Guidance for Industry

Immunogenicity-Related
Considerations for the Approval
of Low Molecular Weight
Heparin for NDAs and ANDAs





DRAFT GUIDANCE

This guidance document is being distributed for comment purposes only.

Comments and suggestions regarding this draft document should be submitted within 90 days of
publication in the Federal Register of the notice announcing the availability of the draft
guidance. Submit comments to the Division of Dockets Management (HFA-305), Food and
Drug Administration, 5630 Fishers Lane, rm. 1061, Rockville, MD 20852. All comments
should be identified with the docket number listed in the notice of availability that publishes in
theFederal Register.

For questions regarding this draft document contact Daniela Verthelyi, 301-827-1702.


U.S. Department of Health and Human Services
Food and Drug Administration
Center for Drug Evaluation and Research (CDER)


April 2014
CMC



Guidance for Industry
Immunogenicity-Related
Considerations for the Approval
of Low Molecular Weight
Heparin for NDAs and ANDAs

Additional copies are available from:
Office of Communications
Division of Drug Information, WO51, Room 2201
10903 New Hampshire Ave.
Silver Spring, MD 20993
Phone: 301-796-3400; Fax: 301-847-8714
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)

April 2014
CMC

Contains Nonbinding Recommendations
Draft Not for Implementation


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/ANDAs) .............................................................. 6
1. Studies Assessing the Interaction of LMWH with PF4 .................................................................... 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
Draft Not for Implementation

1
Guidance for Industry
1
1
Immunogenicity-Related Considerations for the Approval of Low 2
Molecular Weight Heparin for NDAs and ANDAs 3
4
5
This draft guidance, when finalized, will represent the Food and Drug Administrations (FDAs) current 6
thinking on this topic. It does not create or confer any rights for or on any person and does not operate to bind 7
FDA or the public. You can use an alternative approach if the approach satisfies the requirements of the 8
applicable statutes and regulations. If you want to discuss an alternative approach, contact the FDA staff 9
responsible for implementing this guidance. If you cannot identify the appropriate FDA staff, call the 10
appropriate number listed on the title page of this guidance. 11
12
13
14
15
I. INTRODUCTION 16
17
This draft guidance discusses immunogenicity-related approval considerations for low molecular 18
weight heparin (LMWH) products. Section II includes background information. Section IIIA 19
includes recommendations on meeting the requirement for active ingredient sameness for abbreviated 20
new drug applications (ANDAs) for LMWHs which helps to address immunogenicity-related 21
considerations in the context of ANDAs. Section IIIB includes recommendations on addressing 22
impurities and their potential effect on immunogenicity for ANDAs. Section IIIB also includes 23
recommendations on impurities for new drug applications (NDAs) and supplemental NDAs (sNDAs) 24
or supplemental ANDAs (sANDAs)
2
in instances where the source material (Heparin Sodium, USP) 25
or another component is changed, or when there are alterations in the manufacturing process for the 26
LMWH either before approval of the LMWH
3
or after approval of the LMWH. Drug Master File 27
(DMF) holders should also be mindful of the recommendations in this guidance and ensure DMFs are 28
current. DMF holders must notify authorized applicants of changes.
4
29
30
FDAs guidance documents, including this guidance, do not establish legally enforceable 31
responsibilities. Instead, guidances describe the Agencys current thinking on a topic and should be 32
viewed only as recommendations, unless specific regulatory or statutory requirements are cited. The 33
use of the word should in Agency guidances means that something is suggested or recommended, but 34
not required. 35
36

1
This guidance has been prepared by the Office of Pharmaceutical Science 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 generally 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 21 CFR 314.420.
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2
II. BACKGROUND 37
38
LMWH products are anticoagulants used for prevention and treatment of thrombosis (blood clots). 39
These products are produced by depolymerization of the anticoagulant heparin; complex, naturally 40
occurring polysaccharides found in certain animal species and whose backbone consists of repeating 41
disaccharide building blocks.
5
Treatment with heparin or LMWH products is associated with a 42
potentially fatal adverse event, heparin-induced thrombocytopenia (HIT). This occurs when the 43
patient produces antibodies to heparin or LMWH in complex with the chemokine platelet factor 44
(PF4), leading to irreversible aggregation and depletion of blood platelets (thrombocytopenia). In the 45
clinical trials supporting the approval of the LMWH Lovenox, HIT occurred in 1.3 percent of patients 46
receiving Lovenox, 1.2 percent of patients receiving heparin sodium, and 0.7 percent of patients 47
receiving placebo.
6
However, in subsequent reports, the risk of HIT in patients treated with Lovenox 48
and heparin sodium was estimated to be 0.2 percent
7
and 2 3 percent, respectively.
8
Although the 49
rate of HIT is relatively low for LMWHs, there are potentially serious consequences associated with 50
HIT. Because LMWHs can be administered in out-patient settings, it is particularly important that 51
applicants document how the risk of immunogenicity is assessed and managed. The review of 52
LMWH applications includes a review of the immunogenicity-related information. 53
54
In general, clinical trials are used to assess the risk of immunogenicity for products approved under 55
original NDAs. Clinical trials are also used in some cases to address the risk of immunogenicity 56
following postapproval source-material or manufacturing changes for NDAs. This guidance discusses 57
an alternative approach that can be used, once the risk of immunogenicity has been evaluated through 58
clinical trials in the first instance, to assess the effect of certain changes (including postapproval 59
changes) on the products immunogenicity risk. Because it is important that duplicate versions of 60
LMWHs (i.e., ANDAs) are as safe and effective as their brand name counterparts, including 61
immunogenicity, this guidance also provides recommendations on meeting the requirement for active 62
ingredient sameness for ANDAs for LMWHs as well as addressing impurities and their effect on 63
forming complexes with PF4 and eliciting an immune response. 64
65
For a product that is the subject of an ANDA or sANDA, the relevant reference product is the 66
reference listed drug (RLD). For a product that is the subject of an NDA or sNDA, the relevant 67
reference product in the case of postapproval changes is the approved LMWH product; or in the case 68
of an original NDA for which manufacturing changes are made after completion of clinical trials, the 69
product used in clinical trials. 70
71
The risk of immunogenicity for the LMWH products subject to this draft guidance
9
can be adequately 72
characterized in comparison to their relevant reference products by addressing three principal critical 73
elements: (1) the sameness of the active pharmaceutical ingredient (API) (ANDAs only); (2) the 74
impurities in the product that may impact on the association of the LMWH product with the 75

5
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.
6
See WARNINGS AND PRECAUTIONS section of Lovenox Product labeling, NDA-20-164, revised J une 2013.
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 the Introduction.
Contains Nonbinding Recommendations
Draft Not for Implementation

3
chemokine PF4, as well as the size and charge of the complexes formed with PF4 (NDAs/ANDAs); 76
and (3) the impurities in the product that could modify the detection, uptake, processing or 77
presentation of the product (or the complexes it forms with PF4) to the immune system 78
(NDAs/ANDAs). The methods used can vary provided the methods used are sensitive to changes in 79
the LMWH product. Because bioanalytical characterization may be insufficient to confirm these three 80
critical elements as they relate to immunogenicity considerations, we also recommend using in vitro 81
and/or in vivo studies of the immune system to detect differences in the LMWH product as compared 82
to its relevant reference product. Differences in any of the principal elements described above 83
between the LMWH product and its relevant reference product could suggest increased risk for 84
immune responses and the need to perform other studies (e.g., clinical studies). Furthermore, as 85
science and technology evolve, there may be different methods available for evaluating the 86
immunogenicity of LMWH products. Similarly, the Agency's approval considerations may evolve 87
based on greater scientific knowledge, such as a better understanding of potential causes of increased 88
risk of immunogenicity of these products. 89
90
III. SUBMISSION RECOMMENDATIONS 91
92
A. Characterization of Active Ingredient Sameness (ANDAs only) 93
94
A demonstration of the sameness of an active ingredient is critical to addressing the risk of 95
immunogenicity in the context of ANDAs. 96
97
The characterization of sameness of the active ingredient or API
10
contained in the LMWH product 98
and relevant reference product can be established by demonstrating equivalence with respect to the 99
following: (1) physicochemical properties; (2) heparin source material and mode of 100
depolymerization; (3) disaccharide building blocks, fragment mapping and sequence of 101
oligosaccharide species; (4) biological and biochemical assay; and (5) in vivo pharmacodynamic 102
profile.
11,12
The comparative approach we describe below has been shown to be sufficient to 103
characterize the heterogeneity of the LMWH API. These criteria are highly sensitive to minor 104
changes in manufacturing conditions and able to identify differences in a number of attributes
13
105
among LMWH products found to meet relevant compendial standards (e.g., anti-Xa activity, anti-IIa 106
activity, and anti-Xa/anti-IIa). 107
108
Criterion 1: Equivalence of Physicochemical Properties 109
110

10
For purposes of this guidance active ingredient and API are used interchangeably.
11
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 J uly 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.
12
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.
13
These differences included, for example, the levels of modified disaccharide building blocks and sequences of some
short oligosaccharides.
Contains Nonbinding Recommendations
Draft Not for Implementation

4
You should characterize the relative abundance of oligosaccharides of different molecular weights in 111
the LMWH API and the molecular weight distribution of the relevant reference product API to 112
demonstrate equivalence. Such a comparison can be achieved by size exclusion chromatography 113
(SEC), in conjunction with the method commonly referred to as the chain mapping method
14,15
that 114
provides complementary information on a fingerprint profile of oligosaccharide molecular weights at 115
a higher resolution. 116
117
In addition to molecular weight, you should demonstrate equivalence for key features of the LMWH 118
oligosaccharides by analyzing their overall chemical composition. These analyses should include 119
nuclear magnetic resonance (NMR) analysis of characteristic structures, such as the epimerization 120
state of the uronic acid structure (i.e., iduronic versus glucuronic acid) and the modified structures at 121
the non-reducing end of the oligosaccharide chains, ultraviolet (UV) specific absorbance that 122
demonstrates the presence of unique functional groups such as the
4,5
-uronate structure of 123
enoxaparin, and certain USP tests (e.g.,
13
C NMR spectra, sodium content, and the ratio of sulfate to 124
carboxylate). 125
126
Criterion 2: Equivalence of Heparin Source Material and Mode of Depolymerization 127
128
The distribution of sequences of LMWH API is a function of both the sequences found naturally in 129
the parent heparin and the site(s) where the cleavage reaction occurs in the polysaccharide chain. 130
Chemical structures introduced at the terminal ends of the cleaved oligosaccharide chains are a result 131
of the cleavage reaction by which the heparin polysaccharide chains are depolymerized into the 132
LMWH API oligosaccharide chains. Because the diversity of disaccharide building block sequences 133
within heparin results from its biosynthetic pathway, the use of equivalent heparin source material 134
and equivalent methods of depolymerization is expected to ensure that LMWH API will be at least 135
similar with respect to both the distribution of natural sequences of dissacharide units in the 136
oligosaccharide chains and the diversity of the modified disaccharide building blocks at the terminal 137
ends of the oligosaccharide chains. Therefore, as the applicant, you should use equivalent heparin 138
source material (i.e., heparin derived from porcine intestinal mucosa and that meets USP monograph 139
standards for heparin sodium). For equivalent mode of depolymerization, you should utilize the same 140
depolymerization chemistry (e.g., alkaline -elimination of the benzyl ester derivative of heparin for 141
enoxaparin) to cleave the heparin polysaccharides, as used to manufacture the relevant reference 142
product. 143
144
Criterion 3: Equivalence in Disaccharide Building Blocks, Fragment Mapping, and 145
Sequence of Oligosaccharide Species 146
147
You should demonstrate equivalence in the identity and quantitative levels of the disaccharide 148
building blocks,
16
including their modifications, between the LMWH API and its relevant reference 149
product API. This can be achieved by exhaustive digestion of the LMWH API with purified 150

14
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.
15
Thanawiroon, C., Rice, K.G., Toida, T., Linhardt, R.J . 2004, Liquid Chromatography/Mass Spectrometry Sequencing
Approach for Highly Sulfated Heparin Derived Oligosaccharides, The J ournal of Biological Chemistry, 279:2608-2615.
16
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).
Contains Nonbinding Recommendations
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5
digesting enzymes (e.g., heparinases I, II, and III) and/or chemical reagents (e.g., nitrous acid) to 151
yield the disaccharide building blocks comprising the LMWH API. These disaccharide building 152
blocks can then be separated and quantified by a variety of analytical approaches, such as capillary 153
electrophoresis (CE), reversed-phase high performance liquid chromatography (RP-HPLC) and 154
strong anion exchange high performance liquid chromatography (SAX-HPLC).
17,18,19
The identification 155
of these disaccharide building blocks can be achieved by using a combination of several techniques, 156
including (but not limited to) comparison to structurally assigned disaccharide building blocks in the 157
literature, mass spectroscopy (MS), NMR spectroscopy, and/or chemical approaches such as analysis 158
with modifying reagents (e.g., sodium borohydride and nitrous acid) or modifying enzymes (e.g., 2- 159
O-sulphatase, 6-O-sulphatase, and
4,5
-glycuronidase).
20,21, 22,23
160
161
You should demonstrate equivalence in the fragment map of digested oligosaccharides (representing 162
the signature of recurring oligosaccharide sequences within the LMWH) between the LMWH API 163
and its relevant reference product API. This can be achieved by partial digestion using enzymatic 164
reagents that cleave in a structurally specific fashion (e.g., heparinase I), followed by a qualitative 165
and quantitative analysis using sensitive analytical methods (e.g., RP-HPLC or SAX-HPLC).
24,25
166
167
You should analyze sequences of a subset of oligosaccharides in the LMWH API and demonstrate 168
their equivalence to those present in the relevant reference product API. The direct sequencing of 169
oligosaccharides from the LMWH API can be done by isolating particular oligosaccharide species 170
from the mixture through size and/or charge separation, and then analyzing their sequence using 171
high-resolution analytical techniques (e.g., approaches based on property-encoded nomenclature 172
(PEN) in conjunction with matrix-assisted laser desorption/ionization-mass spectrometry (MALDI- 173
MS), iterative chemical and enzymatic digestion of fluorescent tagged oligosaccharides in 174
conjunction with analysis by polyacrylamide gel electrophoresis, and/or enzymatic digestion in 175
conjunction with NMR spectroscopy).
26,27,28,29
In this part of the evaluation, it is not necessary to 176

17
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.
18
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.
19
Mourier P., Viskov C., J une 2, 2005, Method for Determining Specific Groups Constituting Heparins or Low
Molecular Weight Heparins, US Patent Application Publication US2005/0119477 A1.
20
Ibid.
21
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.
22
Stringer, S.E., Balbant, S.K., Pye, D.A., Gallagher, J .T., 2003, Heparin Sequencing, Glycobiology, 13(2):97-103.
23
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.
24
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 J ournal, 254:781-787.
25
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, J ournal of
Chromatography, A 932:65-74.
26
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.
27
Sasisekharan, R., Venkataraman, G., Shriver, Z. & Raman, R., 1999, Sequencing Complex Polysaccharides, Science,
286:537-542.
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6
sequence all oligosaccharides in the LMWH API; the focus should be on sequencing short 177
oligosaccharides that provide a sensitive measure of changes in process conditions.
30
178
179
Criterion 4: Equivalence in Biological and Biochemical Assays 180
181
You should demonstrate that the LMWH API is equivalent to its relevant reference product API with 182
respect to in vitro biological assays for relevant markers of anticoagulant activity, such as the 183
activated partial thromboplastin time (aPPT) and the Heptest prolongation time. To meet the criterion 184
of equivalence with respect to biochemical assays, you should demonstrate that the LMWH API is 185
equivalent to its relevant reference product API in terms of factor Xa inhibition (anti-Xa) and factor 186
IIa inhibition (anti-IIa). 187
188
Criterion 5: Equivalence of In Vivo Pharmacodynamic Profile 189
190
You should demonstrate equivalence in the in vivo pharmacodynamic profiles based upon 191
measurements of in vivo plasma anti-Xa and anti-IIa activities. For this purpose, you should conduct 192
a fasting, single-dose, two-way crossover in vivo study in normal subjects as described in FDAs 193
individual drug product bioequivalence guidance for enoxaparin and dalteparin.
31,32
194
195
B. Impurities and Immunogenicity Risk (NDAs/sNDAs and ANDAs/sANDAs) 196
197
1. Studies Assessing the Interaction of LMWH with PF4 198
199
Heparin-induced thrombocytopenia (HIT) is mediated by antibodies to the LMWH-PF4 complex. 200
The primary immunogenicity risk factor associated with LMWHs is thought to involve the interaction 201
of the active ingredient with PF4 and the presence of impurities may affect the interaction of the 202
LMWH with PF4. Therefore, the association of the LMWH with PF4, as well as the size and charge 203
of the LMWH-PF4 complexes formed under specified conditions, should be assessed and compared 204
to that of the relevant reference product. 205
206
The characteristics of the complexes formed by LMWH with PF4 could be affected by the ratio and 207
concentration of the two components and; therefore, the association of the proposed LMWH and PF4 208
should be characterized at different ratios and concentrations. The ratios and concentrations selected 209
should encompass those previously described in the literature as being immunogenic, including those 210

28
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.
29
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.
30
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.
31
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.
32
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.
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7
that lead to the formation of ultralarge complexes.
33
The methods used to assess association of the 211
LMWH with PF4 (e.g., surface plasmon resonance) should be sensitive to differences in the LMWH 212
and the development and validation studies that support the suitability of the method(s) selected 213
should be submitted to the application. 214
215
Because size and charge of the various complexes formed between PF4 and the LMWH are expected 216
to change depending on the ratio and concentration of the two components, for each set of 217
concentrations, the size and charge and relative concentrations of small, intermediate and ultralarge 218
complexes
34,35
formed should be characterized using suitable bioanalytical methods. Several 219
methods may be needed to accurately characterize both small and large complexes. Suitable methods 220
include SEC-UV and SEC-multi-angle light scattering analysis, photon correlation spectroscopy, 221
analytical ultracentrifugation, field flow fractionation, and atomic force microscopy. The chosen 222
method(s) should be shown to be suitable for identifying differences in the size and charge of the 223
LMWH-PF4 complexes, and the development and validation studies that support the suitability of the 224
method(s) selected should be submitted to the application. The results obtained should be confirmed 225
using an orthogonal method, whenever possible. 226
227
2. Characterization of Impurities 228
229
Impurities in a LMWH can foster product immunogenicity by catalyzing changes in the product, 230
acting as innate immune agonists, or changing the interaction of the LMWH with PF4. Impurities 231
may be either process or product related. They can be of known structure, partially characterized, or 232
unidentified.
36
You should characterize impurities (e.g., residual proteins, nucleic acids, and lipids) 233
present in the LMWH that could potentially modify the detection, uptake, processing, or presentation 234
of the LMWH, or the complexes it forms with PF4, to the immune system. Studies should 235
demonstrate that the LMWH is free of such impurities or contains similar levels and quality of such 236
substances as its relevant reference product. 237
238
FDA recommends three complementary approaches using a variety of suitable bioanalytical methods 239
to address impurities: (1) testing the LMWH, as well as the unfractionated heparin source material 240
and other raw materials for the presence of impurities (e.g., proteins, lipids, and nucleic acids);
37
(2) 241
assessing the capacity of the manufacturing process to remove potential impurities; and (3) 242
characterizing the amount and nature of product impurities in the LMWH relative to those in its 243
relevant reference product. The chosen method(s) should be shown to be suitable for identifying 244
impurities and the development and validation studies supporting the suitability of the method(s) 245
selected should be submitted to the application. 246
247

33
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.
34
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.
35
Suvarna, S, QI R, and Arepally, G M, 2009, Optimization of a Murine Immunization Model for Study of PF4/
Heparin Antibodies, J ournal of Thrombosis and Haemostasis, 7:857864.
36
International Conference on Harmonisation guidances for industry Q3A (R2) Impurities in New Drug Substances and
Q3B (R2) Impurities in New Drug Products.
37
However, these impurities are usually controlled during the preparation of unfractionated heparin sodium.
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8
In addition, information should be provided on extractables and leachables from the container closure 248
system over the shelf life of the LMWH product. 249
250
3. Use of In Vitro and In Vivo Immunological Models 251
252
The immune system can be an effective tool to detect small changes in product impurities and active 253
ingredients that are missed by current analytical methods. Assessment of multiple parameters of 254
immune activation and characterization of the immune response elicited by the LMWH and its 255
relevant reference product using in vitro and/or in vivo models may complement other bioanalytical 256
techniques designed to assess the potential of the LMWH to generate greater immune responses as 257
compared to its relevant reference product. The chosen method(s) should be shown to be suitable for 258
detecting changes in product impurities and the LMWH and the development and validation studies 259
that support the suitability of the method(s) selected should be submitted to the application. 260
261
4. Selection and Specification of Product Lots Used for Studies to Assess the Risk 262
of Immunogenicity 263
264
For each lot of the LMWH used in the experiments in comparison to the relevant reference product, 265
the documentation should include the identification name, date, and site of manufacture; 266
manufacturing process (if more than one exists); container closure system; and results from the 267
release and stability testing. The rationale for the selection of lots should be provided. We also 268
recommend that you provide to FDA freshly manufactured, mid-expiry-cycle, and close-to-expiry 269
product lots for the LMWH, and similar-stage lots of the relevant reference product. 270
271
272