Crosslinking Studies in Gelatin Capsules Treated with

Formaldehyde and in Capsules Exposed to Elevated

Temperature and Humidity

CLYDE M. OFNER III, YU-E ZHANG, VALERIE C. JOBECK, BILL J. BOWMAN

Department of Pharmaceutical Sciences, Philadelphia College of Pharmacy, University of the Sciences in Philadelphia,
600 South 43rd Street, Philadelphia, Pennsylvania 19104

Received 11 August 1999; revised 30 June 2000; accepted 11 July 2000

ABSTRACT: Incomplete in vitro capsule shell dissolution and subsequent drug release
problems have recently received attention. A modified USP dissolution method was
used to follow capsule shell dissolution, and a 2,4,6-trinitrobenzenesulfonic acid (TNBS)
assay was used to follow loss of ␧-amino groups to study this shell dissolution problem
postulated to be due to gelatin crosslinking. The dissolution problems were simulated
using hard gelatin capsule (HGC) shells previously treated with formaldehyde to
crosslink the gelatin. These methods were also used to study the effect of uncrosslinked
HGC stored under stressed conditions (37 °C and 81% RH) with or without the presence
of soft gelatin capsule shells (SGC). A 120 ppm formaldehyde treatment reduced gelatin
shell dissolution to 8% within 45 min in water at 37 °C. A 200 ppm treatment reduced
gelatin ␧-amino groups to 83% of the original uncrosslinked value. The results also
support earlier reports of non-amino group crosslinking by formaldehyde in gelatin.
Under stressed conditions, HGC stored alone showed little change over 21 weeks.
However, by 12 to 14 weeks, the HGC exposed to SGC showed a 23% decrease in shell
dissolution and an 8% decrease in the number of ␧-amino groups. These effects on the
stressed HGC are ascribed to a volatile agent from SGC shells, most likely formalde-
hyde, that crosslinked nearby HGC shells. This report also includes a summary of the
literature on agents that reduce gelatin and capsule shell dissolution and the possible
mechanisms of this not-so-simple problem. © 2001 Wiley-Liss, Inc. and the American Phar-
maceutical Association J Pharm Sci 90: 79–88, 2001
Keywords: gelatin crosslinking, gelatin dissolution, amino group assay, formalde-
hyde crosslinking in proteins

INTRODUCTION tention. This problem was first reported in 1974

After ingestion, gelatin capsule shells must dis-
solve sufficiently and release their drug contents
to produce a therapeutic effect. Incomplete in
vitro capsule shell dissolution and subsequent
drug release problems have recently received at-
Current address: Sanofi Research, 9 Great Valley Park-
way, P.O. Box 3026 Malvern, Pennsylvania 19355
Correspondence to: C.M. Ofner III (Telephone: 215-596-
8881; Fax: 215-895-1100; E-mail: cofner@usip.edu)
Journal of Pharmaceutical Sciences, Vol. 90, 79–88 (2001)
© 2001 Wiley-Liss, Inc. and the American Pharmaceutical Association
for a hard gelatin capsule (HGC) product contain-
ing chloramphenicol1 and in 1977 for a soft gela-
tin capsule (SGC) product containing digoxin.2
These and later cases were associated with ad-
verse storage conditions of elevated temperature,
humidity, or prolonged storage. But in these two
cases, as in almost all others, there was no re-
duced bioavailability of the drug compared with
the readily dissolving product.3–5
Two cases of reduced bioavailability are of his-
torical interest. The first case occurred in 1985,
with an HGC product containing phenytoin that

JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 90, NO. 1, JANUARY 2001 79

80 OFNER ET AL.
was stored at elevated temperature and humidity
and was identified in a patient hospitalized for
loss of seizure control.6 An FDA drug recall of a
SGC product in 1993, because of dissolution prob-
lems and reduced bioavailability,7 is the second
case. The latter led to the formation of an FDA/
Industry Gelatin Capsule Working Group to ex-
amine the effects of the in vitro dissolution prob-
lem on drug bioavailability. The Working Group
simulated this dissolution problem with formal-
dehyde crosslinked HGC and SGC containing
acetaminophen and evaluated their bioequiva-
lence in humans.7,8 It is now generally believed
that this in vitro dissolution problem has little
risk of reduced drug bioavailability because gas-
tric and intestinal enzymes that contribute to
the physiological dissolution process have not
been routinely used in dissolution tests. These
enzymes break down the swollen and poorly
soluble film of the capsule shell, sometimes called
a pellicle, that usually retards or prevents drug
release under in vitro dissolution conditions.
Carstensen and Rhodes,9 as well as others, have
suggested introducing such enzymes into in vitro
dissolution testing. One result of these sugges-
tions and of the Working Group investigation was
the initiation of the regulatory process to allow
so-called two-tier testing; that is, the use of a sec-
ond USP dissolution test, which contains an en-
zyme if the product failed the original monograph
dissolution test because of this problem.7,10
Although this in vitro dissolution problem is
unlikely to produce biological consequences, it re-
mains a theoretical safety issue and its causes are
a concern. There is a growing body of evidence
that this problem is primarily caused by gelatin
crosslinking in the capsule from aldehyde impu-
rities that form the pellicle barrier to drug re-
lease.9,11 Crosslinking gelatin leads to an intri-
cate network of high molecular weight that pro-
duces a swellable hydrogel but substantially
reduces, or even prevents, dissolution of the gela-
tin.12–14 An earlier review of gelatin crosslinking
and capsule dissolution,11 as well as related stud-
ies with a different focus have been reported.15–17
Recently, approaches to minimize this crosslink-
ing and dissolution problem using additives18 and
by accelerated detection testing19 have also been
reported.
This report has three goals. The first is to de-
scribe and disseminate an in vitro evaluation of
gelatin crosslinking in the formaldehyde-treated
capsules used in the Working Group acetamino-
phen biostudy. The second goal is to report the
JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 90, NO. 1, JANUARY 2001
application of the methods just described to
evaluate crosslinking and shell dissolution in
plain, uncrosslinked capsules that have been
“stressed” by exposure to elevated temperature
and humidity. The last goal is to collect the dis-
parate literature on this issue for a discussion
and comparison of these results for this not-so-
simple dissolution problem. The ideal evaluation
of crosslinking in gelatin would include determi-
nation of the crosslinking extent, identification of
the agent or cause and the specific reaction
mechanism, as well as identification of the site,
the molecular weight, and the number of such
crosslinks. A macroscopic and molecular ap-
proach was used in the current investigation to
elucidate some of these aspects. The macroscopic
approach was to evaluate dissolution of the cap-
sule shell alone, without the presence of drug or
excipients, using an assay for the dissolved gela-
tin. This gelatin dissolution is used as an indica-
tion of crosslinking13 without regard to specifics
such as mechanism or site. The molecular ap-
proach was to assay for the potential loss of amino
groups due to crosslinking because formaldehyde
(and other aldehydes) reacts with amino groups
on lysine and hydroxylysine amino acid residues
of gelatin during crosslinking.20–22 Such specific-
ity suggests that this formaldehyde crosslinking
can be quantitatively determined. The formalde-
hyde–gelatin crosslinking mechanism, however,
is not entirely clear; non-amino group crosslink-
ing also has been reported.21 A comparison be-
tween results of the two methods in this investi-
gation could corroborate such non-amino group
crosslinking. Another approach to enhance our
understanding of this dissolution problem is to
apply these methods in an exploratory manner to
stressed, plain, uncrosslinked capsules. Specifi-
cally, the theoretical possibility of self-cross-
linking occurring without an agent11,12 can be ex-
amined. Under the stressed conditions of this
study, a crosslinking agent was generated from a
SGC shell that induced crosslinking in HGC
shells. This stressed-induced crosslinking can be
directly measured, which to the authors’ knowl-
edge has not yet been reported.
EXPERIMENTAL SECTION
Materials
Type B gelatin granules, with a bloom strength of
254 and an approximate moisture content of 11%
(w/w) determined by loss on drying at 105 °C for

72 h, were supplied by Kind and Knox (Sioux
City, IW, sample #T7468, lot #1). Capsugel AG
(Basel, Switzerland) supplied formaldehyde-
treated HGC and controls that were prepared as
described later. Plain uncrosslinked HGC used in
the stressed conditions study were purchased
from Lilly. Untreated SGC were supplied by Ban-
ner Pharmacaps Inc. (High Point, NC) and con-
tained a poly(ethylene glycol) (PEG) fill liquid and
a shell composed of gelatin, glycerin, sorbitol, and
water. The BCA (bicinchoninic acid) protein assay
kit and 2,4,6-trinitrobenzenesulfonic acid (TNBS)
was purchased from Pierce Chemical Company
(Rockford, IL). Water was purified by reverse os-
mosis. All other chemicals were at least ACS re-
agent grade. Formaldehyde-treated HGC were
prepared by Capsugel,23 by mixing formaldehyde
(HCHO) with lactose at different levels. The
HCHO/lactose mixture was filled into empty HGC
capsules that were then exposed briefly to 40 °C
and 75% RH, then to 25 °C and 50% RH for 6
weeks, at which time the HCHO/lactose mixture
was removed from the capsules. The treatment
levels of HCHO in the mixtures were 0, 20, 30,
120, 200, 400, and 1000 ppm. The 0, 20, and 120
ppm levels were evaluated in the Working Group
Biostudy. The control passed the in vitro dissolu-
tion tests in the absence and presence of enzyme
(P/P). The 20 ppm capsule failed the conventional
dissolution tests but passed with the addition of
enzyme (F/P). The 120 ppm capsule failed disso-
lution tests in the absence and presence of en-
zyme (F/F). The 20 ppm capsules were found bio-
equivalent to the uncrosslinked controls, whereas
the 120 ppm capsules were not bioequivalent.
Stressed Hard Gelatin Capsules
A saturated ammonium sulfate solution was used
in glass desiccates to produce 81%RH at 37 °C.
The desiccates were stored in an incubator at 37
°C. Gelatin granules and plain HGC, in separate
petri dishes, were placed in a desiccator desig-
nated as “HGC stored alone”. Gelatin granules,
plain HGC, and untreated SGC, in petri dishes,
were placed in the desiccator designated as “HGC
stored with SGC”. Prior to this exposure, the SGC
were cut in half and rinsed with small amounts of
methanol to remove residue of the PEG fill liquid.
HGC and granule samples were removed at de-
sired times and evaluated by shell dissolution and
amino group assay.
JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 90, NO. 1, JANUARY 2001
CROSSLINKING STUDIES IN GELATIN CAPSULES 81
Capsule Shell Dissolution
A modified USP paddle method was used to
evaluate dissolution of the gelatin from the cap-
sule shell. The Van-Kel apparatus (Chatham, NJ)
used consists of a 200-mL kettle (20-cm height,
4-cm inner diameter), an adapter ring to allow the
“mini” kettle to fit into the water bath system for
conventional kettles, 100 mL of purified water at
37 °C, a 100 rpm speed for the minipaddle, and
Teflon-coated coilers to weight the empty cap-
sules to the bottom of the kettle. After removal of
1-mL samples, this volume was replaced with
warmed purified water. Dissolved gelatin was de-
termined from predetermined calibration plots by
BCA protein assay or ultraviolet (UV) absorbance
measurements at 214 nm. In the resulting cap-
sule dissolution profiles, each point is the mean of
six samples and error bars represent standard de-
viation (SD).
Gelatin Amino Group Assay
The uncrosslinked, or free, ␧-amino groups of gela-
tin were assayed by a method reported previ-
ously24 This method uses TNBS as a UV chromo-
phore that reacts with primary amino groups and,
after acid hydrolysis and organic extraction, re-
mains in the aqueous phase reacted with ␧-amino
groups on lysine residues in peptide fragments.
Statistics
The results were analyzed by a one-way analysis
of variance (ANOVA). Multiple comparisons be-
tween means were evaluated by a Neuman–Keuls
test.25 For the few cases of non-normal variance,
the multiple comparisons between means were
evaluated using a Kruskal–Wallis nonparametric
ANOVA test with log-transformed data.25
RESULTS
Evaluation of Formaldehyde-Treated Capsules
Capsules crosslinked with various amounts of
HCHO were evaluated using gelatin shell disso-
lution as a general indicator of crosslinking and
the ␧-amino group assay as an indicator of specific
functional group participation. The dissolved cap-
sule profiles in Figure 1 show no difference be-
tween the control, untreated capsules and those
treated with the two lower amounts of 20 and 30
ppm HCHO. Additional, but unsuccessful, experi-
82 OFNER ET AL.

Figure 2. Loss of ␧-amino groups in formaldehyde

treated hard gelatin capsules (bars are SD; n ⳱ 5; *, p
Figure 1. Gelatin shell dissolution profiles of formal-
< 0.05).
dehyde treated hard gelatin capsules in 37 °C water
(bars are SD, n ⳱ 6).

with 1000 ppm. This is a 17% loss of ␧-amino

ments were conducted to enhance differences be- groups compared with untreated capsules. The
tween these profiles using different paddle SD for these maximally treated capsules is 0.29.
speeds, an acid medium, capsule strips, fillers,
and different weights of fillers (data not shown).
The next levels of 120 and 200 ppm, however, Evaluation of Stressed Hard Gelatin Capsules
produced substantial reductions leading to 8 and Figure 3 illustrates the conditions of HGC expo-
2%, respectively, of dissolved gelatin by 45 min, sure to 37 °C and 81% RH in the presence of SGC.
and only 15 and 4%, respectively, of dissolved A separate desiccator experiment was conducted
gelatin by 2 h. In addition, the variation among without SGC to evaluate changes induced in gela-
replicates in the higher two levels of crosslinking tin by only the elevated temperature and humid-
was less than that of the controls and two lower ity. Figure 4 shows the percent dissolved of cap-
crosslinking levels. The SD for the controls sule gelatin at 45 min as a function of weeks of
ranged from 4 to 11, that of the 20 ppm capsules exposure to the stressed conditions. The HGCs
ranged from 1 to 7, and that of the 120 and 200 stored alone showed little change in their com-
ppm capsules ranged from 0 to 2. plete dissolution up to 21 weeks of exposure. The
The loss of ␧-amino groups in the HCHO- HGC stored with SGC showed dissolution re-
treated capsules is shown in Figure 2. The un-
treated control capsules have 32.9 × 10−5 mol/g of
free, uncrosslinked ␧-amino groups. This value
corresponds to 32.9 ␧-amino groups on one gelatin
molecule of 1000 amino acid residues with an
ideal molecular weight of 100,000. The value is
virtually identical to the 33.0 × 10−5 mol/g previ-
ously reported for Type B gelatin granules.24 The
variation is also the same with a relative SD
(RSD) of 2.6%.
There is no measurable difference between the
control capsules and those treated with 120 ppm
HCOH, and by inference, the capsules treated
with lower amounts. There is, however, a small
but measurable loss of ␧-amino groups for the cap-
sules treated with 200 ppm. The loss continues Figure 3. Stressing of hard gelatin capsules and
with increasing HCHO treatments to a final gelatin granules at 37 °C and 81% RH in the presence
value of 27.4 × 10−5 mol/g for capsules treated of soft gelatin capsule shells.

JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 90, NO. 1, JANUARY 2001


Figure 4. Dissolution of stressed hard gelatin cap-
sule shells during storage at 37 °C and 81% RH up to 21
weeks in the presence and absence of soft gelatin cap-
sule shells.
duced to 77% at 12 weeks followed by a return to
complete dissolution by 19 weeks.
A very similar trend is found for the loss of
␧-amino groups in the gelatin capsule shells ex-
posed to these conditions. Figure 5 shows little if
any amino group loss from exposure to the el-
evated temperature and humidity in the absence
of SGC. For exposure with SGC at these condi-
tions, there is a small but real (8%) loss of ␧-amino
groups by 14 weeks. As was observed in the dis-
solution experiments, these measurements re-
turn to their normal values at ∼19 weeks. This
return to normal values for both measures is at-
tributed to hydrolytic degradation of the cross-
Figure 5. Loss of ␧-amino groups in stressed hard
gelatin capsule shells during storage at 37 °C and 81%
RH up to 21 weeks in the presence of soft gelatin cap-
sule shells.
JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 90, NO. 1, JANUARY 2001
CROSSLINKING STUDIES IN GELATIN CAPSULES 83
links and regeneration of the amino groups. Dis-
solution and amino assay results for gelatin gran-
ules were very similar to that of the capsules and
are not shown.
DISCUSSION
Evaluation of Formaldehyde-Treated Capsules
Formaldehyde crosslinking in the gelatin shell
forms a three-dimensional molecular network of a
higher average molecular weight than the origi-
nal molecules that dissolve at this temperature.
In addition, this crosslinking reduces hydrophilic-
ity of the molecules by loss of ionizable amino
groups. Both these effects lead to formation of the
nondissolving layer, or pellicle, that hinders or
prevents drug release. The HCHO from the
treated lactose induces crosslinking on the inner
capsule shell surface to form the nondissolving
layer that extends further into the shell as the
HCHO treatment levels are increased. Treatment
of 120 ppm extended the crosslinking throughout
the gelatin shell, which substantially reduced
gelatin shell dissolution to only 15%. The thin
layer from 20 and 30 ppm HCHO exposure prob-
ably represents only a small fraction of the gela-
tin that is not distinguishable from 100% dissolu-
tion of untreated shells using the shell dissolution
method.
The higher levels of crosslinking from 200 to
1000 ppm decrease free ␧-amino groups, which
substantiates the participation of these groups in
a covalent crosslinking reaction. The downward
trend also suggests that not all potential
crosslinking sites reacted with HCHO, even at
the 1000 ppm level. It is interesting to estimate
the HCHO content of these treated capsules and
compare these amounts to the loss of amino
groups. Assuming that all HCHO in the lactose
reacts with the gelatin shell, a lactose fill weight
of 400 mg, a capsule weight of 75 mg, and a mois-
ture content of 10%, the calculated HCHO in the
shells is 0.4, 2.4, and 20 × 10−5 mol/g gelatin for
20-, 120-, and 1000-ppm treatments, respectively.
These values are not far from the amino group
losses of 0.3, 0.9, and 5.5 × 10−5 mol/g gelatin for
the 120-, 200-, and 1000-ppm treatments, respec-
tively. Gold et al.15 measured HCHO crosslinking
in HGC using near-infrared spectrophotometry
after exposing the capsules to 150 ppb HCHO at-
mosphere. These exposures substantially reduced
in vitro release of incorporated amoxicillin. Al-
though this method is more sensitive for detecting
84 OFNER ET AL.
HCHO crosslinking, it does not identify and
quantitate participation of a specific functional
group.
Crosslinking clearly occurs from 20- and 120-
ppm HCHO treatments as shown by results from
the Working Group Biostudy, and in Figure 1 for
the 120 ppm treatment. However, the ␧-amino
group assay only measures a loss of these groups
for treatments >200 ppm. The assay can measure
a loss of one ␧-amino group from the initial 33
groups on lysine and hydroxyl lysine residues per
average gelatin molecule of 1000 residues (see
Figure 2). This number of gelatin residues per
molecule is based on the ideal gelatin molecule of
100,000 g/mol (three molecules from a parent
triple helical collagen molecule).26 Two average
gelatin molecules crosslinked to each other by as
little as one ␧-amino group on each gelatin mol-
ecule should then be detectable. This would be
analogous to the ␧-amino group loss at 200 ppm
shown in Figure 2. These two crosslinked gelatin
molecules would represent a small crosslinked
unit that should be detectable in this situation.
Consequently, and in contrast to these results,
the extensive crosslinked network producing the
substantial dissolution reduction observed from
the 120-ppm treatment would be expected to have
a measurable loss of amino groups involved in
such crosslinking.
Formaldehyde was chosen as the crosslinking
agent in these capsules because it has been iden-
tified as the causative agent of this dissolution
problem in two cases,3,16 and to model the more
complex problem of crosslinking under stressed
conditions. The mechanistic details of HCHO-to-
gelatin crosslinking, however, are not entirely
clear. A likely explanation for the apparent insen-
sitivity of the amino group assay is that HCHO
crosslinks with other functional groups in combi-
nation with, or independent of, ␧-amino groups.
In a 13C nuclear magnetic resonance (NMR) study
of 13C-labeled HCHO reacting with gelatin in so-
lution and the solid state, no evidence was found
of HCHO crosslinking between amino groups on
lysine residues;21 however, strong evidence of ar-
ginine (guanidino functional groups)-to-lysine
crosslinks and arginine-to-arginine crosslinks
was reported. In fact, these investigators reported
that optimum crosslinking of both reactions was
achieved at 30 °C and 60–70% RH. In addition,
only arginine-to-arginine crosslinks were mea-
sured after 24 h at 30 °C and 100% RH. These
conditions are important to the current investiga-
tion because the HCHO-treated capsules were
JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 90, NO. 1, JANUARY 2001
also exposed to elevated humidity as part of their
treatment. Whereas the HCHO concentrations in
the cited study are much higher than those used
in the current study, the earlier study demon-
strates the possibility of the arginine-to-arginine
reaction in the solid state. Gold et al.22 recently
obtained corroborating evidence in solution ex-
periments for the lysine-to-arginine crosslinks for
HCHO concentrations as low as 100ppm, but did
not report any evidence for arginine-to-arginine
crosslinks. The indication of crosslinking in the
current study at the 120-ppm treatment from the
capsule dissolution, but the absence of ␧-amino
groups participating at this HCHO level supports
the presence of arginine-to-arginine crosslinks.
The two methods used in the current investi-
gation are probably less sensitive than the USP
drug dissolution test as an indicator of gelatin
shell HCHO crosslinking. In a report of acetamin-
ophen dissolution using these HCHO treated cap-
sules, the 20-ppm treated capsules demonstrated
impaired dissolution in water and only 50% re-
lease of drug by 45 min.22 Acetaminophen release
from the capsules treated at the higher level of
120 ppm was <1% under these conditions. Such
reductions in the rate and extent of drug release
reflect effects of the crosslinked gelatin layer im-
peding drug diffusion already described. These
cited acetaminophen dissolution tests were con-
ducted in water, as were the current capsule shell
dissolutions. The amoxicillin release reported by
Gold et al.15 also illustrates the sensitivity of the
USP dissolution test to HCHO crosslinking in
gelatin shells.
Evaluation of Stressed Hard Gelatin Capsules
Evaluation of the HCHO-treated capsules illus-
trates the uses and limitations of the current
study methods to evaluate gelatin shell crosslink-
ing. The amino, carboxylic acid, and perhaps
other groups on the gelatin molecule raise the
possibility that gelatin may crosslink with itself
under the so-called stressed conditions of elevated
temperature and humidity. It has been suggested
that amino groups might undergo oxidative
deamination to an aldehyde, which could then in-
duce a crosslink.11 High-temperature exposure of
gelatin (105 °C and reduced pressure) has pro-
duced insoluble, but swellable gelatin that is as-
cribed to a self-crosslinking mechanism.12,13 The
HGC stored alone at 37 °C and 81% RH show no
evidence of self-crosslinking.
Crosslinking is clearly present in the HGC
shells exposed to SGC under these conditions.

The reductions in dissolved gelatin shells and in
␧-amino groups are striking because they occur at
virtually identical times. Because such reductions
are absent in HGC stored alone, it is concluded
that the SGC exposure under these conditions in-
duced the crosslinking. In addition, the crosslink-
ing substance must be volatile because the HGC
and SGC are physically separated but share the
same atmosphere. The magnitude of the ␧-amino
group reduction indicates extensive crosslinking
in the HGC shell. This result is somewhat sur-
prising because a 400 ppm HCHO treatment was
required to produce a similar reduction. Yet the
reduced shell dissolution is not as extensive as
would be expected by this level of HCOH treat-
ment. It is conceivable that the HGC surface in
contact with the petri dish was protected from the
volatile crosslinking agent to allow dissolution of
this portion of the shell, and perhaps dissolution
of the inner surface of the shell. Such an atmo-
spheric transfer of the crosslinking agent is un-
likely to be influenced by the powder fill. In addi-
tion, it should be noted that generation of this
crosslinking agent from the SGC also appears to
crosslink the SGC. These SGC were not analyzed
for crosslinking due to interferences from non-
gelatin components of the shell.
A report of formaldehyde generated from a sur-
factant in a HGC capsule suggests a plausible ex-
planation for the volatile crosslinking agent origi-
3
nating in the SGC. Polysorbate 80 in a gemfibri-
zole HGC formulation was shown to produce
HCHO, insoluble gelatin films, and impaired cap-
sule dissolution after 1 month of storage at 37 °C
and 80% RH. The authors attributed the HCHO
production to auto-oxidation of the end hydroxyl
groups in the polyoxyethylene (POE) chains of
polysorbate 80. The SGC capsules in the current
study initially contained PEG as the fill liquid.
The PEG molecule is essentially identical to the
POE chains of polysorbates. Although the SGC
were opened and washed at the start of the ex-
periments, PEG could have previously migrated
into the SGC shell because of the 6–12% water in
typical SGC shells. In this case, the end groups of
PEG could auto-oxidize under these conditions to
produce HCHO. An earlier report also implicates
HCHO as the causative agent because storage of
the SGC at 40 and 60 °C produced reductions in
SGC shell disintegration, equilibrium swelling,
and shell dissolution to the same extent as that
produced from exposure to HCHO.28 In addition,
it has been suggested that the PEG fill in SGC may
contain formaldehyde and induce crosslinking.16
JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 90, NO. 1, JANUARY 2001
CROSSLINKING STUDIES IN GELATIN CAPSULES 85
Agents That Reduce Gelatin Dissolution and
Possible Mechanisms
Aldehydes and Covalent Crosslinking
The unintended influence of aldehyde impurities
leading to dissolution problems has occurred from
a surprising range of sources. One of the earliest
reports already noted was the generation of
HCHO from polysorbate 80. In addition, a poly-
hydroxyl pentose, as an intermediate from cellu-
lose in rayon coilers, has also been shown to form
the aldehyde furfural and a crosslinked, poorly
soluble gelatin product29after storage at 40 °C and
75% RH for 2 months. Another potential source
of formaldehyde is hexamethylenetetramine,
which may be added to cornstarch as a stabilizer
and which may decompose to formaldehyde under
humid conditions.11 Additional compounds that
have been reported to promote crosslinking in
gelatin include glucose, hydrogen peroxide, ben-
zene, sulfonic acid, p-toluene sulfonic acid, and
guanidine HCl.30
Aldehydes have a long history of crosslinking,
but the exact crosslinking mechanisms are not
entirely clear. For example, the dialdehyde glu-
taraldehyde is recognized to react with primary
amino groups, but its mechanism, first suggested
in 1968,31 was more recently suggested to involve
four different crosslinks from 12 different reac-
tions.32 Gelatin crosslinking studies with formal-
dehyde and the dialdehyde glyoxal have been re-
ported as early as 1963.33 The lysine–arginine
gelatin crosslinks from HCHO already discussed
were first suggested in 1978,34 and studies have
continued to elucidate the process.20–22 The cur-
rent investigation lends support to the lysine–
arginine and arginine–arginine crosslinks sug-
gested earlier.
High Temperatures and Covalent Crosslinking
Exposure to 105 °C and reduced pressure of 10
␮m Hg has produced insoluble gelatin that was
ascribed to an amino group-to-carboxylic acid con-
densation crosslinking mechanism.12 In addition,
it has been suggested that oxidative deamination
between two adjacent amino groups on lysine
residues would form an aldehyde and then a
crosslink during exposure to elevated tempera-
tures.11 However, no measurable loss of gelatin
amino groups was reported for gelatin exposed to
these conditions for up to 5 days,35 which sug-
gests a crosslinking mechanism without an amino
86 OFNER ET AL.
group occurs to produce insolubility at high tem-
peratures.
Dyes, Noncovalent Binding, and Light
Gelatin carboxyl and amino groups have been
shown to interact with functional groups on dyes
via ionic, hydrogen, and van der Waals bonds to
reduce gelatin solubility. As early as 1973, the
dyes FD&C Red No.3 and FD&C Red No.40 were
shown to interact with gelatin and reduce its solu-
bility.36 Gautam and Schott37 reported that dye
binding by ion pairing, hydrogen bonds, and other
secondary valence forces rendered the gelatin less
ionic and less hydrophilic. These interactions cor-
roborate a report of the cationic compounds of ce-
tylpyridinium Cl and dodecylammonium Cl re-
ducing both the initial swelling rate and equilib-
rium swelling of uncrosslinked anionic gelatin.38
A high-dose, water-insoluble drug filled into cap-
sules containing FD&C Red No.3 stored at 80%
RH and 30 °C under fluorescent light produced
only 20% drug dissolution by 60 min.39 Substan-
tial dissolution reductions were also reported for
these capsules exposed to high humidity at ambi-
ent, as well as UV light. Less severe dissolution
reductions were noted for these capsules stored at
35% RH and when protected from light, indicat-
ing the accelerating effect of moisture and light on
this dye interaction. Capsules containing FD&C
Blue No.1 also demonstrated substantial dissolu-
tion reductions but only from exposure to fluores-
cent light at high humidity.40 Storage for 8 days
at 40 °C and 75% RH in the presence of UV and
visible illumination reduced drug dissolution to
<5% for a SGC and <60% for a HGC product by 60
min.19. Also, proteins are frequently crosslinked
by exposure to UV light. This mechanism is as-
cribed to free radical formation and subsequent
crosslinking. However, the participating residues
and the composition, number, and size of
crosslinks are not fully understood.
CONCLUSIONS
The capsule dissolution test measured reduced
dissolution from 120-ppm HCHO-treated cap-
sules. This test is more sensitive than the ␧-amino
group assay that required a 200-ppm HCHO
treatment to measure an effect. However, com-
paring these results with a report of a USP dis-
solution test of such HCHO-treated capsules con-
taining acetaminophen indicated that the USP
JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 90, NO. 1, JANUARY 2001
test was more sensitive to this crosslinking than
the methods used in this study. The inability of
the shell dissolution test to detect crosslinking in
20- and 30-ppm HCHO-treated capsules is prob-
ably due to a thin crosslinked and insoluble film
on the inner shell wall that prevented drug re-
lease. This film may represent only a small frac-
tion of the total soluble gelatin in the shell. The
␧-amino group assay results verify participation
of these groups in HCHO crosslinking and sup-
port earlier reports of HCHO crosslinking in gela-
tin between amino groups of lysine and guanidino
groups of arginine, and between the guanidino
groups in arginine-to-arginine crosslinks. Un-
treated, plain gelatin capsules stored at 37 °C and
81% RH up to 21 weeks show no evidence of self-
crosslinking. This crosslinking mechanism, how-
ever, should not be completely ruled out because
of theoretical possibilities, evidence of high tem-
perature self-crosslinking, and the sensitivity and
mechanism specific limitations of the methods
used in this study. Volatile agent(s) generated
from SGC at 37 °C and 81% RH can cause sub-
stantial and reproducible ␧-amino group cross-
linking and dissolution reductions in nearby
HGC. Auto-oxidation of residual PEG to form
HCHO and induce the crosslinking is a likely ex-
planation. Capsule shell dissolution might be
used to distinguish shell dissolution problems
from other dissolution problems, such as formu-
lation or degradation. Proper containers and stor-
age are important issues to minimize this disso-
lution problem in gelatin capsule products.
ACKNOWLEDGMENTS
Presented in part at the 1997 annual meeting of
the American Association of Pharmaceutical Sci-
entists (AAPS) in Boston, MA. Supported in part
by USPHS Contract FDA-223-95-3006 (to CMO).
We gratefully acknowledge Dr. Ewart T. Cole
(Capsugel AG, Basel, Switzerland) for donation of
the formaldehyde-treated hard gelatin capsules,
Banner Pharmacaps (Highpoint, NC) for donation
of soft gelatin capsules, and Dr. J. Michael Dunn
(Kind & Knox, Sioux City, IO) for donation of the
gelatin granules.
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