molecules

Review
From Farm to Pharma: An Overview of Industrial
Heparin Manufacturing Methods
Jan-Ytzen van der Meer *, Edwin Kellenbach and Leendert J. van den Bos
Development and Technical Support Aspen Oss, Kloosterstraat 6, P.O. Box 98, 5340 AB Oss, The Netherlands;
ekellenbach@nl.aspenpharma.com (E.K.); lvandenbos@nl.aspenpharma.com (L.J.v.d.B.)
* Correspondence: jvandermeer@nl.aspenpharma.com; Tel.: +31-(0)88-277-9191

Academic Editors: Giangiacomo Torri and Jawed Fareed

Received: 19 May 2017; Accepted: 18 June 2017; Published: 21 June 2017

Abstract: The purification of heparin from offal is an old industrial process for which commercial
recipes date back to 1922. Although chemical, chemoenzymatic, and biotechnological alternatives
for this production method have been published in the academic literature, animal-tissue is still the
sole source for commercial heparin production in industry. Heparin purification methods are closely
guarded industrial secrets which are not available to the general (scientific) public. However by
reviewing the academic and patent literature, we aim to provide a comprehensive overview of the
general methods used in industry for the extraction of heparin from animal tissue.

Keywords: heparin; heparin process; manufacturing methods; industrial

1. Introduction
Heparin is a strongly charged polysaccharide anticoagulant which has been used and produced
for nearly a century [1,2]. The heparin manufacturing methods used rely strongly on the unique
molecular properties of heparin, including its acidity, high charge density and stability. These
characteristics enable the purification of heparin despite the low concentration present in the starting
material (~160–260 mg/kg). Therefore a short summary of the old and current views on structure and
biosynthesis of heparin is given below, for more elaborate reviews on these topics see references [2–5].
Discussions on the structure of heparin date back to the 1920s. By the 1940s it was concluded that
heparin consisted of uronic acids and amino sugars with a high content of ester sulfates and that the
amino groups were (partly) acetylated [6]. Further biochemical characterization studies indicated
that desulphonation resulted in loss of heparin activity [7]. Additionally, fractional precipitation of
active material suggested that heparin consisted of a mixture of closely related structures instead
of a single structure. [6,8] More recent studies have shown that these observations and conclusions
were correct. We now know that heparin is indeed a highly sulfated polysaccharide consisting of
alternating glucosamine and uronic acid units. In the biosynthetic pathway towards heparin, these
monosaccharides (i.e., N-acetyl-D-glucosamine (GlcNAc) and D-glucuronic acid (GlcA)) are added to a
tetrasaccharide linkage region (GlcA-Gal-Gal-Xyl-) which is attached to proteins containing Ser-Gly
repeats. After this elongation step, heparin chains of up to 100 kDa are generated. During and after
the elongation, several modifications can occur which include: epimerization of GlcA leading to
L -iduronic acid (IdoA), N-deacetylation, N-sulfation, 2-O-sulfation, 6-O-sulfation, and more rarely
3-O-sulfation of the glucosamine [4]. The most prevalent disaccharide present in heparin is depicted
below in Figure 1.

Molecules 2017, 22, 1025; doi:10.3390/molecules22061025 www.mdpi.com/journal/molecules

Molecules 2017, 22, 025 2 of 13
Molecules 2017, 22, 025
1025 2 of 13

SO4-
SO4-
O
O O O
O O O
HO O
HO CO2H SO4-
HO n

CO2H SO4-
HO n
NHSO3-
NHSO3-

Figure 1.
Figure Major disaccharide
1. Major disaccharide found
found in
in heparin:
heparin: (-4)-α-
(-4)-α-LL-IdoA2S-(1-4)-α-
-IdoA2S-(1-4)-α-DD-GlcNS6S-(1-)
-GlcNS6S-(1-)[5].
[5].
Figure 1. Major disaccharide found in heparin: (-4)-α-L-IdoA2S-(1-4)-α-D-GlcNS6S-(1-) [5].

The
Thecomplete
completebiosynthesis
biosynthesistakes takesplaceplace ininthetheGolgi
Golgi compartment
compartment of mainly mast cells. These are
The complete biosynthesis takes place in the Golgi compartment of of
mainlymainly mast mast cells.
cells. These These
are
aare
type of
a type an
of animmune
immune cells containing
cells containing heparin-rich
heparin-rich granules. As a
granules.AsAsa aresult result
resultof of this
of this biosynthesis
this biosynthesis
biosynthesis and and
and
a type of an immune cells containing heparin-rich granules.
subsequent
subsequentmodifications,
modifications,there thereare are32 32theoretically
theoreticallypossible possibledisaccharides
disaccharideswhich whichmake makeup upheparin,
heparin,
subsequent modifications, there are 32 theoretically possible disaccharides which make up heparin,
making heparin
making heparin more complex than other biopolymers such as proteins and nucleic acids [9].
making heparinmore more complex
complex than other
than biopolymers
other biopolymers such as suchproteins and nucleic
as proteins andacidsnucleic[9]. Moreover,
acids [9].
Moreover,
in contrastin tocontrast
proteinsto proteins and nucleic acids,is heparin is synthesized in a non-template directeddirected
Moreover, in contrast toand nucleic
proteins and acids,
nucleicheparin synthesized
acids, heparin in a non-template
is synthesized in a non-template fashion
directed
fashion
which which is
results results
a high isdegree
a high of degree of heterogeneity
heterogeneity for all for all structural
structural properties. properties.
fashion which results is a high degree of heterogeneity for all structural properties.
The
The anticoagulant
anticoagulant activity activity of of heparin
heparin is is the
the result
result of of its
its potentiating action action on on antithrombin
The anticoagulant activity of heparin is the result of its potentiating
potentiating action on antithrombin
antithrombin
(ATIII)
(ATIII) which
which isisanananti-coagulation
anti-coagulation factor.
factor.Potentiated
Potentiated ATIII,ATIII,subsequently
subsequently inhibits the action
inhibits of pro-
the action of
(ATIII) which is an anti-coagulation factor. Potentiated ATIII, subsequently inhibits the action of pro-
coagulation
pro-coagulation factors IIa
factors (i.e., thrombin)
IIa (i.e., thrombin) and Xa by covalent binding, finally resulting in reduced
coagulation factors IIa (i.e., thrombin) andand XaXa bybycovalent
covalentbinding,
binding,finally finallyresulting
resulting in in reduced
reduced
coagulation.
coagulation. TheThe molecular
molecular mechanism
mechanism byby which
which heparin
heparin potentiates
potentiates ATIII
ATIII differs
differs for
for these
these two
two
coagulation. The molecular mechanism by which heparin potentiates ATIII differs for these two
factors
factors [5,10].
[5,10]. The
The potentiation
potentiation of
of ATIII
ATIII towards
towards factor
factor Xa
Xa mainly
mainly depends
depends onon an
an allosteric
allosteric activation
activation
factors [5,10]. The potentiation of ATIII towards factor Xa mainly depends on an allosteric activation
of
of ATIII
ATIII byby aaa specific
specific pentasaccharide
pentasaccharidesequence sequencein inheparin.
heparin.This Thispentasaccharide,
pentasaccharide, which
which contains
contains a
of ATIII by specific pentasaccharide sequence in heparin. This pentasaccharide, which contains a
unique 3-O-sulfate
a unique3-O-sulfate glucosamine
3-O-sulfateglucosamine
glucosaminetriggers triggers a conformational
triggersa aconformational
conformationalchange change in
changeininATIII ATIII
ATIII uponupon binding,
upon binding,
binding, whichwhich
which
unique
results
results in
in a ~1000-fold increasedaffinity
increasedaffinity of ATIIIfor for
XaXa leading to to increased inhibition of factor Xa
results in aa ~1000-fold
~1000-fold increasedaffinityofofATIII ATIII for leading
Xa leading increased
to increased inhibition
inhibition of factor Xa [10].
of factor Xa
[10]. The pentasaccharide
The pentasaccharide sequence sequence is
is sufficient sufficient
for the Xa for the
inhibitionXa inhibition activity
activity of heparin. of heparin.
Forheparin. For
the inhibition theof
[10]. The pentasaccharide sequence is sufficient for the Xa inhibition activity of For the
inhibition
IIa, however,of IIa,
a however,
heparin chaina heparin
forms chain
a bridgeforms a bridge
between ATIIIbetween
and ATIIIIIa
factor and by factor IIa by electrostatic
electrostatic interactions
inhibition of IIa, however, a heparin chain forms a bridge between ATIII and factor IIa by electrostatic
interactions
resulting in resulting in a stable
a stable ternary ternary
complex complex
[10]. To enable [10].this
To enable
‘bridge’ this ‘bridge’ chain
a heparin a heparin shouldchain be should
at least
interactions resulting in a stable ternary complex [10]. To enable this ‘bridge’ a heparin chain should
be at
15–16 least 15–16
saccharide saccharide
units in units
length in length
[11]. [11].
Besides Besides
chain chain
length, length,
also the also the
overall overall
charge charge
(i.e., high(i.e., high
sulfate
be at least 15–16 saccharide units in length [11]. Besides chain length, also the overall charge (i.e., high
sulfate to carboxylate
to carboxylate ratio orratio S/C or S/C ratio)
ratio) of a heparin
chain ischain is important
for this for this mechanism since it since it
sulfate to carboxylate ratio or S/Cofratio)a heparin
of a heparin important
chain is important mechanism
for this mechanism enables
since it
enables strong
strong interactions interactions
betweenbetweenbetween heparin
heparin heparin
and ATIII and andATIII and
heparin heparin and factor IIa.
enables strong interactions and ATIII and and heparinfactor and IIa.factor IIa.
The
The objective
objective of
of the
the heparin
heparin manufacturing
manufacturing process
process is,
is, therefore,
therefore, to
to maximize
maximizethe theyield
yieldof ofhighly
highly
The objective of the heparin manufacturing process is, therefore, to maximize the yield of highly
charged,
charged, high
high molecular
molecular weight
weight heparin
heparin chains
chains present
present in
in the
the starting
starting material
material without
without affecting
affecting the
the
charged, high molecular weight heparin chains present in the starting material without affecting the
material
material by by degradation
by degradation (e.g., depolymerization and/or desulfation) caused by the applied process
material degradation (e.g., (e.g., depolymerization
depolymerization and/or desulfation) caused
and/or desulfation) caused by by thethe applied
applied process
process
conditions.
conditions. Typical
Typical industrial processes
industrial processescan canbe bedivided
dividedinto intofive fivedistinctive
distinctivesections sections(Figure
(Figure 2).2). Each
Each of
conditions. Typical industrial processes can be divided into five distinctive sections (Figure 2). Each
of these
these section
section willwill be
be be discussed
discussed in
in thisthis review.
review.
of these section will discussed in this review.

Figure
Figure 2.
2. (a),
(a), Schematic
Schematic representation
representationof
of an
an industrial
industrial heparin
heparin purification
purification process;
process; (b)
(b) Discussed
Discussed
Figure 2. (a), Schematic representation of an industrial heparin purification process; (b) Discussed
topics
topicsper
persection;
section; (c)
(c) General
General process
process conditions
conditions and
and reagents;
reagents; (d)
(d) Removed
Removed impurities per section.
topics per section; (c) General process conditions and reagents; (d) Removed impurities per section.
Molecules 2017, 22, 1025 3 of 13

1.1. Collection and Stabilization Starting Material

1.1.1. Regulatory Aspects Related to Sourcing

The biological material used for heparin production (i.e., mucosa or bovine lungs) should be
derived from animals which meet the requirements for health suitable for human consumption [12].
This ensures the slaughtered animals are healthy and free of medication such as antibiotics.
To guarantee this, several heparin producers provide full traceability of their starting material to the
slaughterhouses and farms. Additionally, a polymerase chain reaction (PCR) or an immunochemical
analysis is suggested by the FDA and European Pharmacopoeia to demonstrate the absence of any
ruminant material in the starting material to mitigate the risk for contamination with BSE [12,13].
Moreover, from 2013 onwards, the complete supply chain of heparin, starting with the collection of the
starting material, falls under EudraLex volume 4, annex 2 of the EU guidelines for “GMP for medicinal
products for human and veterinary use” [14]. This indicates that the entire process falls under GMP
control, albeit at different levels depending on the stage of the manufacturing process.

1.1.2. Sources: Porcine and Bovine

The first heparin production protocols used canine or bovine livers as a source. Later, mainly
porcine mucosa and bovine lungs were used [15]. However, since porcine intestinal mucosa was found
to be a much ‘cleaner’ source which required less degradation compared to bovine lungs and also as a
result of the outbreak of bovine spongiform encephalopathy in the 1990s [3,16], heparin production
from bovine material has decreased significantly. In fact, the only FDA-approved source of heparin is
currently porcine mucosa [13,16]. However, several countries, including Brazil, Argentina and India,
still allow bovine-derived heparin. Bovine heparin is preferred by some for religious reasons. Heparin
is second only to insulin in application as a biological. To meet the annual heparin need, the offal
of about 1.109 pigs are required, therefore there is a substantial risk for future heparin shortages.
To prevent these potential shortages there is currently a strong debate ongoing to re-introduce bovine
heparin in the USA [15]. Because strong drop of BSE prevalence in cows, the strongly increased
knowledge on the disease and the prion reduction during the heparin purification process [17] the risks
with respect to patient safety are now better understood [15]. This may facilitate the re-introduction of
bovine heparin to the US market. There are however significant differences between porcine and bovine
heparin. For instance, bovine heparin has substantially lower activity compared to porcine heparin [18]
(see Table 1). A large amount of scientific literature is available describing structural differences
between porcine and bovine derived heparin which might explain the difference in activity [15,17,19].

1.1.3. Other Mammalian Sources

Besides bovine and porcine heparin, sheep (ovine) intestines have been used in the past to produce
pharmaceutical heparin. Ovine heparin is currently still available from chemical manufacturers for
research purposes. An elaborate overview of physiochemical characteristics of commercially available
ovine heparin compared to both porcine and bovine heparin has been given by Li Fu et al. [16].
Ovine heparin resembles porcine heparin more closely then bovine heparin based on the disaccharide
composition, antithrombin affinity and MW . Because of this resemblance, it was mentioned that
programs to manufacture fractionated heparin from ovine sources have been initiated [20]. There are
no religious restrictions on using sheep as a heparin-source however, a transmissible prion disease
(scrapie) does occur in sheep. Although scrapie is not transmissible to humans, infected sheep are
typically not used for consumption.
Dromedary (Camelus dromedaries) has been suggested as a heparin source since it is free of
any religious and health reason concerns. To investigate this source heparin was isolated from
dromedary intestines [21]. The disaccharide analysis in this study indicated that non- and monosulfated
disaccharides were more abundant in dromedary heparin compared to porcine heparin. Consistent
with this low degree of overall sulfation, dromedary raw heparin had a specific aXa activity of
Molecules 2017, 22, 1025 4 of 13

~50–60 IU/mg which is approximately half of the activity of porcine heparin which was purified as a
reference in that same study (see Table 1).

Table 1. Overview of characteristic of heparin derived from different sources.

Source aXa Activity (IU/mg) aPTT Activity Average Mol. Weight (kDa) S/C Ratio Yield (mg/kg) Refs
Porcine 148–219 168–277 15.0–19.0 a 2.31–2.57 160–260 [15,16,18,22]
Bovine b 123–156 103–181 16.2–16.5 2.29–2.40 n.d. [16,18,19]
Ovine 142 150 22.9 3.66 n.d. [16]
Dromedary 50–60 n.d. 24.0 2.0 400 [21]
Chicken 111 133 n.d. 2.26 n.d. [18]
Turkey 16.6 n.d. n.d. n.d. 300 [23]
Salmon 110–137 n.d. <8.0 c 2.20 n.d. [24]
Shrimp 95–100 n.d. 8.5 n.d. 32 [25]
Clam 317 347 14.9 n.d. 2100 [26]
a Pharmacopoeial specification; b intestinal mucosa; c 96% was ≤8.0 Da.

1.1.4. Non-Mammalian Sources

By-products of the poultry industry might seem an obvious source for heparin production
because of the high global chicken and turkey meat production. Active heparin can be derived from
chicken intestines with a specific aXa activity of 111 IU/mg and slightly lower degree of sulfation
compared to porcine heparin produced according to the same method (S/C ratio resp.: 2.26 and 2.40) [18].
In this study, however no yields have been reported. Heparin derived from chicken intestines
appears to approach porcine-derived heparin based on disaccharide composition and aXa activity
(111 IU/mg) [18]. The specific aXa activity of heparin derived from turkey intestines was extremely
low (16.6 IU/mg) [23]. Although based on this information, chicken might be a potential source of
biologically active heparin, to the best of our knowledge there is currently no heparin production on
an (semi-) industrial scale using poultry-derived starting material. The obtained GAGs from turkey
tissue mainly consisted of heparan sulfate. Differences between mammalian and avian immunological
mechanisms might be an explanation for the absence of active heparin species in turkey [23].
A relatively recent report on heparin purification from salmon (Salmo salar) gills and intestines
describes a partial purification where heparin was obtained with a low molecular weight
(96% ≤ 8000 Da). The aXa activity of the salmon-derived heparin was in the range of clinically
approved fractionated heparin (LMWH) [24].
Shrimp (Penaeus brasiliensis) heads can also be used as a source of natural LMWH with a yield of
32 mg/kg starting material [25]. This shrimp-derived heparin had a molecular weight of 8500 Da and
an aXa activity of 95 IU/mg which is also comparable (on the low end) to LMWH. Nonetheless, in vivo
experiments indicated that shrimp heparin had a slightly lower antithrombotic activity compared to
pharmaceutical grade LMWH.
Heparin derived from clams (Tapes phylippinarum) was found to have substantially better aXa
activity (317 IU/mg) compared to typical porcine heparin and an average molecular weight (Mw)
of 14,900 Da, which is comparable to unfractionated heparin [26]. The yield of the clamp derived
heparin was ~2.1 g/kg dry tissue which is a high yield. However, since the whole animal is used
here, there is a strong competition with the food industry, which explains the high price of the starting
material. To the best of our knowledge, there is currently no commercial heparin production from any
of these sources.

1.1.5. Obtaining and Stabilizing Source Material

This section focusses on porcine intestinal mucosa, since this is the major source of the globally
produced heparin. However, the processing steps also apply to bovine heparin. The mucosa production
is highly linked to the production of casings for the sausage industry. A typical procedure starts with
the removal of the content from the intestine and subsequent soaking of the intestine in a salt solution.
After soaking, the mucosa is scraped from the intestines, yielding approximately 0.8 kg of mucosa
Molecules 2017, 22, 1025 5 of 13

per pig [22]. The emptied intestines are further processed as casings for the sausage industry and
the mucosa is collected for further processing to heparin. Besides mucosa, whole porcine intestines
can be used for heparin production and are referred to as “hashed porcine guts” [27,28]. It is known
that chemical and/or enzymatic desulfation occurs after prolonged storage (mainly glucosamine
6-O-desulfation) resulting in decreased activity [18], therefore some sort of stabilization or preservation
of the material is required. Typically, mucosa is preserved using an oxygen scavenger such as sodium
bisulphite e.g., 1.5–2.5% w/w until further processing to limit microbiological growth [29]. Other
preservatives which can be added to the mucosa include calcium propionate or phenol [22].

1.1.6. Heparin on Resin

To circumvent the transport of large volumes of mucosa and to reduce the risk of degradation
during transport, a method was described [26] where the mucosa is hydrolyzed at the slaughterhouses
and the resulting heparin subsequently loaded on an anion exchange resin. For this procedure,
3000–4000 L of intestinal mucosa (daily yield of a typical pig slaughterhouse) was enzymatically
hydrolyzed using a subtilisin alkaline protease at 50–55 ◦ C and at alkaline pH. After 3–4 h or when
the viscosity was sufficiently reduced (threshold of 14.8 mPa·s) the enzyme was heat-inactivated and
the hydrolysate was filtered. To this filtrate 24–30 kg of anion-exchange resin was added (examples
described in the patent include Amberlite, Dowex, Duolite and Lewatit) and mixed for 10–13 h.
The loaded resin was sieved off, dried and transferred to a container. As a preservative, 50 g/L of
sodium bisulphite was added, and the container was shipped to a specialized chemical plant for
further processing. Using this method the yield could be improved from 30,000 units per kg of mucosa
to 48,000 units per kg mucosa, possibly because of the short processing times. Therefore, this approach
can be highly advantageous, especially when sourcing mucosa at distant slaughterhouses. However, it
does require more complex equipment, logistics and technical expertise at the slaughterhouses.

1.2. Digestion and Release of Heparin from Proteoglycans

The early heparin production procedures already included (e.g., [8]) a digestion step aiming to
liberate the heparin from the (mast-) cells and proteoglycans. This digestion step can be done by
autolysis, addition of pancreas extract, saliva, proteolytic enzymes or by chemical means. Chemical
hydrolysis can be performed under acidic or alkaline conditions at high temperatures. This might affect
the structure of the heparin. For instance, alkaline proteolysis at pH 11 leads to slight 2-O-desulfation
of the uronic acids through base-catalyzed epoxide formation. [18] For an enzymatic digestion, a wide
range of proteolytic enzymes (i.e., proteases) can be applied including trypsin, chymotrypsin [30],
papaine [23] or subtilisin-type enzymes such as Alcalase® or Maxatase® [26,29,31]. For this enzymatic
digestion, multiple tons of mucosa should be heated until the optimal temperature of the used enzyme
(e.g., 50–60 ◦ C for alcalase). Subsequently the pH should be set (e.g., 8.6 for subtilisins) [26,29] and
the enzyme can be added at a typical enzyme:substrate ratio is 0.2–2 g/kg mucosa [31]. The reaction
mixture is incubated for 4–16 h to ensure heparin is fully released from the proteoglycans.

1.3. Capture of the Heparin

Prior to the capture step, the heparin concentration in the digested starting material is extremely
low (~0.01% w/w). Therefore, the aim of this step is to enrich the heparin content to enable further
purification, at a higher concertation later in the process. Initial heparin extraction protocols describe
the precipitation of heparin from autolyzed starting material by applying a low pH (2–2.5) and a high
concentration of ammonium sulfate [7,8]. This step, most likely, precipitated mainly protein-bound
heparins instead of free heparin, as the proteolytic (trypsin) digestion was conducted after this acid
precipitation. The heparin crude obtained after this step still contained high amounts of protein [32].
Moreover, it was realized that this acid precipitation did not ‘drop out all of the heparin’ [33]. This is
probably because heparin itself does not precipitate under these conditions. Therefore, in that latter
patent a method was described using hydrophobic primary amines (such as hexylamine) which can
Molecules 2017, 22, 025 6 of 13
Molecules 2017, 22, 1025 6 of 13

form an insoluble complex with heparin under slightly acidic conditions due to their positive charge.
This neutral heparin-amine complex was subsequently collected as a precipitate on the interface
form an insoluble complex with heparin under slightly acidic conditions due to their positive charge.
between the aqueous phase and an organic phase (e.g., methyl isobutyl ketone).
This neutral heparin-amine complex was subsequently collected as a precipitate on the interface
The principle of using ammonium cations such as in the example above is currently still the most
between the aqueous phase and an organic phase (e.g., methyl isobutyl ketone).
widely used method for capturing heparin from digestion mixtures according to (patent-) literature.
The principle of using ammonium cations such as in the example above is currently still the
However, currently the quaternary ammonium cations are usually either immobilized on a resin (i.e.,
most widely used method for capturing heparin from digestion mixtures according to (patent-)
anion exchange resin) [29,31] or designed in such a way that they selectively form insoluble
literature. However, currently the quaternary ammonium cations are usually either immobilized
complexes with heparin (quaternary ammonium salts) [34,35]. Both methods exploit the polymeric
on a resin (i.e., anion exchange resin) [29,31] or designed in such a way that they selectively form
nature and uniquely high charge density of heparin (~3.7 negative charges/disaccharide),
insoluble complexes with heparin (quaternary ammonium salts) [34,35]. Both methods exploit the
distinguishing heparin from other biopolymers such as chondroitin sulfate (CS) (S/C ratio of ~1),
polymeric nature and uniquely high charge density of heparin (~3.7 negative charges/disaccharide),
dermatan sulfate (DS) (S/C ratio of ~1) or DNA/RNA (1 negative charge per disaccharide) present in
distinguishing heparin from other biopolymers such as chondroitin sulfate (CS) (S/C ratio of ~1),
the digestion mixture [36]. This allows a strong charge-based cooperative binding.
dermatan sulfate (DS) (S/C ratio of ~1) or DNA/RNA (1 negative charge per disaccharide) present in
the digestion mixture [36]. This allows a strong charge-based cooperative binding.
2.3.1. Precipitation with Quaternary Ammonium Salts
1.3.1.Several
Precipitation
patentswith
fromQuaternary Ammonium
the 1960s describe Salts
the precipitation of heparin with quaternary ammonium
salts Several
[34,35]. patents
The general formula of such a salt is depicted below
from the 1960s describe the precipitation of heparin (Figurewith
3a) where the Xammonium
quaternary represents
any anion that does not render the salt water-insoluble, (e.g., chloride) and
salts [34,35]. The general formula of such a salt is depicted below (Figure 3a) where the X represents anythe 1R and 2R group

represents
anion that does an aliphatic
not renderhydrocarbon chain of at(e.g.,
the salt water-insoluble, leastchloride)
eight carbons
and the optionally interrupted
1 R and 2 R group by:
represents
aromatic rings, double bounds, oxygen- or nitrogen atoms and 2R–4R are groups consisting of 1–7
an aliphatic hydrocarbon chain of at least eight carbons optionally interrupted by: aromatic rings,
carbon atoms [35].
double bounds, Afterorincubation
oxygen- nitrogen atoms of theanddigestion mixture
2 R–4 R are groupswith such aofsalt,
consisting a water-insoluble
1–7 carbon atoms [35].
complex is formed with the heparin chains. These insoluble complexes can be
After incubation of the digestion mixture with such a salt, a water-insoluble complex is formed with obtained as solids. One
example of an applicable quaternary ammonium salt is Hyamine ® 1622 (see Figure 3b) [34]. This
the heparin chains. These insoluble complexes can be obtained as solids. One example of an applicable
patent describes
quaternary ammoniuma method
salt were Hyamine
is Hyamine ® 16221622 is Figure
(see added 3b)to digested
[34]. Thisground lungs, subsequently
patent describes a method were the
precipitated Hyamine-heparin salt was filtered off and suspended in
Hyamine 1622 is added to digested ground lungs, subsequently the precipitated Hyamine-heparin salt an organic solvent. This
suspension
was filtered was extracted
off and suspendedwithinan anaqueous solution,This
organic solvent. andsuspension
from this solution
was extractedthe potassium salt of
with an aqueous
heparin
solution,precipitated
and from this bysolution
increasingthe the potassium
potassium salt acetate
of heparinconcentration
precipitatedtoby 30% (w/v). Approximately
increasing the potassium
0.6 g of crude material was obtained with an activity of 110
acetate concentration to 30% (w/v). Approximately 0.6 g of crude material was IU/mg from 2 kg of ground
obtained lungs using
with an
this method. Alternatively, the precipitated Hyamine-heparin complex
activity of 110 IU/mg from 2 kg of ground lungs using this method. Alternatively, the precipitated can be extracted using a
concentrated
Hyamine-heparin NaClcomplex
solutioncan(i.e.,
be 2M) to replace
extracted using the heparin andNaCl
a concentrated subsequently precipitated
solution (i.e., with
2M) to replace
MeOH to obtain the heparin sodium salt [30]. Neither patents describe an
the heparin and subsequently precipitated with MeOH to obtain the heparin sodium salt [30]. Neitherextensive characterization
on the obtained
patents describeheparin, therefore
an extensive there is no data
characterization on obtained
on the the amount of impurities
heparin, therefore such
thereas isnucleic acids
no data on
and CS/DS.
the amount of impurities such as nucleic acids and CS/DS.

a b

Figure 3.
Figure Structuresofofquaternary
3. Structures quaternaryammonium
ammonium salts
salts used
used forfor heparin
heparin capture
capture by precipitation.
by precipitation. (a)
(a) General structure of an applicable salt; (b) Structure of benzethonium chloride (Hyamine ® 1622).
General structure of an applicable salt; (b) Structure of benzethonium chloride (Hyamine® 1622).

1.3.2. Ion Exchange Resins

2.3.2.
Like Hyamine, the ion exchange resin can be added directly to the digestion mixture. Typical
amounts of anion exchange resin include 2–4 L per 100 kg of mucosa mucosa [31].
[31]. Adsorption is typically
hours during
done over several hours during which
which temperature,
temperature, pH
pH and
and salt
saltconcentration
concentrationarearecritical
criticalparameters.
parameters.
Deviations on any of these parameters might result in increased binding of unwanted contaminants
After binding,
such as nucleic acids or other GAGs. After binding, the loaded resin has to be be separated
separated from
from the now
now
heparin-free digestion
digestion mixture
mixturebybysieving.
sieving.To
Toenable
enablethis
thissieving
sievingthethe
anion exchanger
anion exchanger (see Table
(see 1, for
Table 1,
for functional groups) should be immobilized on a matrix which allows to be sieved. Examples
 Molecules 2017, 22, 1025 7 of 13

Molecules
Molecules 2017,
2017, 22,
22, 025
025 77 of 13
Molecules
Molecules 2017,
2017, 22, 025
22,groups)
025 7 of
of 13
13
functional should be immobilized on a matrix which allows to be sieved. Examples7 of 13
thereof
include
thereof
thereof crosslinked
include
include crosslinked
crosslinkedacrylic or polystyrene
acrylic
acrylic or
or polystyrenebeads.
polystyrene After After
beads.
beads. sieving
After the resulting
sieving
sieving the waste waste
the resulting
resulting streamstream
waste (peptone)
stream
thereof
thereof include
include crosslinked
crosslinked acrylic
acrylic or or polystyrene
polystyrene beads.
beads. AfterAfter sieving
sieving the
the resulting
resulting wastewaste stream
stream
could be
(peptone)
(peptone) usedbe
could
could beasused
a fertilizer
used as for farmlands,
as aaa fertilizer
fertilizer for animalanimal
for farmlands,
farmlands, feed offeed
animal for medical
feed of
of for formulations
for medical
medical for enteral
formulations
formulations for or
for
(peptone)
(peptone) could
could be be used
used as as a fertilizer
fertilizer forfor farmlands,
farmlands, animal
animal feed feed ofof for
for medical
medical formulations
formulations for for
parental
enteral
enteral or nutrition
parental
or parental [37].
nutrition The
[37].
nutrition [37]. loaded
The resin
loaded
The loaded may
resin be
may
resin may washed
be with
washed
be washed water
with or a
water
with water solution
or a with
solution
or aa solution a relatively
with
with aaa
enteral
enteral or
or parental
parental nutrition
nutrition [37].
[37]. The
The loaded
loaded resin
resin may
may be be washed
washed with with water
water or or a solution
solution with
with a
low ionic
relatively
relatively lowstrength
low ionic (e.g., 5.8%
ionic strength
strength (e.g., NaCl
(e.g., 5.8% m/v)m/v)
5.8% NaCl
NaCl to eliminate
m/v) to
to eliminate
eliminateunbound
unbound
unbound material and and
material
material to reduce
and to lessless
to reduce
reduce tightly
less
relatively
relatively low
low ionic
ionic strength
strength (e.g.,
(e.g., 5.8%
5.8% NaCl
NaCl m/v)
m/v) toto eliminate
eliminate unbound
unbound material
material and and toto reduce
reduce less
less
bound
tightly
tightly bound
boundcompounds
compounds
compounds suchsuch
as nucleic
such as acids
as nucleic
nucleic and and
acids
acids other
and less less
other
other charged
less charged GAGs
charged [38]. [38].
GAGs
GAGs ThisThis
[38]. step step
This can also
step can be
can also
also used
tightly
tightly bound
bound compounds
compounds such such as as nucleic
nucleic acids
acids andand other
other lessless charged
charged GAGs
GAGs [38].[38]. This
This step
step can
can also
also
be
be to
usedincrease
used toto the
increase activity
increase thethe of
activity heparin
activity ofof (vide
heparin infra)
(vide
heparin (vide by
infra) removing
by
infra) by removing heparin
heparin
removing heparin withwitha low
a low
with aa low affinity to
affinity the
to
affinity to the
theresin.
be
be used
used to
to increase
increase the
the activity
activity ofof heparin
heparin (vide
(vide infra)
infra) by
by removing
removing heparin
heparin withwith a lowlow affinity
affinity to
to the
the
Subsequent
resin.
resin. Subsequent
Subsequent elution
elution
elutionwith
witha high
with saltsalt
aa high
high concentration
salt concentration
concentration (i.e.,(i.e.,
>14%)
(i.e., >14%)released
>14%) the heparin
released
released the
the fromfrom
heparin
heparin the column
from the
the
resin. Subsequent elution with a high salt concentration (i.e., >14%) released
resin. Subsequent elution with a high salt concentration (i.e., >14%) released the heparin from the the heparin from the
and and
column
column enables
and further
enables
enables processing.
further
further processing.
processing. There
There
There areare
several
are several
several suitable
suitableanion
suitable anionexchange
anion exchangeresins
exchange resinsavailable
resins available on
available on the
on
column
column andand enables
enables further
further processing.
processing. ThereThere areare several
several suitable
suitable anion
anion exchange
exchange resinsresins available
available on on
the
the market (Table
market (Table 2).
(Table 2). These
2). These resins
resins might
resins might
might be be the
be the same
the same type
same type
type as as the
as the ones
the ones used
ones used for
used for water
for water treatment.
water treatment.
treatment.
the
the market
market (Table
(Table 2).
2). These
These resins
resins might
might be be the
the same
same type
type as as the
the ones
ones used
used forfor water
water treatment.
treatment.
Table
Table 2. 2. Selected examples of functional groups usedanion
on anion exchange resins for heparin capture.
Table
Table 2. Selected
Selected examples
examples of
of functional
functional groups
groups used
used on
on anion exchange
exchange resins
resins for
for heparin
heparin capture.
capture.
Table 2.
2. Selected
Selected examples
examples of
of functional
functional groups
groups used
used on
on anion
anion exchange
exchange resins
resins for
for heparin
heparin capture.
capture.
Resin
Resin
Resin Name
Name
Name Functional
Functional
Functional Group
Group
Group References
References
References
Resin
Resin Name
Name Functional
Functional Group
Group References
References
Matrix
Matrix
Matrix
Matrix
Amberlite
Amberlite IR-120,
IR-120, FPA98/,
FPA98/, IRA900/CG-45
IRA900/CG-45 [24,29,31,36]
[24,29,31,36]
Amberlite
Amberlite IR-120,
AmberliteIR-120, FPA98/,
IR-120,FPA98/, IRA900/CG-45
FPA98/,IRA900/CG-45
IRA900/CG-45 [24,29,31,36]
[24,29,31,36]
[24,29,31,36]

Dowex
Dowex 22CL
22CL Matrix
Matrix [23]
[23]
Dowex
Dowex 22CL
Dowex 22CL
22CL Matrix
Matrix [23]
[23][23]

[26,31]
[26,31]
[26,31]
Matrix
Matrix [26,31]
[26,31]
Matrix
Matrix
Lewatitt CA9249
Lewatitt
Lewatitt CA9249
CA9249
Lewatitt
Lewatitt CA9249
CA9249
Matrix [39]
[39] [39]
Matrix
Matrix [39]
[39]
Matrix

Matrix
Matrix
Matrix
Matrix
DEAE
DEAE [39,40]
[39,40]
[39,40]
DEAE
DEAE [39,40]
[39,40]

2.3.3.
2.3.3. Resins
1.3.3.
Resins or
or Fractional
Resins or Fractional
Fractional Elution
Elution
Elution for
for Activity
for Activity
Activity Increase
Increase
Increase
2.3.3.
2.3.3. Resins
Resins or or Fractional
Fractional ElutionElution for for Activity
Activity Increase
Increase
Heparin’s
Heparin’s
Heparin’s affinity
affinity
affinity to to
to anionexchange
anion
anion exchangeresin
exchange resin depends
resin depends both
depends bothon
both onitsits
on itsoverall
overall
overall charge
charge
charge andand its charge
and its
its chargedensity.
charge
Heparin’s
Heparin’s affinity
affinity to to anion
anion exchange
exchange resin resin depends
depends both both on on its
its overall
overall charge
charge and and itsits charge
charge
These
density.
density. are
These both
These are are closely
both related
closely
both closely to degree
related
related to to of sulfation
degree
degree of of and chain
sulfation
sulfation and length
and which
chain
chain length are
length also
whichimportant
which are are for its
also
also
density.
density. TheseThese are are both
both closely
closely related
related to to degree
degree of of sulfation
sulfation and and chain
chain length
length which which are are also
also
important
heparin
important for its
for its heparin
activity. As aactivity.
result,
heparin activity. As
the a result,
binding
As aa result, the binding
affinity
the binding of affinity
heparin to
affinity ofof heparin
anion
heparin to to
exchange anion exchange
resin
anion exchange correlates resin
resinwith
important
important for for its
its heparin
heparin activity.
activity. As As a result,
result, the the binding
binding affinity
affinity of of heparin
heparin to to anion
anion exchange
exchange resin resin
correlates
correlates with
its activity.
with its
its activity.
Therefore
activity. Therefore
fractional
Therefore fractional
elution
fractional elution
or a washing
elution or
or aa washing
step step
with a relatively
washing step with
with aa relatively
low low
ionic strength
relatively low ionic
(<3.5%
ionic
correlates with its activity. Therefore fractional elution or a washing
correlates with its activity. Therefore fractional elution or a washing step with a relatively low ionic step with a relatively low ionic
strength
NaCl)
strength (<3.5%
help NaCl)
to
(<3.5% NaCl) enrich help to
highly
help to enrich
active highly
enrich highly heparin active
chains heparin
active heparin [39–41]. chains
Loaded[39–41].
chains [39–41]. resin Loaded resin (N-N-diethyl-
(N-N-diethyl-2-hydroxypropyl
Loaded resin (N-N-diethyl-
strength
strength (<3.5%
(<3.5% NaCl)
NaCl) help help to to enrich
enrich highly
highly activeactive heparin
heparin chainschains [39–41].
[39–41]. Loaded
Loaded resin resin (N-N-diethyl-
(N-N-diethyl-
2-hydroxypropyl
ammonium functionalized
2-hydroxypropyl ammonium
ammonium functionalized
cellulose)
functionalized was washedcellulose)
cellulose) with was
was washed
a ~2.3%
washed NaCl with aa ~2.3%
solution.
with ~2.3% NaCl
Subsequent
NaCl solution.
fractional
solution.
2-hydroxypropyl
2-hydroxypropyl ammonium ammonium functionalized
functionalized cellulose) cellulose) was was washed
washed with with aa ~2.3%
~2.3% NaCl NaCl solution.
solution.
Subsequent
elution with
Subsequent fractional
fractional elution
a gradient
elutionfrom with
with2.3% aa gradient
to 8.2%
gradient from
fromNaCl 2.3%
2.3% to
to 8.2%
enabled 8.2%theNaCl
NaCl enabled
enabledtothe
inventors the inventors
obtain
inventorsheparin to
to obtain
fractions
obtain
Subsequent
Subsequent fractional
fractional elution
elution withwith aa gradient
gradient from from 2.3% 2.3% to to 8.2%
8.2% NaCl
NaCl enabled
enabled the the inventors
inventors to to obtain
obtain
heparin
with
heparin fractions
a 2.5–5
fractions with with
fold a 2.5–5
increase fold
of
a 2.5–5 fold increase
specific
increase of of
activity specific
relativeactivity
specific activityto the relative
starting
relative to to the starting
material
the starting material
yielding heparinyielding
material yielding with up
heparin
heparin fractions
fractions with with aa 2.5–5
2.5–5 fold
fold increase
increase of of specific
specific activity
activity relative
relative to to the
the starting
starting material
material yielding
yielding
heparin
to 500with
heparin IU/mg
with up
up to aXa
to 500
500 IU/mg
activity
IU/mgand aXa
aXaup activity
to 400 and
activity IU/mg
and up to
to 400
up aIIa IU/mg
activity.
400 aIIa
aIIa activity.
IU/mgAlthough for this
activity. Although
invention
Although for this
forpurified
this
heparin
heparin with with up up to to 500
500 IU/mg
IU/mg aXa aXa activity
activity and and up up to to 400
400 IU/mg
IU/mg aIIa aIIa activity.
activity. Although
Although for for this
this
invention
heparin
invention purified
was
purified heparin
fractionated
heparin was
instead
was fractionated
of the
fractionated more instead
crude
instead of
form
of the
the ofmore
heparin
more crude
which
crude form
formis of
bound
of heparin
to
heparin anionwhich
which is
exchange
is
invention
invention purified heparin was fractionated instead of the more crude form of heparin which is
purified heparin was fractionated instead of the more crude form of heparin which is
bound
bound to
resin,
to anion
this
anion exchange
patent
exchange resin,
illustrates
resin, this
the
this patent
potential
patent illustrates
of applying
illustrates the
thethepotential
anion
potential of applying
exchanger
of applying step the
to
the anion
improve
anion exchanger
the
exchanger activity,
bound
bound to to anion
anion exchange
exchange resin, resin, this
this patent
patent illustrates
illustrates the the potential
potential of of applying
applying the the anion
anion exchanger
exchanger
step
step to
to improve
however
improve the
at the activity,
theexpense
activity,of however
yield since
however at
at thelow
the expense
activityof
expense yield
yield since
ofheparin still low
since added
low activity
to theheparin
activity still
still added
initial yield.
heparin Otherto
added toresin
step
step to
to improve
improve the the activity,
activity, however
however at at the
the expense
expense of of yield
yield since
since low
low activity
activity heparin
heparin still still added
added to to
the
the initial
which yield.
can
initial yield. be Other
used resin
to
Other resin improvewhich can
heparin
which can be used
activity
be used to
on improve
lab
to improve scale heparin
include activity on lab
antihrombin-sepharose
heparin activity on lab scale include
scale include [42] and
the
the initial
initial yield.
yield. Other
Other resinresin which
which can can be be usedused to to improve
improve heparinheparin activity
activity on on lablab scale
scale include
include
antihrombin-sepharose
gel filtration
antihrombin-sepharose columns. [42] and
The
[42] and gel
first filtration
of these
gel filtration twocolumns.
selects
columns. The The first
heparin of these
chains
first of two
based
these two selects
on heparin
anti-thrombin
selects heparin chains
affinity
chains
antihrombin-sepharose
antihrombin-sepharose [42] [42] and
and gelgel filtration
filtration columns.
columns. The The first
first of
of these
these twotwo selects
selects heparin
heparin chains
chains
based
based on
whichon anti-thrombin
is largely determined
anti-thrombin affinity
affinity bywhich
which is
the presence
is largely
largely determined
of the “high affinity
determined by
by the presence
pentasaccharide”
the presence of
of the
the and“high affinity
the second
“high affinity type
based on anti-thrombin affinity which is largely determined
based on anti-thrombin affinity which is largely determined by the presence of the “high affinity by the presence of the “high affinity
pentasaccharide”
pentasaccharide” and
and thethe second
second type type separates
separates heparin heparin chains based
chains based on chain
on chain length.
length. BothBoth methods
methods
pentasaccharide”
pentasaccharide” and and thethe second
second type type separates
separates heparin heparin chains
chains based
based on on chain
chain length.
length. Both Both methods
methods
are
are highly
highly useful
useful analytical
analytical tools
tools but
but due
due to
to scaling
scaling issues
issues and
and considerable
considerable costs
costs of
of the
the column
column
are
are highly
highly useful
useful analytical
analytical tools tools but but duedue to to scaling
scaling issues
issues and and considerable
considerable costs costs of of the
the column
column
material
material not
not practical
practical for
for industrial
industrial scale
scale heparin
heparin production.
production.
material not practical for industrial scale
material not practical for industrial scale heparin production. heparin production.
Molecules 2017, 22, 1025 8 of 13

separates heparin chains based on chain length. Both methods are highly useful analytical tools but
due to scaling issues and considerable costs of the column material not practical for industrial scale
Molecules 2017,
heparin 22, 025
production. 8 of 13

2.4. Purification
1.4. Purification and
and Bleaching
Bleaching
At this
At this stage
stage ofof the
the production
production process,
process, we
we have
have aa heparin
heparin crude
crude solution
solution which
which isis relatively
relatively
pure. The
pure. The main
main remaining
remaining contaminants
contaminants atat this
this stage
stage are
are nucleic
nucleic acids
acids and
and non-heparin
non-heparin GAGs,
GAGs, such
such
as chondroitin sulfate (CS) and
as and dermatan
dermatan sulfate
sulfate (DS),
(DS),which
whichlike
likeheparin
heparinarearebiopolymers
biopolymershave havea
arelatively
relativelyhigh
highnegative
negativecharge-density
charge-densityandandtherefore
thereforealso
alsoadsorb
adsorb to
to the
the anion exchange resin or or
precipitate with
precipitate with the
the quaternary
quaternary ammonium
ammonium salt. salt. Moreover
Moreover a variety of pathogens, including: viruses,
bacteria and
bacteria and in
in the
the case
case of
of bovine
bovine starting
starting material
material bovine
bovine spongiform
spongiform encephalopathy
encephalopathy (BSE)
(BSE) related
related
prions
prions may not have been adequately reduced to ensure patient safety. Therefore, the
not have been adequately reduced to ensure patient safety. Therefore, the following following steps
are designed
steps to reduce
are designed the the
to reduce inactive material
inactive material such
suchasasDNA/RNA
DNA/RNAand andtotosufficiently
sufficiently reduce the
the
infectious agents
infectious agents listed
listed above.
above.

1.4.1.
2.4.1. (Fractional) Precipitation
Precipitation
Precipitation
Precipitation is both both conducted
conducted to isolate the heparin
heparin or heparin
heparin crude from aa solution
solution and
and toto
remove impurities such as non-heparin GAGs and
remove impurities such as non-heparin GAGs and nucleic acids. nucleic acids. It also serves to remove metal
serves to remove metal ions ions
and
and small
small and/or
and/or less polar molecules such as peptide peptide fragments
fragments generated
generated during
during digestion
digestion andand
residues of chemicals used in processing: these will remain in the
residues of chemicals used in processing: these will remain in the supernatant. supernatant. For this step, different
different
organic
organic solvents such as methanol,
methanol, ethanol,
ethanol,propanol,
propanol,or oracetone
acetonecan canbebeused
used[36,43].
[36,43].AsAs a result
a result of
of decreasing
decreasing polarity
polarity byby additionofofthe
addition theantisolvent,
antisolvent,the
thehighly
highlycharged
charged heparin
heparin chains
chains precipitate.
Most
Most protocols
protocols in in the
the literature
literature apply
apply either
either methanol
methanol or ethanol
ethanol for this
this step
step with
with aa percentage
percentage of of
~50%
~50% (v/v).
(v/v). TheThe concentration
concentrationof of the
the organic
organic solvent
solvent is
is paramount
paramount at at this stage as well as NaCl NaCl
concentration
concentration and temperature.
temperature. ThisThis was demonstrated
demonstrated by Volpi [36] [36] who
who performed
performed aa fractional
fractional
precipitation
precipitation on a mixture of heparin, CS and DS with methanol, ethanol and propanol. Here itit was
on a mixture of heparin, CS and DS with methanol, ethanol and propanol. Here was
observed
observed that
that the
the order
order of of precipitation
precipitation ofof these
thesecompounds
compounds reflects
reflectstheir
theircharge
chargedensity
density(i.e.,
(i.e.,S/C
S/C
ratios of resp. 2.4, 1.1 and 0.98). Using a relative volume of 1 methanol (i.e., ~50%
ratios of resp. 2.4, 1.1 and 0.98). Using a relative volume of 1 methanol (i.e., ~50% v/v) precipitated v/v) precipitated
nearly
nearly all
all the
the heparin
heparin from
from thethe mixture
mixture whereas
whereas aa substantial
substantial concentration
concentration of CS CS and
and DS
DS was
was still
still
present
present inin the
the supernatant
supernatant(see (seeFigure
Figure4).
4).

45 Heparin
amount of percipitated GAG (mg)

40 DS
CS
35
30
25
20
15
10
5
0
0 0.5 1 1.5 2
relative volume of MeOH

Figure 4. Fractional
Figure Fractionalprecipitation
precipitationofof heparin, DSDS
heparin, andand
CS CS
at different concentrations
at different of methanol.
concentrations Data
of methanol.
derived
Data fromfrom
derived VolpiVolpi
et al.et[36].
al. [36].

2.4.2. Bleaching
1.4.2.
The last
The last step
step in
inmost
mostheparin
heparinprocesses
processesusually
usuallyincludes
includesananoxidation/bleaching
oxidation/bleaching step
step meant
meant to
to
remove/reduce color, endotoxins (depyrogenization), bacteria, mold, viruses and prions, followed
remove/reduce color, endotoxins (depyrogenization), bacteria, mold, viruses and prions, followed by by
solvent precipitation
solvent precipitation sequence
sequence and
and aa 0.22
0.22 µ
µ sterile
sterile filtration
filtration to
to remove
remove microbes
microbes and
and drying
drying [43–47].
[43–47].
The oxidation is performed using e.g., potassium permanganate (KMnO4) hydrogen peroxide (H2O2),
peracetic acid (CH3CO3H), sodium hypochlorite (NaClO) or ozone (O3) [48]. Prion reduction by
hydrogen peroxide oxidation has been used by the FDA as a justification for the re-introduction of
bovine heparin [15]. The term ‘oxidation’ is misleading here and ‘bleaching’ is more appropriate since
the aim of the step is not to affect the heparin molecule by oxidation but rather to purify the heparin.
Molecules 2017, 22, 1025 9 of 13

The oxidation is performed using e.g., potassium permanganate (KMnO4 ) hydrogen peroxide (H2 O2 ),
peracetic acid (CH3 CO3 H), sodium hypochlorite (NaClO) or ozone (O3 ) [48]. Prion reduction by
hydrogen peroxide oxidation has been used by the FDA as a justification for the re-introduction of
bovine heparin [15]. The term ‘oxidation’ is misleading here and ‘bleaching’ is more appropriate since
the aim of the step is not to affect the heparin molecule by oxidation but rather to purify the heparin.
However, this step can still result in the inadvertent modification of the heparin chains. 9 This
Molecules 2017, 22, 025 of 13
can
resultMolecules
in damage to the
2017, 22, 025 heparin chain and/or a reduced biological activity [46,49,50]. In the wake
9 of 13
of theresult
2008incontaminated
damage to the heparin crisis,and/or
heparin chain 1D 1H-NMR
a reducedwas introduced
biological activityas a pharmacopoeial
[46,49,50]. In the wake release
of
result
the
test. 1D in contaminated
12008 damage
H-NMR hadtoproved
theheparin
heparin chain1D
crisis,
successful and/or a reduced
in 1H-NMR
detecting was biological activity
theintroduced
contaminant [46,49,50].chondroitin
as aoversulfated
pharmacopoeial In the wake
release of
test.
sulfate
which the
1Dhas2008
1H-NMRcontaminated
had proved
a distinctive heparin
signal crisis,
successful
due to the 1D 1H-NMR
in detecting
glucosamine was introduced
the contaminant
N-acetyl groupas a pharmacopoeial
oversulfated
[51–53].chondroitinrelease
This resulted test.
sulfate
in close
1D 1H-NMR
which had proved
hasheparin
a distinctive successful
signal to in
duespectrathedetecting the contaminant
glucosamine N-acetyl oversulfated
group chondroitin
[51–53].spectral
This resulted sulfate
inspection to 1D 1 H-NMR for unknown signals in specific areas.inPotassium
close
which has a distinctive signal due to the glucosamine N-acetyl group [51–53].
inspection to heparin 1D H-NMR spectra for unknown signals in specific spectral areas. Potassium
1 This resulted in close
permanganate oxidation has been shown to oxidize the reducing end hydroxyl of N-acetyl glucosamine
inspection to heparin 1D 1H-NMR spectra for unknown signals in specific spectral areas. Potassium
permanganate oxidation has been shown to oxidize the reducing end hydroxyl of N-acetyl
to yield a carboxylate
permanganate group has
oxidation resulting in
beengroup a 2.10
shown toppm signal
oxidize from
the ppm the N-acetyl
reducing function in the NMR
glucosamine to yield a carboxylate resulting in a 2.10 signal end
fromhydroxyl of function
the N-acetyl N-acetyl
spectrum
in theof potassium
glucosamine
NMR to yieldpermanganate
spectrum aofcarboxylate bleached
group
potassium permanganate heparin
resulting in a (see Figure
2.10heparin
bleached ppm 5, [54–57]).
signal
(see from the
Figure N-acetyl function
5, [54–57]).
in the NMR spectrum of potassium permanganate bleached heparin (see Figure 5, [54–57]).

Figure 5. Reducing end oxidized N-acetylglucosamine heparin modification as a result of potassium

Figure 5. Reducing end oxidized N-acetylglucosamine heparin modification as a result of potassium
Figure 5. Reducing
permanganate end oxidized N-acetylglucosamine heparin modification as a result of potassium
bleaching.
permanganate bleaching.
permanganate bleaching.
Potassium permanganate oxidation also results in the reduction of the residual (glycol) serine at
Potassium
the reducing
Potassium permanganate
end of the heparin
permanganate oxidation
oxidation also results
chain also
[50]. ininthe
Oxidation
results byreduction
the potassium
reduction of the
of residual
permanganate (glycol)
the residual serine
results in an
(glycol) at
serine
the reducing
at theincrease
reducing endofofthe
in carboxylate
end the heparin
groups
heparin chain [50].
in chain
heparin[50].
andOxidation
concomitant
Oxidation byby potassium
chain breakspermanganate
potassium [50]. results
This should
permanganate be in anin an
taken
results
increase
into in carboxylate groups in heparin and concomitant chain breaks [50]. Thisasshould be taken
increase inaccount when groups
carboxylate using the
in sulfate/carboxylate
heparin ratio which
and concomitant chainis breaks
commonly[50].used
This a measure
should be for into
taken
into account
heparin when[57].
sulfation using the sulfate/carboxylate
Oxidation by peracetic acid ratio which
results is commonly
in the formation used as a measureacid
of 3-acetyluronic for
account when using the sulfate/carboxylate ratio which is commonly used as a measure for heparin
heparindisplays
which sulfation [57]. Oxidation
a signal at 2.18 ppmby peracetic acid results
in the 1H-NMR in the
spectrum of formation of 3-acetyluronic
peracetic acid bleached heparinacid
sulfation
which[57]. Oxidation by peracetic acid results in the formation of 3-acetyluronic acid which displays
displays
(Figure 6 [58]). a signal1 at 2.18 ppm in the 1H-NMR spectrum of peracetic acid bleached heparin
a signal at 2.18 ppm in the H-NMR spectrum of peracetic acid bleached heparin (Figure 6 [58]).
(Figure 6 [58]).

Figure 6. 3-Acetyluronic acid heparin modification as a result of peracetic acid bleaching.

Figure 6. 3-Acetyluronic acid heparin modification as a result of peracetic acid bleaching.
Figure 6. 3-Acetyluronic acid heparin modification as a result of peracetic acid bleaching.
Oxidation by other oxidation agents such as hydrogen peroxide and hypochlorite bleached
Oxidation
heparin have not bybeen
otherreported
oxidation agents
to have such as hydrogen
a distinctive signals in peroxide
its 1H-NMRand hypochlorite
spectrum. Thus, bleached
these
Oxidation
heparin have
‘process by other
not been
signatures’ oxidation
allowreported agents such
to have a between
to discriminate as
distinctivehydrogen peroxide
signals methods
oxidation in its 1H-NMR and
used hypochlorite
spectrum.
based on theThus,
1H-NMR bleached
these
‘process
havesignatures’ allow products
to discriminate between oxidation methods 1 used based
heparin
spectroscopy not [59].
beenOther
reported to have duea to distinctive
oxidation signals
have been in its H-NMR
described theon
in spectrum. the H-NMR
1
literatureThus,[50].these
spectroscopy
Oxidation
‘process signatures’ [59]. Other
as purification
allow products
tostep is only
discriminate due to oxidation
feasible
betweenbecause have
of thebeen
oxidation high described
intrinsic
methods inbased
the literature
stability
used ofonthethe 1[50].
heparin
H-NMR
Oxidation
molecule as
whichpurification
is evident step
from isa only
numberfeasible
of because
studies. of the
[28,60,61] high
Less intrinsic
stable
spectroscopy [59]. Other products due to oxidation have been described in the literature [50]. Oxidation stability
(bio-) of
moleculesthe heparin
such as
moleculewould
proteins which beis evident
degraded from
by athese
number of studies.
strongly [28,60,61]
oxidizing Less stable
conditions, hence(bio-)
the molecules
strong such as
virus/prion
as purification step is only feasible because of the high intrinsic stability of the heparin molecule which
proteins would
reductions be degraded by [3,15].
these strongly
The highoxidizing conditions, hence the strong virus/prion
is evident from achieved
a number in this step
of studies. [28,60,61] Lessstability
stable of the heparin
(bio-) molecules molecule
such as together
proteins with its
would be
reductions
charged achieved
polymeric in this
nature step
[60] [3,15].
enable The
it to be high stability
isolated in spiteof of
thebeing
heparin molecule
present together
at relatively low with its
levels
degraded
charged
by polymeric
these strongly
nature
oxidizing
[60] enable
conditions, hence the strong virus/prion reductions achieved
in the complex starting material [27]. it to be isolated in spite of being present at relatively low levels
in this
in step [3,15]. starting
the complex The high stability
material [27].of the heparin molecule together with its charged polymeric
nature [60] enable it to
2.5. Isolation and Drying be isolated in spite of being present at relatively low levels in the complex
2.5.material
starting Isolation and Drying
[27].
After this bleaching step, most properties of the heparin including: molecular composition,
Afterprofiles,
impurity this bleaching
microbial step, most
safety andproperties of the heparin
color are determined. including:
Therefore thesemolecular composition,
steps are optimized to
impurity
reach profiles,
maximal microbial
yields and a safety
minimum and color are determined.
of residual Therefore
solvents. There these steps
are several are optimized
methods possible to
reach maximal
isolate yields
the heparin. andofa these
Some minimum methods of residual
includesolvents. There are
a precipitation stepseveral
using amethods possible of
high percentage to
isolate theorheparin.
methanol ethanolSome of these
ensuring that methods
all heparin include a precipitation
is precipitated. step using is
The precipitate a high
then percentage
collected and of
methanol or ethanol ensuring that all heparin is precipitated. The precipitate is then collected and
Molecules 2017, 22, 1025 10 of 13

1.5. Isolation and Drying

After this bleaching step, most properties of the heparin including: molecular composition,
impurity profiles, microbial safety and color are determined. Therefore these steps are optimized to
reach maximal yields and a minimum of residual solvents. There are several methods possible to
isolate the heparin. Some of these methods include a precipitation step using a high percentage of
methanol or ethanol ensuring that all heparin is precipitated. The precipitate is then collected and
dried under vacuum at elevated temperatures (e.g., 40–75 ◦ C) [62]. Alternatively, the heparin can be
directly isolated from the solution by spray drying [63] or barrel drying [62].

2. Concluding Remark
Sourcing, isolation and purification of this 100 year old drug is still evolving for optimal efficacy
and patient safety as well as production efficiency. The strict GMP regulations and pharmacopoeial
criteria on the complete supply chain from farm to pharma will continue to assure the safety and
efficacy of this WHO essential drug [64].

Acknowledgments: We thank Gijs van Dedem for critical reading of the paper and for his constructive comments.
Author Contributions: J.-Y.v.d.M., E.K. and L.J.v.d.B. constructed the idea for the manuscript, all authors
contributed to the writing of the paper.
Conflicts of Interest: All authors hold positions in an enterprise which commercially manufactures heparin.

References
1. Torri, G.; Naggi, A. Heparin centenary—An ever-young life-saving drug. Int. J. Cardiol. 2016, 212, S1–S4.
[CrossRef]
2. Yates, E.A; Rudd, T.R. Recent innovations in the structural analysis of heparin. Int. J. Cardiol. 2016, 212,
S5–S9. [CrossRef]
3. Barrowcliffe, T.W. History of heparin. In Heparin-A Century of Progress; Lever, R., Mulloy, B., Page, C.P., Eds.;
Springer: Berlin/Heidelberg, Germany, 2012; pp. 3–22.
4. Carlsson, P.; Kjellén, L. Heparin biosynthesis. In Heparin—A Century of Progress; Lever, R., Mulloy, B.,
Page, C.P., Eds.; Springer: Berlin/Heidelberg, Germany, 2012; pp. 23–41.
5. Mulloy, B.; Hogwood, J.; Gray, E.; Lever, R.; Page, C.P. Pharmacology of heparin and related drugs. Pharmacol. Rev.
2016, 68, 76–141. [CrossRef] [PubMed]
6. Jorpes, E. On the chemistry of heparin. Biochem. J. 1942, 36, 203–213. [CrossRef] [PubMed]
7. Charles, A.F.; Scott, D.A. Studies on heparin: Observations on the chemistry of heparin. Biochem. J. 1936, 30,
1927–1933. [CrossRef] [PubMed]
8. Kruizenga, M.H.; Spaulding, L.B. The preparation of highly active barium salt of heparin and its fractionation
into two chemically and biologically different constituents. J. Biol. Chem. 1943, 148, 641–647.
9. Shriver, Z.; Raman, R.; Venkataraman, G.; Drummond, K.; Turnbull, J.; Toida, T.; Linhardt, R.; Biemann, K.;
Sasisekharan, R. Sequencing of 3-O sulfate containing heparin decasaccharides with a partial antithrombin
III binding site. Proc. Natl. Acad. Sci. USA 2000, 97, 10359–10364. [CrossRef] [PubMed]
10. Olson, S.T.; Richard, B.; Izaguirre, G.; Schedin-Weiss, S.; Gettins, P.G. Molecular mechanisms of
antithrombin-heparin regulation of blood clotting proteinases. A paradigm for understanding proteinase
regulation by serpin family protein proteinase inhibitors. Biochimie 2010, 92, 1587–1596. [CrossRef] [PubMed]
11. Petitou, M.; Hérault, J.P.; Bernat, A.; Driguez, P.A.; Duchaussoy, P.; Lormeau, J.C.; Herbert, J.M. Synthesis of
thrombin-inhibiting heparin mimetics without side effects. Nature 1999, 398, 417–422. [CrossRef] [PubMed]
12. European Pharmacopeia. Monograph for Heparin Sodium Heparinum Natricum, 9th ed.; 01/2017:0333; Council
of Europe: Strasbourg, France, 2017.
13. Guidance for Industry-Heparin for Drug and Medical Device Use: Monitoring Crude Heparin for Quality.
Available online: https://www.fda.gov/downloads/Drugs/GuidanceComplianceRegulatoryInformation/
Guidances/UCM291390.pdf (accessed on 19 April 2017).
Molecules 2017, 22, 1025 11 of 13

14. EudraLex. The Rules Governing Medicinal Products in the European Union, Volume 4: EU Guidelines for
Good Manufacturing Practice for Medicinal Products for Human Van Veterinary Use, Annex 2 Manufacture
of Biological Active Substances and Medicinal Products for Human Use. Ref. Ares(2012)118531-28/06/2012.
Available online: https://ec.europa.eu/health/sites/health/files/files/eudralex/vol-4/vol4-an2__2012-
06_en.pdf (accessed on 16 June 2017).
15. Keire, D.; Mulloy, B.; Chase, C.; Al-Hakim, A.; Cairatti, D.; Gray, E.; Hogwood, J.; Morris, T.; Mourão, P.A.S.;
da Luz Carvalho Soares, M.; et al. Diversifying the Global Heparin Supply Chain: Reintroduction of Bovine
Heparin in the United States? Pharm. Technol. 2015, 28, 36.
16. Fu, L.; Li, G.; Yang, B.; Onishi, A.; Li, L.; Sun, P.; Zhang, F.; Linhardt, R.J. Structural Characterization of
Pharmaceutical Heparins Prepared from Different Animal Tissues. J. Pharm. Sci. 2013, 102, 1447–1457.
[CrossRef] [PubMed]
17. Bett, C.; Grgac, K.; Long, D.; Karfunkle, M.; Keire, D.A.; Asher, D.M.; Gregori, L. A Heparin Purification
Process Removes Spiked Transmissible Spongiform Encephalopathy Agent. AAPS J. 2017, 1–7. [CrossRef]
[PubMed]
18. Bianchini, P.; Liverani, L.; Mascellani, G.; Parma, B. Heterogeneity of unfractionated heparins studied in
connection with species, source, and production processes. Semin. Thromb. Hemost. 1997, 23, 3–10. [CrossRef]
[PubMed]
19. St. Ange, K.; Onishi, A.; Fu, L.; Sun, X.; Lin, L.; Mori, D.; Zhang, F.; Dordick, J.S.; Fareed, J.; Hoppensteadt, D.;
et al. Analysis of heparins derived from bovine tissues and comparison to porcine intestinal heparins.
Clin. Appl. Thromb. Hemost. 2016, 22, 520–527. [CrossRef] [PubMed]
20. Hoppensteadt, D.; Maia, P.; Silva, A.; Kumar, E.; Guler, N.; Jeske, W.; Kahn, D.; Walenga, J.M.; Coyne, E.;
Fareed, J. Resourcing of Heparin and Low Molecular Weight Heparins from Bovine, Ovine, and Porcine
Origin. Studies to Demonstrate the Biosimilarities. Blood 2015, 126, 4733. [CrossRef]
21. Warda, M.; Gouda, E.M.; Toida, T.; Chi, L.; Linhardt, R.J. Isolation and characterization of raw heparin from
dromedary intestine: Evaluation of a new source of pharmaceutical heparin. Comp. Biochem. Physiol. C
Toxicol. Pharmacol. 2003, 136, 357–365. [CrossRef] [PubMed]
22. Vreeburg, J.W.; Baauw, A. Method for Preparation of Heparin from Mucosa. Patent No. WO2010/110654 A1,
24 March 2009.
23. Warda, M.; Mao, W.; Toida, T.; Linhardt, R.J. Turkey intestine as a commercial source of heparin? Comparative
structural studies of intestinal avian and mammalian glycosaminoglycans. Comp. Biochem. Physiol. B Biochem.
Mol. Biol. 2003, 134, 189–197. [CrossRef]
24. Flengsrud, R.; Larsen, M.L.; Ødegaard, O.R. Purification, characterization and in vivo studies of salmon
heparin. Thromb. Res. 2010, 126, e409–e417. [CrossRef] [PubMed]
25. Dietrich, C.P.; Paiva, J.F.; Castro, R.A.; Chavante, S.F.; Jeske, W.; Fareed, J.; Gorin, P.A.; Mendes, A.; Nader, H.B.
Structural features and anticoagulant activities of a novel natural low molecular weight heparin from the
shrimp Penaeus brasiliensis. Biochim. Biophys. Acta 1999, 1428, 273–283. [CrossRef]
26. Cesaretti, M.; Luppi, E.; Maccari, F.; Volpi, N. Isolation and characterization of a heparin with high
anticoagulant activity from the clam Tapes phylippinarum: Evidence for the presence of a high content
of antithrombin III binding site. Glycobiology 2004, 14, 1275–1284. [CrossRef] [PubMed]
27. Liu, H.; Zhang, Z.; Linhardt, R.J. Lessons learned from the contamination of heparin. Nat. Prod. Rep. 2009,
26, 313–321. [CrossRef] [PubMed]
28. Linhardt, R.J.; Gunay, N.S. Production and chemical processing of low molecular weight heparins.
Semin. Thromb. Hemost. 1999, 25, 3–10.
29. Griffin, C.C.; Linhardt, R.J.; van Gorp, C.L.; Toida, T.; Hileman, R.E.; Schubert, R.L.; Brown, S.E. Isolation and
characterization of heparan sulfate from crude porcine intestinal mucosal peptidoglycan heparin. Carbohydr. Res.
1995, 276, 183–197. [CrossRef]
30. Vidic, H.J. Process for Preparation of Heparin. U.S. Patent 4,283,530 A, 12 November 1976.
31. Van Houdenhoven, F.A.E.; Sanders, A.L.M.; van zuthpen, P.J.J. Process for the Purification of Heparin.
U.S. Patent 6,232,093, 3 January 2000.
32. Homan, J.D.H.; Lens, J. A simple method for the purification of heparin. Biochim. Acta 1948, 2, 333–337.
[CrossRef]
33. Bush, J.A.; Freeman, S.; Hagerty, E.B. Process for Preparing Heparin. U.S. Patent 2,884,358, 22 April 1957.
Molecules 2017, 22, 1025 12 of 13

34. Nomine, G.; Pierre, B. Process of Purifying Heparin, and Product Produced Therefrom. U.S. Patent 2,989,438,
20 June 1961.
35. Mozen, M.M.; Evans, T.D. Process for Purifying Heparin. U.S. Patent 3,058,884, 14 September 1959.
36. Volpi, N. Purification of heparin, dermatan sulfate and chondroitin sulfate from mixtures by sequential
precipitation with various organic solvents. J. Chromatogr. B Biomed. Sci. Appl. 1996, 685, 27–34. [CrossRef]
37. Van Gorp, C.L.; Vosburgh, F.; Schubert, R.L. Protein Hydrolysate from Mucosal Tissue. U.S. Patent 5,607,840,
30 November 1992.
38. Yamamoto, R.; Bellomo, E.G.; Kim, Y.; Rachana, V.Y.A.S. Method for Enhanced Heparin Quality. Patent No.
WO2016/137471 A1, 26 February 2015.
39. Sache, E.; Maman, M.; Bertrand, H. New Heparin fractions Having Increased Anticoagulant Activities.
Patent No. GB 2,051,103 A, 10 April 1980.
40. Toccaceli, N. Chromatographic Purification of Heparin. U.S. Patent 3,099,600, 30 July 1963.
41. Green, J.P. Fractionation of heparin on an anion exchanger. Nature 1960, 186, 472. [CrossRef] [PubMed]
42. Höök, M.; Björk, I.; Hopwood, J.; Lindahl, U. anticoagulant activity of heparin: Separation of high-activity
and low-activity heparin species by affinity chromatography on immobilized antithrombin. FEBS Lett. 1976,
66, 90–93. [CrossRef]
43. Volpi, N. Fractionation of heparin, dermatan sulfate, and chondroitin sulfate by sequential precipitation:
A method to purify a single glycosaminoglycan species from a mixture. Anal. Biochem. 1994, 218, 382–391.
[CrossRef] [PubMed]
44. Arthur, B.E.; Ernest, H.J.; William, R.L. Process of Removing Colored Impurities from Heparin. U.S. Patent
2,830,931, 28 June 1954.
45. Bush, J.A.; Freeman, L.D.; Hagerty, E.B. Method for Purifying Sulfated Carbohydrates with Oxidizing Agents.
U.S. Patent 31,356,660 A, 9 November 1956.
46. Ewards, F.R.; Horner, A.A. Purification of Heparin. U.S. Patent 3,179,566 A, 16 May 1963.
47. Celsus Website. Available online: https://www.heparin.com/manufacture_controls.php (accessed on
14 April 2017).
48. Coyne, E. Heparin—Past, present and future. In Chemistry and Biology of Heparin; Lundblad, R.L.,
Virgil Brow, W., Mann, K.G., Roberts, H.R., Eds.; Elsevier/North-Holland: Amsterdam, The Netherlands,
1981; pp. 9–17.
49. Yongjun, G. Refining Optimization Technology for Crude Heparin Sodium. Patent No. CN105,001,353 A,
17 August 2015.
50. Mourier, P.; Viskov, C. Process for Oxidizing Unfractionated Heparins and Detecting Presence or Absence of
Glycoserine in Heparin and Heparin Products. Patent No. WO2005090411 A1, 24 March 2004.
51. Guerrini, M.; Beccati, D.; Shriver, Z.; Naggi, A.; Viswanathan, K.; Bisio, A.; Capila, I.; Lansing, J.C.; Guglieri, S.;
Fraser, B.; et al. Oversulfated chondroitin sulfate is a contaminant in heparin associated with adverse clinical
events. Nat. Biotechnol. 2008, 26, 669–675. [CrossRef] [PubMed]
52. Szajek, A.Y.; Chess, E.; Johansen, K.; Gratzl, G.; Gray, E.; Keire, D.; Linhardt, R.J.; Liu, J.; Morris, T.;
Mulloy, B.; et al. The US regulatory and pharmacopeia response to the global heparin contamination crisis.
Nat. Biotechnol. 2016, 34, 625–630. [CrossRef] [PubMed]
53. Szajek, A.Y.; Morris, T.S.; Koch, W.F.; Abernethy, D.R.; Williams, R.L. Inside USP: Heparin Monographs
Further Revised. Pharm. Technol. 2009, 33, 136–137.
54. Beccati, D.; Roy, S.; Yu, F.; Gunay, N.S.; Capila, I.; Lech, M.; Linhardt, R.J.; Venkataraman, G. Identification of
a novel structure in heparin generated by potassium permanganate oxidation. Carbohydr. Polym. 2010, 82,
699–705. [CrossRef] [PubMed]
55. Kellenbach, E.; Sanders, K.; Michiels, P.J.A.; Girard, F.C. 1H NMR signal at 2.10 ppm in the spectrum of
KMnO4-bleached heparin sodium: Identification of the chemical origin using an NMR-only approach.
Anal. Bioanal. Chem. 2011, 399, 621–628. [CrossRef] [PubMed]
56. Mourier, P.A.; Guichard, O.Y.; Herman, F.; Viskov, C. Heparin sodium compliance to the new proposed USP
monograph: Elucidation of a minor structural modification responsible for a process dependent 2.10 ppm
NMR signal. J. Pharm. Biomed. Anal. 2011, 54, 337–344. [CrossRef] [PubMed]
57. Casu, B.; Gennaro, U. A conductimetric method for the determination of sulphate and carboxyl groups in
heparin and other mucopolysaccharides. Carbohydr. Res. 1975, 39, 168–176. [CrossRef]
Molecules 2017, 22, 1025 13 of 13

58. Mourier, P.A.; Guichard, O.Y.; Herman, F.; Viskov, C. Heparin sodium compliance to USP monograph:
Structural elucidation of an atypical 2.18 ppm NMR signal. J. Pharm. Biomed. Anal. 2012, 67, 169–174.
[CrossRef] [PubMed]
59. Lee, S.E.; Chess, E.K.; Rabinow, B.; Ray, G.J.; Szabo, C.M.; Melnick, B.; Miller, R.L.; Nair, L.M.; Moore, E.G.
NMR of heparin API: Investigation of unidentified signals in the USP-specified range of 2.12–3.00 ppm.
Anal. Bioanal. Chem. 2011, 399, 651–662. [CrossRef] [PubMed]
60. Jandik, K.A; Kruep, D.; Cartier, M.; Linhardt, R.J. Accelerated stability studies of heparin. J. Pharm. Sci. 1996,
85, 45–51. [CrossRef] [PubMed]
61. Liu, L.; Linhardt, R.J.; Zhang, Z. Quantitative analysis of anions in glycosaminoglycans and application in
heparin stability studies. Carbohydr. Polym. 2014, 106, 343–350. [CrossRef] [PubMed]
62. Huang, L.; Tang, J.; Wang, Z.; Zhou, J.; Yan, H. Method for Drying Heparin by Atomization. Patent No.
CN1,218,058, 26 November 1997.
63. Raghavan, R.; Jett, J.L. Method to Produce a Solid Form of Heparin, U.S. Patent 2004/0176581, 27 February 2004.
64. WHO Model Lists of Essential Medicines. Available online: http://www.who.int/medicines/publications/
essentialmedicines/EML_2015_FINAL_amended_NOV2015.pdf?ua=1 (accessed on 8 May 2017).

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