1. 2 Pharmaceutical Technology November 2015 PharmTech.com
Special Report: Global Supply Chain
Image:CourtesyofUSPharmacopeialConvention
H
eparin regulates hemostasis at
various points of the coagulation
cascade mainly through its inter-
action with antithrombin and heparin
cofactor II. Because of these properties,
heparin is a life-saving anticoagulant
drug used in renal dialysis, cardiac
surgery, and treatment for deep vein
thrombosis. The drug also binds to
platelets, inhibiting platelet function
and contributing to the hemorrhagic
effects of heparin. Bovine heparin,
first approved in 1939, was widely used
in the United States for more than
50 years (see Figure 1). Like all drugs,
heparin can cause adverse effects, but
overall, bovine heparin products were
found to be safe and effective during
that period.
In the late 1980s, bovine spongi-
form encephalopathy (BSE, or “mad
cow disease”) was reported first in the
United Kingdom and later in several
other countries, raising concerns about
the use of bovine-sourced heparin
products in humans. Because of these
concerns, manufacturers of bovine
heparin products voluntarily withdrew
them from the US market in the 1990s.
Since then, heparin products approved
for use in the US and Europe have
been sourced solely from pigs, with
approximately 60% of the supply of
the drug coming from China. Figure 2
illustrates steps involved in manufac-
turing heparin from porcine intestinal
mucosa and potential impurities that
are inactivated and/or removed from
each manufacturing step.
Heparin is a lifesaving drug that was
safely used since the 1940s. However,
in 2007, contaminated heparin caused
a number of deaths in the US and hun-
dreds of adverse reactions worldwide
(1). The contaminated heparin was
found to contain over-sulfated chon-
droitin sulfate (OSCS). OSCS was an
inexpensive synthetic adulterant that
had some anticoagulant activity and
was presumably added to heparin to
increase profit when the drug was
in short supply due to a pig disease
outbreak. This “heparin crisis” dem-
onstrated the vulnerability of drug
supplies produced from increasingly
global manufacturing chains and high-
lighted the risks inherent in reliance on
one country and one animal species as
the primary source for a crucial drug.
To mitigate these concerns by diversi-
fying the sources of heparin drugs, FDA
is considering reintroduction of bovine
heparin drug product to the US market.
In August 2015, the US Pharmacopeial
Convention (USP) hosted the 6th Work-
shop ontheCharacterization ofHeparin
Products in São Paulo, Brazil, with co-
sponsors, the National Institute for Bio-
logical Standards and Control (NIBSC,
UK), National Health Surveillance
Agency(ANVISA,Brazil),andSaoPaulo
State Pharmaceutical Manufacturers As-
sociation(SINDUSFARMA,Brazil). The
focus of the workshop was an examina-
tion of the global heparin supply chain,
specificallytherisksofheparinshortages,
adulteration, and contamination.
The following is an overview of sci-
entific research and clinical experience
presented at the workshop to generate
improved understanding of the differ-
ences between porcine and bovine hepa-
rins, the clinical implications of reintro-
ducingbovineheparinintheUS,andthe
broader ramifications of bovine heparin
in the US market and worldwide.
David Keire is acting laboratory chief, Branch I,
Division of Pharmaceutical Analysis, FDA;
Barbara Mulloy is visiting professor at Institute of
Pharmaceutical Sciences, King’s College London;
Christina Chase is senior scientific writer at US
Pharmacopeial Convention (USP);
Ali Al-Hakim is acting director of Division of New
Drug API at the FDA; Damian Cairatti is head of
Latin American Regulatory Affairs at USP;
Elaine Gray is principal scientist at National Institute
of Biological Standards and Control (NIBSC) in the UK;
John Hogwood is research scientist at NIBSC;
Tina Morris is senior VP of Science Global
Biologics at USP;
Paulo Mourão is professor at Federal University
of Rio de Janeiro;
Monica Da Luz Carvalho Soares is a member of the
Deliberative Council of the Brazilian Pharmacopeia
at ANVISA and a visiting fellow at the University of
Maryland Baltimore County (UMBC); and
Anita Szajek is principal scientific liaison at USP.
The global supply
chain for bovine
and porcine heparin
and regulatory
considerations are
examined.
Diversifying the Global
Heparin Supply Chain:
Reintroduction of
Bovine Heparin in
the United States?
David Keire, Barbara Mulloy, Christina Chase, Ali Al-Hakim,
Damian Cairatti, Elaine Gray, John Hogwood, Tina Morris,
Paulo A.S. Mourão, Monica da Luz Carvalho Soares, and Anita Szajek

2. 4 Pharmaceutical Technology November 2015 PharmTech.com
Porcine vs. bovine heparins
Heparin is a natural product, extracted
from animals. Just as pork and beef
are different from each other, heparin
products made from pigs and cattle
are similar but not identical. Two ses-
sions of the USP workshop focused on
laboratory tests used to understand the
differences in structure and biological
activity between bovine and porcine
heparin.
The structure of heparin is that of
a linear polysaccharide consisting
of repeating disaccharide motifs in
which uronic acids alternate with glu-
cosamine. The polysaccharide chains
can vary in length and in substitu-
tion with sulfates and N-acetyl groups.
Structural analysis techniques range
from relatively simple, straightforward
spectroscopic and chromatographic
analyses to sophisticated applications
of techniques in nuclear magnetic res-
onance and mass spectrometry.
The biological activity of heparin,
including its abilities to inhibit the en-
zymes of blood clot formation in vivo
and in vitro, can be quantified by sev-
eral methods. Research suggests that
molecular weight and disaccharide
composition both play an important
role in biological activity. High mo-
lecular weight fractions of heparin, for
example, have a greater effect than do
lower molecular weight fractions on
anticoagulation potency.
Overall, the combination of struc-
tural and functional information avail-
able provides a clear picture of heparin
products from different species. Nu-
merous samples have been tested by
heparin manufacturers and academic
and regulatory labs revealing clear and
consistent differences in the structures
(see Figure 3) and biological activity
profiles of porcine mucosal and bovine
mucosal heparin. In addition, the few
samples of bovine lung heparin tested
were quite distinct from either mucosal
sample type.
Importantly, the data presented
show that bovine heparin was signifi-
Special Report: Global Supply Chain
Figure1:HistoricaldevelopmenttimelineoftherapeuticheparininUnitedStates.
Figure2:Heparinmanufacturingprocess.

3. 6 Pharmaceutical Technology November 2015 PharmTech.com
Special Report: Global Supply Chain
cantly less potent, weight for weight,
than porcine heparin. The relationship
between laboratory testing and clini-
cal experience is not straightforward
for such complex products, yet a differ-
ence in potency could have important
clinical relevance (2). Therefore, fur-
ther investigation including clinical
research may be warranted.
Bovine heparin and safety
There are two main safety concerns as-
sociated with bovine heparin. The first
is heparin-induced thrombocytopenia
(HIT), an infrequent but potentially
devastating adverse event (3,4). HIT
is an immune response in which the
body makes antibodies to large com-
plexes formed between the highly sul-
fated heparin chains and platelet factor
4 (5). HIT occurs in 0.2–5% of patients
regardless of the type of heparin ad-
ministered; porcine and bovine hepa-
rin appear similar in terms of HIT risk
(6,7). Another common adverse event
associated with heparin is bleeding,
which can be controlled through the
neutralization of heparin by protamine
sulfate (8).
The second safety concern, specific
for bovine heparin, is the possible pres-
ence of BSE infectious agents (9). Dur-
ing the BSE epidemic in the UK, some
people consumed BSE-infected beef.
From 1999–2000, after a long incuba-
tion period, some of these individuals
developed variant Creutzfeldt-Jakob
disease (vCJD); 229 cases have oc-
curred worldwide as of May 28, 2015
(10). Since its peak in 2000, vCJD has
declined significantly but has not been
eradicated, as a few cases are still de-
tected every year. No known cases of
vCJD, however, have been linked to
use of bovine heparin. In addition, in
India, Brazil, and Argentina—where
bovine heparin products have been in
continuous use—no cows have tested
positive for BSE and no cases of vCJD
have been observed. In the US, only
three atypical BSE cases (i.e., different
from the distinct BSE strain from the
UK that causes vCJD) in cattle have
been identified (11). Since the 1990s,
much has been learned about how BSE
leads to vCJD in humans. In addition,
methods for minimizing BSE risk in
bovine materials have advanced (12,
13). Generally, risks from tissue spon-
giform encephalopathy (TSE) agents
are controlled in three steps: animal
origin of species and supply chain
control, tissue harvest controls, and
chemical treatments that remove in-
fectious agents (11). If bovine heparin
is reintroduced, these steps will be in-
strumental for ensuring patient safety.
Because of the safety concerns noted
previously, bovine heparin is likely
to have its own USP monograph and
a separate label (Physician Labelling
Rule) that differentiates bovine hepa-
rin from porcine heparin.
Possible reintroduction of bovine
heparin into the US market
The only approved source of heparin in
most of the world is pig intestine, but
the global pig supply is limited geo-
graphically. In addition, there is little
growth potential for porcine heparin
products to be manufactured in other
parts of the world. Thus, FDA is con-
cerned about potential shortages due
to pig disease or possible geo-political
instability.
After considering the available op-
tions, FDA hosted a meeting with its
Science Board in June 2014 to discuss
the possible reintroduction of bovine
heparin in the US. Reintroduction of
bovine heparin would no longer limit
the source to one animal species and
would extend the geographic distri-
bution of source animals. If disease
occurs in one animal source and/or
there is geo-political instability in a
major source country, the risk of sup-
ply shortages could be more readily
mitigated.
The original US-approved hepa-
rin drugs from the 1930s were from
a bovine source (cow lung) and upon
approval of porcine heparin products,
both were used interchangeably until
the 1990s without major safety risks
or concerns. Notably, bovine mucosa
heparin drug product is currently
available and manufactured in South
Figure3:PartoftheprotonNMRspectraof(lower,redspectrum)porcine
mucosaland(upper,bluespectrum)bovinemucosalheparin.Thoughthe
twospectraaresimilartheyarenotexactlythesame;somedifferences
areindicatedbyarrows.TheNMRspectrareflectthechemicalstructuresof
porcineandbovineheparin,showingthatheparinsamplesfromthetwo
sourcesaresimilarbutnotidentical.
Bovine intestine
Porcine intestine
PPM 5.70 5.60 5.50 5.40 5.30 5.20 5.10 5.00 4.90 4.80 4.70

4. Pharmaceutical Technology November 2015 7
Table I: Pharmacopeial requirements for porcine and bovine heparin. RS is reference standard.
Monograph section Test Porcine heparin Bovine heparin
Identification 1
H NMR No unidentified signals greater than 4% of the
mean of signal height of 1 and 2 are present in
the following ranges: 0.10–2.00, 2.10–3.20, and
5.70–8.00 ppm. No signals greater than 200%
signal height of the mean of the signal height
of 1 and 2 are present in the 3.75–4.55 ppm for
porcine heparin.
• New acceptance criteria need to be
generated based on batch data.
Strong anion exchange
high performance
liquid chromatography
(SAX-HPLC) as
chromatographic identity
The retention time of the major peak from the
Sample solution corresponds to that of the
Standard solution.
• Chrom ID method needs to be qualified/
validated for bovine heparin. Depending
on resolution, a new method may be
needed.
• New RS may be needed
Anti-factor Xa to Anti-
factor IIa ratio
Acceptance criteria: 0.9–1.1 • New acceptance criteria may be needed.
• New potency standard may be needed.
• Potency method needs to be qualified/
validated for bovine heparin.
Molecular weight (MW)
determinations
Acceptance criteria: M24000
is NMT 20%, Mw
is
between 15,000 Da and 19,000 Da, and the
ratio of M8000–16000
to M16000–24000
is NLT 1.0.
• MW method needs to be qualified/
validated for bovine heparin.
• New acceptance criteria may be needed
Sodium It meets the requirements of the flame test for
sodium.
It meets the requirements of the flame test
for sodium.
Species and tissue
identification
Disaccharide analysis Add for species identification Add for species and tissue identification
Assay Anti-factor IIa Potency Not less than (NLT) 180 USP Heparin Units/mg • Needs to be determined using batch data
• New RS may be needed
• New acceptance criteria are needed.
Other components Nitrogen Determination,
Method I 461
1.3%–2.5% 1.3%–2.5%
Impurities Residue on Ignition 281 28.0%–41.0% 28.0%–41.0%
Galactosamine in Total
Hexosamine
Not more than (NMT) 1% NMT 1%
Nucleotidic Impurities and
Protein Impurities
NMT 0.1% NMT 0.1%
Absence of OSCS References Identification Test A and B References Identification Test A and B
BSE/TSE N/A
Specific tests Bacterial Endotoxins Test
85
NMT 0.03 USP Endotoxin Unit/USP Heparin
Unit
NMT 0.03 USP Endotoxin Unit/USP
Heparin Unit
Loss on Drying 731 Loss of NMT 5.0% of weight Loss of NMT 5.0% of weight
pH 791 5.0–7.5 in a solution (1:100) 5.0–7.5 in a solution (1:100)
Sterility Tests 71 Where it is labeled as sterile, it meets the
requirements
Where it is labeled as sterile, it meets the
requirements
Additional
requirements
Packaging and Storage Preserve in tight containers, and
store below 40 ˚C, preferably at room
temperature.
Preserve in tight containers, and
store below 40 ˚C, preferably at room
temperature.
Labeling Label to indicate the tissue and the animal
species from which it is derived
Label to indicate the tissue and the
animal species from which it is derived
USP Reference
Standards 11
USP Heparin Sodium Identification RS
USP Oversulfated Chondroitin Sulfate RS
USP Dermatan Sulfate RS
USP Galactosamine Hydrochloride RS
USP Glucosamine Hydrochloride RS
USP Heparin Sodium for Assays
USP Heparin Sodium Molecular Weight
Calibrant RS
USP Adenosine RS
New RS needed:
USP Bovine Heparin Sodium
Identification RS

5. 8 Pharmaceutical Technology November 2015 PharmTech.com
Special Report: Global Supply Chain
America (particularly Brazil and Argen-
tina) and India. In some countries, bo-
vine heparin is preferred for religious rea-
sons.Thus,therehavebeenmorethan50
years of safe and effective use of bovine
lung heparin in patients in the US, and
bovine mucosal heparin has been used
safely in South America and India. How-
ever, because the heparin characteriza-
tion technology has advanced in the in-
terim,thespecificationsfortheproposed
bovineheparinshouldbemodernizedin
a manner similar to the recent update of
the USP porcine heparin monograph.
Bovineheparinuseworldwide
Heparin derived from bovine lungs was
ingeneraluseinEuropeandtheUSuntil
itgraduallyfelloutofusefortworeasons:
the commercial advantages of the more
potent porcine product, and concerns
in the 1990s about transmission of BSE
to humans through contaminated beef
products.
Workshop speakers from Argentina,
Brazil,andIndiaemphasizedthatbovine
heparin has been approved and used for
decades in their countries. The Argen-
tinian Pharmacopeia is discussing how
to manage bovine and porcine heparins,
and the Brazilian Pharmacopeia is cur-
rently devising specific monographs for
bovine and porcine mucosal heparin.
The availability of bovine and porcine
heparinsinBrazilhasvariedconsiderably
in recent years. In 2008, 42% of heparin
was of bovine origin, but this was fol-
lowed by a decrease and then total re-
movalfromthemarketin2013.InArgen-
tina, the bovine source accounts for 70%
of the total heparin; this has remained
unchanged in recent years.
No serious adverse effects have been
associatedwiththeuseofbovineheparin
in these two countries or in India. Some
excess adverse events associated with
heparin in cardiovascular surgery, how-
ever, were reported to ANVISA in 2008
when, on short notice, bovine heparin
replaced porcine heparin for cardiovas-
cular surgery in Brazil. The Brazilian So-
ciety of Cardiovascular Surgery (14) and
ANVISA (15) published warning notes
suggesting careful monitoring of antico-
agulantlevelswhenusingbovineheparin.
There are no reports of clinical trials
comparing heparins from bovine versus
porcine intestine. Therefore, although bo-
vineheparinhasbeenusedsuccessfullyin
several countries for many years, caution
may be required when porcine heparin
is replaced with bovine heparin without
warning.
Bovineheparinuseinthe
manufactureofLMWHs
Lowmolecularweightheparins(LMWHs)
are manufactured from heparin sodium
(also known as unfractionated heparin
sodium, or UFH) using chemical or en-
zymatic depolymerization methods (16).
Currently, in the US, all forms of LMWH
are made from porcine UFH. Therefore,
their composition and properties are
known based on their biological starting
material.
The structure and composition of bo-
vine UFHs differ from that of porcine,
therefore a LMWH product made from
bovine UFH could have different prop-
erties from the same product made from
porcine UFH.Forexample,ifenoxaparin
(aLMWH)isproducedfrombovinehepa-
rin in the future, this product could not
becalledenoxaparinbecausethestructure
andpropertieswouldbedifferentandthe
activitywouldprobablybedifferentaswell.
Implicationsforpublicstandards
The current USP Heparin Sodium mono-
graph describes system suitability and ac-
ceptance criteria for identity, purity, and
strength that are designed strictly for por-
cine heparin (17, see TableI).
The workshop presentations showed
clear differences in the structures and
biological activity profiles of porcine mu-
cosal, bovine lung, and bovine mucosal
heparin samples, thereby suggesting the
need to have a separate monograph for
bovine heparin sodium. The structural
differences would necessitate separate
identificationreferencestandards(RS)for
1
H NMR, chromatographic identity, and
molecular weight determination tests for
bovine heparin (see TableI). Depending
on the tissue source(s) of bovine heparin,
there may be a need to establish separate
identification RS for bovine lung and bo-
vine mucosal heparins.
Basedonthetestingofnumeroussam-
ples, the anticoagulant activity of bovine
heparin is significantly lower than that
of porcine in the laboratory. Most of the
currentbovineproductsgavepotenciesof
about 100 IU/mg and some batches were
estimated to be as low as 70 IU/mg. Con-
sideringthemonographspecificationfor
porcine heparin is 180 IU/mg, it is likely
that the clinicians will need to give more
bovine heparin than porcine heparin by
weight. Many years of safe bovine hepa-
rin use suggest that the higher amounts
needed, as compared with porcine hepa-
rin, do not impact clinical efficacy. Clini-
cal issues with bovine heparin, however,
will need to be monitored carefully be-
cause wider clinical use could identify
unforeseen differences between porcine
and bovine heparin. Furthermore, bo-
vine heparin requires higher doses of
protamine for neutralization.
Future bovine heparin production
sites (both drug substance and drug
product) should be under cGMP com-
pliance. Supply chains including farms,
slaughterhouses,andfacilitiesthatisolate,
treat, store, and ship the bovine tissue
need to follow the same steps and tests
describedforporcineheparin(18).These
steps include, but are not limited to:
• Determine the species origin to ver-
ify that the ingredient comes only
from the correct species.
• Confirm the absence of OSCS and
ruminant material contaminants
from another species.
• Ensurethatallheparinsuppliersare
audited and inspected regularly re-
garding their documentation prac-
tices and compliance with cGMP.
• Reject any lots containing mucosa
from another species.
Conclusion
Heparin is an essential, life-saving drug
that is needed worldwide. To avoid drug
shortages, FDA is considering reintro-
duction of bovine heparin, which would
diversify the supply chain by adding
a bovine source to the currently used
porcine heparin. Sourcing heparin from
two species could greatly reduce vulner-
ability to shortages when disease strikes
one species, and could also reduce reli-

6. Pharmaceutical Technology November 2015 9
ance on one country as the primary
source. The risks involved in reliance
on one species from one country were
clearly illustrated by the heparin crisis
of 2007–2008, when adulterated porcine
heparin from China caused numerous
deaths and hundreds more adverse ef-
fects (1). Currently, China is the source
for roughly 60% of crude porcine hepa-
rin used in the US and Europe.
Bovine heparin is currently being
used in some countries and was used
safely for more than 50 years in the US
before manufacturers voluntarily with-
drew it from the market during the BSE
crisis in the UK. Despite concerns about
bovine products, there are no known
cases of BSE contamination of bovine
heparin. If bovine heparin is reintro-
duced in the US, methods for inacti-
vating BSE could be applied to further
reduce any risk. The other main safety
issue with heparin is a severe adverse ef-
fect called HIT, but bovine heparin does
not appear to have higher rates of HIT
than does porcine heparin.
Porcine and bovine heparins are dis-
tinctly different in terms of their struc-
tures and biological activities. These
complexproductsmaybehavedifferently
in clinical use than in laboratory testing,
but if potency does in fact differ signifi-
cantly in clinical use, this will need care-
ful evaluation and perhaps dosage adjust-
menttoavoidgivingpatientstoomuchor
too little heparin. If bovine-sourced hep-
arinisreintroduced,supply-chaincontrol
will be critical, with frequent inspections
of slaughterhouses and processing facili-
tiesforcGMPcompliance.Fromthedata
presentedatthemeeting,bovineheparin
and porcine heparin are not equivalent
drugs, and therefore, they will require
two different compendial monographs.
The next steps are for manufacturers to
bring bovine products to the regulatory
agencies for evaluation and possible rein-
troduction to the market after a 15-year
absence.
Acknowledgements
The participants in the USP 6th Work-
shop on the Characterization of Hep-
arin Products are acknowledged with
gratitude.
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Disclaimer
This article reflects the views of the
authors and should not be construed
to represent US FDA’s views or policies.