Immunogenicity-
Related Considerations 
for Low Molecular 
Weight Heparin  
 
Guidance for Industry 
 
 
 
 
 
 
 
 
 
 
 
 
U.S. Department of Health and Human Services 
Food and Drug Administration 
Center for Drug Evaluation and Research (CDER) 
 
 
 February 2016  
Pharmaceutical Quality/CMC 
   
Immunogenicity-
Related Considerations 
for Low Molecular 
Weight Heparin  
 
Guidance for Industry 
 
 
Additional copies are available from: 
Office of Communications, Division of Drug Information  
Center for Drug Evaluation and Research 
Food and Drug Administration 
10001 New Hampshire Ave., Hillandale Bldg., 4th Floor  
Silver Spring, MD 20993-0002  
Phone: 855-543-3784 or 301-796-3400; Fax: 301-431-6353 
Email: druginfo@fda.hhs.gov  
http://www.fda.gov/Drugs/GuidanceComplianceRegulatoryInformation/Guidances/default.htm 
 
 
 
U.S. Department of Health and Human Services 
Food and Drug Administration 
Center for Drug Evaluation and Research (CDER) 
 
February 2016 
Pharmaceutical Quality/CMC 
 
 
  Contains Nonbinding Recommendations 
TABLE OF CONTENTS 
 
 
 
I.  INTRODUCTION............................................................................................................. 1 
II.  BACKGROUND ............................................................................................................... 2 
III.  SUBMISSION RECOMMENDATIONS........................................................................ 3 
A. Characterization of Active Ingredient Sameness (ANDAs only) ............................................... 3 
B. Impurities and Immunogenicity Risk (NDAs/sNDAs and ANDAs/sANDAs)........................... 6 
1. Studies Assessing the Interaction of LMWH with Platelet Factor 4 ................................................ 6 
2. Characterization of Impurities ......................................................................................................... 7 
3. Use of In Vitro and In Vivo Immunological Models ........................................................................ 8 
4. Selection and Specification of Product Lots Used for Studies to Assess the Risk of Immunogenicity 
   ......................................................................................................................................................... 8 
 
  Contains Nonbinding Recommendations 
Immunogenicity-Related Considerations for Low Molecular Weight 
Heparin  
1
Guidance for Industry  
 
 
 
This guidance represents the current thinking of the Food and Drug Administration (FDA or Agency) on this 
topic. It does not establish any rights for any person and is not binding on FDA or the public. You can use an 
alternative approach if it satisfies the requirements of the applicable statutes and regulations. To discuss an 
alternative approach, contact the FDA staff responsible for this guidance as listed on the title page.   
 
 
 
 
I.  INTRODUCTION  
 
This guidance discusses immunogenicity-related considerations for low molecular weight heparin 
(LMWH) products and finalizes the draft guidance titled Immunogenicity-Related Considerations for 
the Approval of Low Molecular Weight Heparin for NDAs and ANDAs that was published on April 9, 
2014 (79 FR 19621). Section II includes background information. Section IIIA includes 
recommendations on meeting the requirement for active ingredient sameness for abbreviated new 
drug applications (ANDAs) for LMWHs, which helps to address immunogenicity-related 
considerations in the context of ANDAs. Section IIIB includes recommendations on addressing 
impurities and their potential effect on immunogenicity for ANDAs. Section IIIB also includes 
recommendations on impurities for new drug applications (NDAs) and supplemental NDAs (sNDAs) 
or supplemental ANDAs (sANDAs)2  in instances where the source material (Heparin Sodium, USP) 
or another component is changed, or when there are alterations in the manufacturing process for the 
LMWH either before or after approval of the LMWH drug application.3,4 Drug Master File (DMF) 
holders should also be mindful of the recommendations in this guidance and ensure DMFs are 
current.  DMF holders must notify authorized applicants of changes.5       
 
In general, FDAs guidance documents do not establish legally enforceable responsibilities.  Instead, 
guidances describe the Agencys current thinking on a topic and should be viewed only as 
recommendations, unless specific regulatory or statutory requirements are cited. The use of the word 
should in Agency guidances means that something is suggested or recommended, but not required.  
 
                                                 
1 This guidance has been prepared by the Office of Pharmaceutical Quality in the Center for Drug Evaluation and 
Research (CDER) at FDA.  
2 If you make changes to an approved NDA or ANDA, you can submit those changes in one of three ways, depending on 
whether the change is a minor change, a moderate change, or a major change (section 506A of the Federal Food, Drug, 
and Cosmetic Act and 21 CFR 314.70 and 21 CFR 314.97).  For LMWH products, generally any changes to the source 
material (Heparin Sodium USP) or other components are considered major changes and require a prior approval 
supplement (PAS). This type of change must be approved by FDA before you can distribute the modified drug product.    
3 This would occur, for example, when you have conducted clinical studies of a LMWH product for an NDA with a 
 
particular source of heparin, and, before approval of the NDA by FDA, you change the source of the heparin.  
4 See the guidances for industry Immunogenicity Assessment for Therapeutic Protein Products and the International 
Conference on Harmonization, Q9 Quality Risk Management. 
5 See 21 CFR 314.420. 
  1  Contains Nonbinding Recommendations 
II.  BACKGROUND 
 
LMWH products are anticoagulants used for the prevention and treatment of thrombosis (blood 
clots). These products are produced by depolymerization of the anticoagulant heparin, a complex, 
naturally occurring polysaccharide found in certain animal species and whose backbone consists of 
repeating disaccharide building blocks.6 Treatment with heparin or LMWH products can be 
associated with a potentially fatal adverse event, heparin-induced thrombocytopenia (HIT). This 
occurs when the patient produces antibodies to heparin or LMWH in complex with the chemokine 
platelet factor (PF4), leading to irreversible aggregation and depletion of blood platelets 
(thrombocytopenia). The risk of HIT in patients treated with Lovenox (enoxaparin) and heparin 
sodium has been estimated to be 0.2 percent7 and 2  3 percent, respectively.8 Although the rate of 
HIT is relatively low for LMWHs, there are potentially serious consequences associated with HIT.  
Because LMWHs can be administered in out-patient settings, it is particularly important that 
applicants document how the risk of immunogenicity is assessed and managed. The review of 
LMWH applications includes a review of the immunogenicity-related information.  
 
In general, clinical trials are used to assess the risk of immunogenicity for products approved under 
original NDAs. Clinical trials are also used in some cases to address the risk of immunogenicity 
following postapproval source-material or manufacturing changes for NDAs.9 This guidance 
discusses an alternative approach that can be used, once the risk of immunogenicity has been 
evaluated through clinical trials in the first instance, to assess the effect of certain changes (including 
postapproval changes) on the products immunogenicity risk. Because it is important that generic 
versions of LMWHs (i.e., products approved in ANDAs) are as safe and effective as their brand name 
counterparts, including immunogenicity, this guidance also provides recommendations on meeting 
the requirement for active ingredient sameness for ANDAs for LMWHs as well as addressing 
impurities and their effect on forming complexes with PF4 and eliciting an immune response. 
  
For a product that is the subject of an ANDA or sANDA, the relevant reference product is the 
reference listed drug (RLD). For a product that is the subject of an NDA or sNDA, the relevant 
reference product in the case of postapproval changes is the approved LMWH product, or in the case 
of an original NDA for which manufacturing changes are made after completion of clinical trials, the 
product used in clinical trials.   
   
The risk of immunogenicity for the LMWH products subject to this guidance10 can be adequately 
characterized in comparison to their relevant reference products by addressing three principal critical 
elements:  (1) the sameness of the active pharmaceutical ingredient (API) (ANDAs only); (2) the 
impurities in the product that may impact on the association of the LMWH product with the 
chemokine PF4, as well as the size and charge of the complexes formed with PF4 (NDAs/ANDAs); 
and (3) the impurities in the product that could modify the detection, uptake, processing or 
presentation of the product (or the complexes it forms with PF4) to the immune system 
                                                 
6 Depolymerization refers to the breaking (or cleavage) of polysaccharide chains into smaller oligosaccharide fragments 
by chemical or enzymatic means.  Because LMWH chains are shorter than the parent heparin chains, in this draft 
guidance we generally use the term oligosaccharides in connection with LMWHs and the term polysaccharides in 
connection with heparin. 
7 Martel N, Lee J, Wells PS, 2005, Risk for Heparin-induced Thrombocytopenia with Unfractionated and Low-Molecular-
Weight Heparin Thromboprophylaxis: A Meta-Analysis, Blood, 106:2710-2715. 
8 Arepally GM, and Ortel TL, 2010, Heparin-Induced Thrombocytopenia, Annual Review of Medicine, 61:77-90. 
9 See footnote 4. 
10 See the Introduction. 
  2  Contains Nonbinding Recommendations 
(NDAs/ANDAs). The methods used can vary provided the methods used are sensitive to changes in 
the LMWH product. Because bioanalytical characterization may be insufficient to confirm these three 
critical elements as they relate to immunogenicity considerations, we also recommend using in vitro 
and/or in vivo studies of the immune system to detect differences in the LMWH product as compared 
to its relevant reference product. Differences in any of the principal elements described above 
between the LMWH product and its relevant reference product could suggest increased risk for 
immune responses and the need to perform other studies (e.g., clinical studies). Furthermore, as 
science and technology evolve, there may be different methods available for evaluating the 
immunogenicity of LMWH products. Similarly, the Agency's approval considerations may evolve 
based on greater scientific knowledge, such as a better understanding of potential causes of increased 
risk of immunogenicity of these products. 
 
III.  SUBMISSION RECOMMENDATIONS 
 
A.  Characterization of Active Ingredient Sameness (ANDAs only) 
 
A demonstration of the sameness of an active ingredient is critical to addressing the risk of 
immunogenicity in the context of ANDAs. 
 
The characterization of sameness of the active ingredient or API11 contained in the LMWH product 
and relevant reference product can be established by demonstrating equivalence with respect to the 
following: (1) physicochemical properties; (2) heparin source material and mode of 
depolymerization; (3) disaccharide building blocks, fragment mapping and sequence of 
oligosaccharide species; (4) biological and biochemical assay; and (5) in vivo pharmacodynamic 
profile.12,13 The comparative approach we describe below has been shown to be sufficient to 
characterize the heterogeneity of the LMWH API. These criteria are highly sensitive to minor 
changes in manufacturing conditions and are able to identify differences in a number of attributes14 
among LMWH products found to meet relevant compendial standards (e.g., anti-Xa activity, anti-IIa 
activity, and anti-Xa/anti-IIa).  
 
Criterion 1: Equivalence of Physicochemical Properties  
 
You should characterize the relative abundance of oligosaccharides of different molecular weights in 
the LMWH API and the molecular weight distribution of the relevant reference product API to 
demonstrate equivalence. Such a comparison can be achieved by size exclusion chromatography 
(SEC), in conjunction with the method commonly referred to as the chain mapping method,15,16 that 
                                                 
11 For purposes of this guidance active ingredient and API are used interchangeably. 
12 These five criteria and the basis by which they ensure sameness of the active ingredient were previously addressed in 
FDAs response to the citizen petition pertaining to the approval of a generic version of Lovenox (enoxaparin sodium) 
injection.  See pages 11  23 of the letter dated July 23, 2010, to Peter Safir, Covington & Burling, from Douglas 
Throckmorton, Deputy Director, CDER, available at 
http://www.fda.gov/downloads/Drugs/DrugSafety/PostmarketDrugSafetyInformationforPatientsandProviders/UCM22008
3.pdf. 
13 Lee, S., Raw, A.,Yu, L., Lionberger, R., Ya, Naiqi, Verthelyi, D., Rosenberg, A., Kozlowski, S., Webber, K., 
Woodcock, J., 2013, Scientific Considerations in the Review and Approval of Generic Enoxaparin in the United States, 
Nature Biotechnology, 31:220-226. 
14 These differences included, for example, the levels of modified disaccharide building blocks and sequences of some 
short oligosaccharides. 
15 Mourier, P.A.J., Viskov, C. 2004, Chromatographic Analysis and Sequencing Approach of Heparin Oligosaccharides 
Using Cetyltrimethylammonium Dynamically Coated Stationary Phases, Analytical Biochemistry, 332:299-313. 
  3  Contains Nonbinding Recommendations 
provides complementary information on a fingerprint profile of oligosaccharide molecular weights at 
a higher resolution.  
 
In addition to molecular weight, you should demonstrate equivalence for key features of the LMWH 
oligosaccharides by analyzing their overall chemical composition. These analyses should include 
nuclear magnetic resonance (NMR) analysis of characteristic structures, such as the epimerization 
state of the uronic acid structure (i.e., iduronic versus glucuronic acid) and the modified structures at 
the non-reducing end of the oligosaccharide chains, ultraviolet (UV) specific absorbance that 
demonstrates the presence of unique functional groups such as the 4,5-uronate structure of 
enoxaparin, and certain USP tests (e.g., 13C NMR spectra, sodium content, and the ratio of sulfate to 
carboxylate).17 
 
Criterion 2: Equivalence of Heparin Source Material and Mode of Depolymerization 
 
The distribution of sequences of LMWH API is a function of both the sequences found naturally in 
the parent heparin and the site(s) where the cleavage reaction occurs in the polysaccharide chain. 
Chemical structures introduced at the terminal ends of the cleaved oligosaccharide chains are a result 
of the cleavage reaction by which the heparin polysaccharide chains are depolymerized into the 
LMWH API oligosaccharide chains. Because the diversity of disaccharide building block sequences 
within heparin results from its biosynthetic pathway, the use of equivalent heparin source material 
and equivalent methods of depolymerization is expected to ensure that LMWH API will be at least 
similar with respect to both the distribution of natural sequences of disaccharide units in the 
oligosaccharide chains and the diversity of the modified disaccharide building blocks at the terminal 
ends of the oligosaccharide chains. Therefore, you should use equivalent heparin source material (i.e., 
heparin derived from porcine intestinal mucosa and that meets USP monograph standards for heparin 
sodium). For equivalent mode of depolymerization, you should utilize the same depolymerization 
chemistry (e.g., alkaline -elimination of the benzyl ester derivative of heparin for enoxaparin) to 
cleave the heparin polysaccharides, as used to manufacture the relevant reference product.   
 
Criterion 3: Equivalence in Disaccharide Building Blocks, Fragment Mapping, and 
Sequence of Oligosaccharide Species 
 
You should demonstrate equivalence in the identity and quantitative levels of the disaccharide 
building blocks,18 including their modifications, between the LMWH API and its relevant reference 
product API. This can be achieved by exhaustive digestion of the LMWH API with purified digesting 
enzymes (e.g., heparinases I, II, and III) and/or chemical reagents (e.g., nitrous acid) to yield the 
disaccharide building blocks comprising the LMWH API. These disaccharide building blocks can 
then be separated and quantified by a variety of analytical approaches, such as capillary 
electrophoresis (CE), reversed-phase high performance liquid chromatography (RP-HPLC) and 
                                                                                                                                                                     
16 Thanawiroon, C., Rice, K.G., Toida, T., Linhardt, R.J. 2004, Liquid Chromatography/Mass Spectrometry Sequencing 
Approach for Highly Sulfated Heparin Derived Oligosaccharides, The Journal of Biological Chemistry, 279:2608-2615. 
17 Keire DA, Buhse LF and al-Hakim, A, 2013, Characterization of currently marketed heparin products: Composition 
analysis by 2D-NMR, Analytical Methods, 5, 2984-2994, Ye H., Toby T.K., Sommers C.D., Ghasriani H., Trehy M.L., 
Ye W., Kolinski R.E., Buhse L.F., al-Hakim A. and Keire D.A., 2013, Characterization of currently marketed heparin 
products: key tests for LMWH quality assurance. J. Pharm. Biomed. Anal., 85C:99-107. 
18 Other small oligosaccharide units that should be considered in this type of compositional analysis include trisaccharide 
units (which derive from LMWH oligosaccharides having an odd number of saccharide units) and tetrasaccharide units 
(which occur due to the inherent resistance of some tetrasaccharide units to further cleavage).  
  4  Contains Nonbinding Recommendations 
strong anion exchange high performance liquid chromatography (SAX-HPLC).19,20,21 The identification 
of these disaccharide building blocks can be achieved by using a combination of several techniques, 
including (but not limited to) comparison to structurally assigned disaccharide building blocks in the 
literature, mass spectroscopy (MS), NMR spectroscopy, and/or chemical approaches such as analysis 
with modifying reagents (e.g., sodium borohydride and nitrous acid) or modifying enzymes (e.g., 2-
O-sulphatase, 6-O-sulphatase, and 4,5-glycuronidase).22,23, 24,25  
 
You should demonstrate equivalence in the fragment map of digested oligosaccharides (representing 
the signature of recurring oligosaccharide sequences within the LMWH) between the LMWH API 
and its relevant reference product API. This can be achieved by partial digestion using enzymatic 
reagents that cleave in a structurally specific fashion (e.g., heparinase I), followed by a qualitative 
and quantitative analysis using sensitive analytical methods (e.g., RP-HPLC or SAX-HPLC).26,27  
 
You should analyze sequences of a subset of oligosaccharides in the LMWH API and demonstrate 
their equivalence to those present in the relevant reference product API. The direct sequencing of 
oligosaccharides from the LMWH API can be done by isolating particular oligosaccharide species 
from the mixture through size and/or charge separation, and then analyzing their sequence using 
high-resolution analytical techniques (e.g., approaches based on property-encoded nomenclature 
(PEN) in conjunction with matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-
MS), iterative chemical and enzymatic digestion of fluorescent tagged oligosaccharides in 
conjunction with analysis by polyacrylamide gel electrophoresis, and/or enzymatic digestion in 
conjunction with NMR spectroscopy).28,29,30,31 In this part of the evaluation, it is not necessary to 
                                                 
19 Sundarem, M., Qi, Y., Shriver, Z., Liu, D., Zhao, G., Venkataraman, G., Langer, R., Sasisekharan, R., 2003, Rational 
Design of Low-Molecular Weight Heparins with Improved In Vivo Activity, Proc Natl Acad Sci USA, 100:651-656. 
20 Toyoda, H., Yamamoto, H., Ogino, N., Toida, T., Imanari, T., 1999, Rapid and Sensitive Analysis in Heparin and 
Heparin Sulfate of Reversed-Phase Ion-Pair Chromatography on a 2 mm Porous Silica Gel Column, J Chromatography, A 
830:197-201. 
21  Mourier P., Viskov C., June 2, 2005, Method for Determining Specific Groups Constituting Heparins or Low 
Molecular Weight Heparins, US Patent Application Publication US2005/0119477 A1.  
22 Ibid. 
23 Saad, O.M., Leary, J.A., 2003, Compositional Analysis and Quantification of Heparin and Heparin Sulfate by 
Electrospray Ionization Ion Trap Mass Spectrometry, Analytical Chemistry, 75:2985-2995. 
24 Stringer, S.E., Balbant, S.K., Pye, D.A., Gallagher, J.T., 2003, Heparin Sequencing, Glycobiology, 13(2):97-103. 
25 Myette, J.R., Shriver, Z., Kisiltepe, T., McLean, M.W., Venkataraman, G., Sasisekharan, R., 2002, Molecular Cloning 
of the Heparin/Heparin Sulfate  4,5 Unsaturated Glycuronidase From Flavobacterium Heparinum, Its Recombinant 
Expression in Escherichia Coli, and Biochemical Determination of Its Unique Substrate Specificity, Biochemistry, 
41:7424-7434. 
26 Linhardt, R.J., Rice, K.O., Kim, Y.S., Lohse, D.L., Wang, H.M., Loganathan, D., 1988, Mapping and Quantification of 
the Major Oligosaccharide Components of Heparin, Biochemical Journal, 254:781-787. 
27 Chuang, W.L., McAllister, H., Rabenstein, D.L., 2001, Chromatographic Methods for Product-Profile Analysis and 
Isolation of Oligosaccharides Produced by Heparinase-Catalyzed Depolymerization of Heparin, Journal of 
Chromatography, A 932:65-74. 
28 Sasisekharan, R. et al., 2000, Sequencing of 3-O Sulfate Containing Heparin Decasaccharides with a Partial 
Antithrombin III Binding Site, P Natl Acad Sci USA, 97:10359-10364. 
29 Sasisekharan, R., Venkataraman, G., Shriver, Z. & Raman, R., 1999, Sequencing Complex Polysaccharides, Science, 
286:537-542. 
30 Turnbull, J.E., Hopwood, J.J. & Gallagher, J.T., 1999, A Strategy for Rapid Sequencing of Heparan Sulfate and 
Heparin Saccharides, P Natl Acad Sci USA, 96:2698-2703. 
31 Sugahara, K. et al., 1999, Structural Studies of Octasaccharides Derived from the Low-sulfated Repeating Disaccharide 
Region and Octasaccharide Serines Derived from the Protein Linkage Region of Porcine Intestinal Heparin, 
Biochemistry, 38:838-847. 
  5  Contains Nonbinding Recommendations 
sequence all oligosaccharides in the LMWH API; the focus should be on sequencing short 
oligosaccharides that provide a sensitive measure of changes in process conditions.32  
 
Criterion 4: Equivalence in Biological and Biochemical Assays 
 
You should demonstrate that the LMWH API is equivalent to its relevant reference product API with 
respect to in vitro biological assays for relevant markers of anticoagulant activity, such as the 
activated partial thromboplastin time (aPTT) and the Heptest prolongation time. To meet the criterion 
of equivalence with respect to biochemical assays, you should demonstrate that the LMWH API is 
equivalent to its relevant reference product API in terms of factor Xa inhibition (anti-Xa) and factor 
IIa inhibition (anti-IIa).  
 
Criterion 5:  Equivalence of In Vivo Pharmacodynamic Profile 
 
You should demonstrate equivalence in the in vivo pharmacodynamic profiles based upon 
measurements of in vivo plasma anti-Xa and anti-IIa activities. For this purpose, you should conduct 
a fasting, single-dose, two-way crossover in vivo study in normal subjects as described in FDAs 
individual drug product bioequivalence guidance for enoxaparin and dalteparin.33,34   
 
B.  Impurities and Immunogenicity Risk (NDAs/sNDAs and ANDAs/sANDAs) 
 
1.  Studies Assessing the Interaction of LMWH with Platelet Factor 4  
 
Heparin-induced thrombocytopenia (HIT) is mediated by antibodies to the LMWH-PF4 complex.  
The primary immunogenicity risk factor associated with LMWHs is thought to involve the interaction 
of the active ingredient with PF4 and the presence of impurities may affect the interaction of the 
LMWH with PF4. Therefore, the association of the LMWH with PF4, as well as the size and charge 
of the LMWH-PF4 complexes formed under specified conditions, should be assessed and compared 
to that of the relevant reference product. 
 
The characteristics of the complexes formed by LMWH with PF4 could be affected by the quality of 
the PF4 itself (e.g., its ability to form tetramers) and the ratio and concentration of the two 
components. Therefore, the association of the proposed LMWH and PF4 should be characterized at 
different ratios and concentrations. The ratios and concentrations selected should encompass those 
previously described in the literature as being immunogenic, including those that lead to the 
formation of ultra large complexes.35 The methods used to assess association of the LMWH with PF4 
                                                 
32 Ozug, J.; Wudyka S.; Gunay N.S. et al. 2012, Structural elucidation of the tetrasaccharide pool in enoxaparin sodium,  
Anal Bioanal Chem  403:2733-2744. 
33 We update guidances periodically.  To make sure you have the most recent version of a guidance, check the FDA 
Drugs guidance Web page at 
http://www.fda.gov/Drugs/GuidanceComplianceRegulatoryInformation/Guidances/default.htm.  
34 The general study design for the in vivo pharmacodynamic study described in the individual drug product 
bioequivalence guidances for enoxaparin and dalteparin are applicable to other similar LMWH products.  See 
http://www.fda.gov/Drugs/GuidanceComplianceRegulatoryInformation/Guidances/ucm075207.htm; Cochran S., Li P., 
Ferro V., 2009, A surface plasmon resonance-based solution affinity assay for heparan sulfate-binding proteins, 
Glycoconj J. 26:577587; and Brandt S., Krauel1, K. Gottschalk K. E.; Renne T., Helm C.A., Greinacher A., Block S., 
2014, Characterisation of the conformational changes in platelet factor 4 induced by polyanions: towards in vitro 
prediction of antigenicity, Thromb Haemost, 112: 5364. 
35 Rauova, L, Poncz, M, McKenzie, SE et al., 2005, Ultra Large Complexes of PF4 and Heparin are Central to the 
Pathogenesis of Heparin-induced Thrombocytopenia. Blood, 05:131-8, and Suvarna S, Espinasse, B, Qi R, Lubica R, 
 
  6  Contains Nonbinding Recommendations 
(e.g., electron microscopy, surface plasmon resonance or circular dichroism) should be sensitive to 
differences in the LMWH and the development and validation studies that support the suitability of 
the method(s) selected should be submitted to the application. 
 
Because size and charge of the various complexes formed between PF4 and the LMWH are expected 
to change depending on the ratio and concentration of the two components, for each set of 
concentrations, the size and charge and relative concentrations of small, intermediate and ultra large 
complexes36,37 formed should be characterized using suitable bioanalytical methods. Several methods 
may be needed to accurately characterize both small and large complexes. Examples of suitable 
methods include SEC-UV and SEC-multi-angle light scattering analysis, photon correlation 
spectroscopy, analytical ultracentrifugation, field flow fractionation, circular dichroism, and atomic 
force microscopy. The chosen method(s) should be shown to be suitable for identifying differences in 
the size and charge of the LMWH-PF4 complexes, and the development and validation studies that 
support the suitability of the method(s) selected should be submitted to the application. The results 
obtained should be confirmed using an orthogonal method, whenever possible. 
 
2.  Characterization of Impurities  
 
Impurities in a LMWH can foster product immunogenicity by catalyzing changes in the product, 
acting as innate immune agonists, or changing the interaction of the LMWH with PF4. Impurities 
may be either process- or product-related. They can be of known structure, partially characterized, or 
unidentified.38 You should characterize impurities (e.g., residual proteins, nucleic acids, and lipids) 
present in the LMWH that could potentially modify the detection, uptake, processing, or presentation 
of the LMWH, or the complexes it forms with PF4, to the immune system. Studies should 
demonstrate that the LMWH is free of such impurities or contains similar levels and quality of such 
substances as its relevant reference product.  
 
FDA recommends three complementary approaches using a variety of suitable bioanalytical methods 
to address impurities: (1) testing the LMWH, as well as the unfractionated heparin source material 
and other raw materials for the presence of impurities (e.g., proteins, lipids, and nucleic acids);39 (2) 
assessing the capacity of the manufacturing process to remove potential impurities; and (3) 
characterizing the amount and nature  of product impurities in the LMWH relative to those in its 
relevant reference product. The chosen method(s) should be shown to be suitable for identifying 
impurities and the development and validation studies supporting the suitability of the method(s) 
selected should be submitted to the application.   
 
In addition, information should be provided on extractables and leachables from the container closure 
system over the shelf life of the LMWH product.  
                                                                                                                                                                     
Poncz M, Cines D B, Wiesner M R, and Arepally G M, 2007, Determinants of PF4/heparin immunogenicity,  Blood, 110: 
4253. 
36 Greinacher, A, Alban, S, Omer-Adam, M A et. al., 2008, Heparin-Induced Thrombocytopenia: A Stoichiometry-Based 
Model to Explain the Differing Immunogenicities of Unfractionated Heparin, Low-Molecular-Weight Heparin, and 
Fondaparinux in Different Clinical Settings, Thrombosis Research, TR-03320. 
37 Suvarna, S, QI R, and Arepally, G M, 2009, Optimization of a Murine Immunization Model for Study of PF4/ 
Heparin Antibodies, Journal of Thrombosis and Haemostasis, 7:857864.  
38 International Conference on Harmonisation guidances for industry Q3A (R2) Impurities in New Drug Substances and 
Q3B (R2) Impurities in New Drug Products. 
39 While these impurities are usually controlled during the preparation of unfractionated heparin sodium, the data should 
be submitted to FDA for review.    
  7  Contains Nonbinding Recommendations 
 
3.  Use of In Vitro and In Vivo Immunological Models  
 
The immune system can be an effective tool to detect small changes in product impurities and active 
ingredients that are missed by current analytical methods. Assessment of multiple parameters of 
immune activation and characterization of the immune response elicited by the LMWH and its 
relevant reference product using in vitro and/or in vivo models may complement other bioanalytical 
techniques designed to assess the potential of the LMWH to generate greater immune responses as 
compared to its relevant reference product. The chosen method(s) should be shown to be suitable for 
detecting changes in product impurities and the LMWH and the development and validation studies 
that support the suitability of the method(s) selected should be submitted to the application.   
 
4.  Selection and Specification of Product Lots Used for Studies to Assess the Risk of 
Immunogenicity 
 
For each lot of the LMWH used in the experiments in comparison to the relevant reference product, 
the documentation should include the identification name, date, and site of manufacture; 
manufacturing process (if more than one exists); container closure system; and results from the 
release and stability testing. The rationale for the selection of lots should be provided.  We also 
recommend that you provide to FDA data on multiple freshly manufactured, mid-expiry-cycle, and 
close-to-expiry product lots for the LMWH, and similar-stage lots of the relevant reference product.   
 
 
  8