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<section class="hero-banner" style="background-image:url(https://www.thermofisher.com/blog/behindthebench/wp-content/uploads/sites/9/2023/09/iStock-867398442_vialssyringe.jpg)"> </section> <div class="site-inner"><div class="content-sidebar-wrap"><main class="content"><article class="post-18081 post type-post status-publish format-standard has-post-thumbnail category-general category-pharmamfg tag-drug-development tag-drug-manufacturing tag-pharma-quality tag-quality-management tag-uv-vis-spectroscopy entry" aria-label="Heparin Quality Should be Verified Before Distribution to Help Ensure It Will Behave as Expected"><header class="entry-header"><h1 class="entry-title">Heparin Quality Should be Verified Before Distribution to Help Ensure It Will Behave as Expected</h1> <span class="post-info-author"> By <a href="https://www.thermofisher.com/blog/behindthebench/tf-author/jennifer-empey/" title="Posts by Jennifer Empey" rel="author">Jennifer Empey, Product Applications Specialist</a> </span> <span class="post-info-date"> 09.19.2023 </span> </header><div class="entry-content"><p><span data-contrast="auto">To ensure the drugs used in clinical environments behave as expected and do not produce unwanted side effects, it is important the quality of the product is verified prior to distribution. For manufactured heparin, it is vital that the activity of the heparin-antithrombin complex meets designated standards to ensure the quality of the final product. UV-Visible absorption spectrophotometer can be an effective tool in the analysis of heparin anti-factor IIa and Xa activity.</span><span data-ccp-props="{"201341983":0,"335559739":200,"335559740":276}"> </span></p> <p><span data-contrast="auto">Heparin, a polysaccharide compound, is an anti-coagulant used to treat thromboembolisms and other conditions.¹- ³ The compound is often administered prior to kidney dialysis.⁴ Heparin functions as it does due to a particular pentasaccharide region that consists of sulfated glucosamine residues not commonly found elsewhere in the compound. This pentasaccharide region will bind with antithrombin, a protein in the blood stream which functions as a naturally occurring mild blood thinner.⁵ The resulting heparin-antithrombin complex inhibits serine proteases such as factor IIa and factor Xa at much greater rates than antithrombin alone does. These proteases, factor IIa and factor Xa, are enzymes which aid in coagulation, and their inhibition by the heparin-antithrombin complex (known as anti-factor IIa and anti-factor Xa activity), prevents blood coagulation.¹, ² </span><span data-ccp-props="{"201341983":0,"335559739":200,"335559740":276}"> </span></p> <h2><span data-contrast="none">Avoiding Unwanted Side Effects</span><span data-ccp-props="{"134245418":true,"134245529":true,"201341983":0,"335559738":40,"335559739":0,"335559740":276}"> </span></h2> <p><span data-contrast="auto">For safe usage, the quality of pharmaceuticals must be properly vetted to help ensure they do not produce undesired side effects. For example, some batches of manufactured heparin have been found to be contaminated by the presence of oversulfated chondroitin sulfate.⁴ The presence of this moiety in the produced drug has been linked to allergic reactions. Adverse effects like this highlight the need to thoroughly test the quality of drug products prior to distribution.</span><span data-ccp-props="{"201341983":0,"335559739":200,"335559740":276}"> </span></p> <p><span data-contrast="auto">For manufactured heparin, it is vital that the activity of the heparin-antithrombin complex meets designated standards to ensure the quality of the final product.</span><span data-ccp-props="{"201341983":0,"335559739":200,"335559740":276}"> </span></p> <p><span data-contrast="auto">The <a href="https://www.usp.org/">US Pharmacopeia</a> (USP) has developed a quality test to ensure the activity of the manufactured heparin meets required specifications.⁶ In this method, the activities of the produced heparin and of USP heparin standards for both factor IIa and factor Xa are determined using a colorimetric assay. This assay requires the use of a <a href="https://www.thermofisher.com/us/en/home/industrial/spectroscopy-elemental-isotope-analysis/molecular-spectroscopy/uv-vis-spectrophotometry/resources.html?icid=CAD_blog_LSM_2023Sept">UV-Visible absorption spectrophotometer</a>.</span><span data-ccp-props="{"201341983":0,"335559739":200,"335559740":276}"> </span></p> <p><span data-contrast="auto">The test compares the measured absorbance of a set of heparin sample solutions to a set of standard solutions. The ratio of the activity of the heparin samples versus the activity of the standards, for both anti-factor IIa and anti-factor Xa, is then compared to acceptance criteria outlined by USP.</span><span data-ccp-props="{"201341983":0,"335559739":200,"335559740":276}"> </span></p> <h2><span data-contrast="none">UV-Vis Spectrophotometer Principles</span></h2> <p><a href="https://www.thermofisher.com/us/en/home/industrial/spectroscopy-elemental-isotope-analysis/molecular-spectroscopy/uv-vis-spectrophotometry/resources.html?icid=CAD_blog_LSM_2023Sept"><span data-contrast="none">UV-Visible spectroscopy</span></a><span data-contrast="auto"> is a well-established analytical technique used in the pharmaceutical industry for testing in the research and quality control stages of drug development. UV-Visible spectroscopy measures the absorption or reflectance of light in the ultraviolet and /or visible region of the spectrum.</span><span data-ccp-props="{"201341983":0,"335559739":200,"335559740":276}"> </span></p> <p><span data-contrast="auto">In the most basic terms, spectrophotometers enable photometric comparisons of relative light intensities across the ultraviolet and visible spectrums. Directing a controlled, constant intensity light source (halogen, deuterium, xenon) across the spectrum or at a specific wavelength through a sample easily can confirm known or calculate unknown characteristics of the sample. The incident light (I0) can be redirected backward as reflection, suffer an energy loss as absorption, and pass through transparent or translucent samples as transmission. (Read more about </span><a href="https://www.thermofisher.com/us/en/home/industrial/spectroscopy-elemental-isotope-analysis/molecular-spectroscopy/uv-vis-spectrophotometry/resources.html?icid=CAD_blog_LSM_2023Sept"><span data-contrast="none">UV-Vis spectrophotometers.</span></a><span data-contrast="auto">)</span><span data-ccp-props="{"201341983":0,"335559739":200,"335559740":276}"> </span></p> <h2><span data-contrast="none">Experiment</span><span data-ccp-props="{"134245418":true,"134245529":true,"201341983":0,"335559738":40,"335559739":0,"335559740":276}"> </span></h2> <p><span data-contrast="auto">To see if UV-Vis technology is a suitable option to verify the quality of heparin, a </span><a href="https://www.thermofisher.com/order/catalog/product/840-341400?icid=CAD_blog_LSM_2023Sept"><span data-contrast="auto">UV-Vis Spectrophotometer</span></a><span data-contrast="auto"> was used to measure the absorption of the prepared heparin standards and samples in the presence of a chromogenic substrate. This instrument meets USP requirements outlined in USP .⁷ The sample preparation and measurements were carried out based on USP monograph . Data analysis was performed using the built-in analysis functionalities in the instrument software. The procedure known as the anti-factor IIa assay was carried out based on the USP  procedure.⁶ </span><span data-ccp-props="{"134233117":false,"134233118":false,"201341983":0,"335551550":1,"335551620":1,"335559685":0,"335559737":0,"335559738":0,"335559739":200,"335559740":276}"> </span></p> <p><span data-contrast="auto">In this case, the UV-Vis spectrophotometer was useful in confirming that the particular heparin samples analyzed in one experiment did not meet established USP standards. You can read the details about the experiment and why the sample did not fall in the acceptable range — including absorption measurements, collected and calculated results, concentration charts, slope ratios, and equations used — in the </span><span data-contrast="auto">application note </span><a href="https://assets.thermofisher.com/TFS-Assets/MSD/Application-Notes/anti-factor-xa-and-IIa-assays-for-unfractioned-heparin-activity-an53623-en.pdf"><i><span data-contrast="none">Anti-Factor Xa and IIa Assays for Unfractioned Heparin Activity</span></i></a><span data-ccp-props="{"201341983":0,"335559739":200,"335559740":276}"> </span></p> <h2><span data-contrast="none">Additional Resources:</span></h2> <p><span data-contrast="auto"><br /> Application Note: </span><a href="https://assets.thermofisher.com/TFS-Assets/MSD/Application-Notes/anti-factor-xa-and-IIa-assays-for-unfractioned-heparin-activity-an53623-en.pdf"><i><span data-contrast="none">Anti-Factor Xa and IIa Assays for Unfractioned Heparin Activity</span></i></a><span data-ccp-props="{"201341983":0,"335559739":200,"335559740":276}"> </span></p> <p><i><span data-contrast="auto">References:</span></i><span data-ccp-props="{"201341983":0,"335559739":200,"335559740":276}"> </span></p> <ol> <li><span data-contrast="auto"> Suzuki, T.; Ishii-Watabe, A.; Hashii, N.; Nakagawa, Y.; Takahashi, T.; Ebisawa, A.; Nishi, S.; Fujita, N.; Bando, A.; Sekimoto, Y.; Miyata, K.; Endo, T.; Otsu, T.; Sugimoto, S.; Kondou, T.; Fujita, Y.; Miyanaga, N.; Mashimo, M.; Shimada, N.; Yoden, H.; Shimamura, H.; Kurata, Y.; Koyama, S.; Kawasaki, N., The Establishment and Validation of Efficient Assays for Anti-IIa and Anti-Xa Activities of Heparin Sodium and Heparin Calcium, Biologicals, 2013, 41, 214 – 423.</span></li> <li><span data-contrast="auto"> Linhardt, R.J., Rice, K.C.; Merchant, Z.M.; Kim, Y.S.; Lohse, D.L, Structure and Activity of a Unique Heparin-derived Hexasaccharide, J. Biol. Chem., 1986, 261, 14448 – 14454.</span></li> <li><span data-contrast="auto"> Egan, G.; Ensom, M.H.H., Measuring Anti-Factor Xa Activity to Monitor LowMolecular-Weight Heparin in Obesity: A Critical Review, Can. J. Hosp. Pharm., 2015, 1, 33 – 47.</span></li> <li><span data-contrast="auto"> Guerrini, M.; Beccati, D.; Shriver, Z.; Naggi, A.; Viswanathan, K.; Bisio, A.; Capila, I.; Lansing, J.C.; Guglieri, S.; Fraser, B.; Al-Hakim, A.; Gunay, N. S.; Zhang, Z.; Robinson, L.; Buhse, L.; Nasr, M.; Woodcock, J.; Langer, R.; Venkataraman, G.; Linhardt, R.J.; Casu, B.; Torri, G.; Sasisekharan, R., Oversulfated Chondroitin Sulfate is a Contaminant in Heparin Associated with Adverse Clinical Events, Nat. Biotechnol., 2008, 26, 669 – 675.</span></li> <li><span data-contrast="auto"> Oscarsson, L.G.; Pejler, G.; Lindahl, U., Location of the Antithrombin-binding Sequence in the Heparin Chain, J. Biol. Chem., 1989, 264, 296 – 304.</span></li> <li><span data-contrast="auto"> USP. Anti-factor Xa and Anti-Factor IIa Assays for Unfractioned and Low Molecular Weight Heparins. In: USP–NF. Rockville, MD: USP.</span></li> <li><span data-contrast="auto"> USP. Ultraviolet-Visible Spectroscopy. In: USP–NF. Rockville, MD: USP.</span></li> </ol> <p><span data-contrast="none">For Research Use Only. 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