski, and Dr Moheb Nasr for reviewing the manuscript. The assertions herein are the private ones from the authors and are not to be construed as official or reflecting the views of the Food and Drug Administration. Appendix. Supplementary material Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.biomaterials.2008.08. 024.
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