See discussions, stats, and author profiles for this publication at: https://www.researchgate.

net/publication/6836928

GMP Production of Liposomes—A New Industrial Approach

Article  in  Journal of Liposome Research · February 2006

DOI: 10.1080/08982100600851086 · Source: PubMed

CITATIONS READS
83 2,444

9 authors, including:

Andreas Wagner Gabriela Stiegler

Polymun Scientific Immunbiologische Forschung GmbH University of Natural Resources and Life Sciences Vienna
32 PUBLICATIONS   1,000 CITATIONS    51 PUBLICATIONS   9,669 CITATIONS   

SEE PROFILE SEE PROFILE

Karola Vorauer-Uhl
University of Natural Resources and Life Sciences Vienna
91 PUBLICATIONS   1,957 CITATIONS   

SEE PROFILE

Some of the authors of this publication are also working on these related projects:

Development of efficient drug carrier systems and functional characterization View project

PAT Plant - PAT & QbD in mammalian cell culture cultivations View project

All content following this page was uploaded by Andreas Wagner on 01 December 2014.

The user has requested enhancement of the downloaded file.

 This article was downloaded by:[St George's Medical School Library]
On: 3 October 2007
Access Details: [subscription number 773566046]
Publisher: Informa Healthcare
Informa Ltd Registered in England and Wales Registered Number: 1072954
Registered office: Mortimer House, 37-41 Mortimer Street, London W1T 3JH, UK

Journal of Liposome Research

Publication details, including instructions for authors and subscription information:
http://www.informaworld.com/smpp/title~content=t713597272
GMP Production of Liposomes - A New Industrial
Approach
Andreas Wagner a; Mirko Platzgummer a; Günther Kreismayr a; Heribert Quendler
b
; Gabriela Stiegler a; Boris Ferko b; Gabriela Vecera a; Karola Vorauer-Uhl b;
Hermann Katinger ab
a
Polymun Scientific Immunbiologische Forschung GmbH, Vienna, Austria
b
Institute of Applied Microbiology, University of Agricultural Sciences, Vienna,
Austria

Online Publication Date: 01 September 2006

To cite this Article: Wagner, Andreas, Platzgummer, Mirko, Kreismayr, Günther,
Quendler, Heribert, Stiegler, Gabriela, Ferko, Boris, Vecera, Gabriela,
Vorauer-Uhl, Karola and Katinger, Hermann (2006) 'GMP Production of Liposomes -
A New Industrial Approach', Journal of Liposome Research, 16:3, 311 - 319
To link to this article: DOI: 10.1080/08982100600851086
URL: http://dx.doi.org/10.1080/08982100600851086

PLEASE SCROLL DOWN FOR ARTICLE

Full terms and conditions of use: http://www.informaworld.com/terms-and-conditions-of-access.pdf

This article maybe used for research, teaching and private study purposes. Any substantial or systematic reproduction,
re-distribution, re-selling, loan or sub-licensing, systematic supply or distribution in any form to anyone is expressly
forbidden.
The publisher does not give any warranty express or implied or make any representation that the contents will be
complete or accurate or up to date. The accuracy of any instructions, formulae and drug doses should be
independently verified with primary sources. The publisher shall not be liable for any loss, actions, claims, proceedings,
demand or costs or damages whatsoever or howsoever caused arising directly or indirectly in connection with or
arising out of the use of this material.
 Journal of Liposome Research, 16:311–319, 2006
Copyright © Informa Healthcare
Downloaded By: [St George's Medical School Library] At: 13:01 3 October 2007

ISSN: 0898-2104
DOI: 10.1080/08982100600851086

1532-2394
0898-2104
LLPR
Journal of Liposome Research
Research, Vol. 16, No. 3, July 2006: pp. 1–20

GMP Production of Liposomes—A New

Industrial Approach

ANDREAS WAGNER,1 MIRKO PLATZGUMMER,1

GMP
A. Wagner
Production
et al. of Liposomes

GÜNTHER KREISMAYR,1 HERIBERT QUENDLER,2

GABRIELA STIEGLER,1 BORIS FERKO,2
GABRIELA VECERA,1 KAROLA VORAUER-UHL,2
AND HERMANN KATINGER PROF1,2
1
Polymun Scientific Immunbiologische Forschung GmbH, Vienna, Austria
2
Institute of Applied Microbiology, University of Agricultural Sciences, Vienna,
Austria

A new scalable liposome production system is presented, which is based on the ethanol
injection technique. The system permits liposome manufacture regardless of production
scale, as scale is determined only by free disposable vessel volumes. Once the parame-
ters are defined, an easy scale up can be performed by just changing the process vessels.
These vessels are fully sterilizeable and all raw materials are transferred into the sani-
tized and sterilized system via 0.2 µm filters to guarantee an aseptic production.
Liposome size can be controlled by the local lipid concentration at the injection
point depending on process parameters like injection pressure, lipid concentration and
injection rate. These defined process parameters are furthermore responsible for highly
reproducible results with respect to vesicle diameters and encapsulation rates Compared
to other technologies like the film method which is normally followed by size reduction
through high pressure homogenization, ultrasonication or extrusion, no mechanical
forces are needed to generate homogeneous and narrow distributed liposomes.
Another important advantage of this method is the suitability for the entrapment
of many different drug substances such as large hydrophilic proteins by passive encap-
sulation, small amphiphilic drugs by a one step remote loading technique or mem-
brane association of antigens for vaccination approaches

Keywords liposomes, GMP, large scale, drug delivery, Vaccine

Introduction
Recent advances in biotechnological science have resulted in design and synthesis of hundreds
of new agents for pharmaceutical use. In search of new ways for application and galenic for-

Received 1 May 2006; accepted 30 May 2006.

Address correspondence to Andreas Wagner, Polymun Scientific Immunbiologische Forschung
GmbH, Nussdorfer Lände 11, A-1190 Vienna, Austria; Tel.: +43/1/36006-6594; Fax: +43/1/
3697615; E-mail: Andreas.Wagner@boku.ac.at

311
 312 A. Wagner et al.
Downloaded By: [St George's Medical School Library] At: 13:01 3 October 2007

mulation of these biopharmaceuticals, liposomes appear to be an interesting tool for the treat-
ment of patients, especially for topical and dermatological products. Liposomes have been
reported to provide more efficient recruitment of therapeutic compounds and furthermore offer
the advantage of continuous drug release, thus compensating for the short half-life of the drug.
In the case of topical drug application, liposomes may act both as drug transporters and drug
localizers. Furthermore, liposomes can be used as vaccine carriers whereby they protect the
antigen from the environment especially in the case of DNA vaccines, and additionally can
have adjuvant activity as described by Gregoriadis in 1974 (Allison, Gregoriadis, 1974).
Over the past few years the developed liposomal drug preparations have been increasingly
used in clinical trials. These clinical trials have guided liposomes from laboratory research to
clinical reality with very encouraging results. For all these products a GMP-conform produc-
tion technique and facility is necessary. Therefore, liposomes must be of defined size and sta-
ble during storage. The process used for liposome preparation must be reproducible and
process conditions must allow the production of sterile and pyrogene-free liposomes.
Based on the ethanol injection technique, we developed a scalable and sterile produc-
tion technique, whereby substantial progress was achieved, leading from the conventional
batch process to a novel continuous procedure (Wagner et al., 2002).
Herein, the principal item is the cross-flow injection module (Wagner et al., 2002), espe-
cially designed for this purpose. This specially conceived unit has the benefit of defined and
characterized injection streams and permits liposome manufacture regardless of production
scale, as scale is determined only by free disposable vessel volumes as shown in Fig. 1a,b. By
this, process development is performed in lab scale at a volume of about 20 mL. Once the
parameters are defined, an easy scale-up can be performed by just changing the process vessels.
In addition, these process vessels are fully sterilizeable, either by steam sterilization or autoclav-
ation and all raw materials like buffer solutions, the lipid ethanol solution and even the N2 for
administering the injection pressure are transferred into the sanitized and sterilized system via
0.2 μm filters to guarantee an aseptic production (Wagner, Vorauer-Uhl, Katinger, 2002).
Liposome size can be controlled by the local lipid concentration at the injection point
which is defined by the lipid concentration in ethanol, the injection whole diameter, the
injection pressure, and the flow rate of the aqueous phase. By varying these parameters
different liposome sizes suitable for the intended purpose can be prepared, as shown in
Fig. 2a-c. These defined process parameters are furthermore responsible for highly
reproducible results with respect to vesicle diameters and encapsulation rates (Wagner,
Vorauer-Uhl, Katinger, 2002). Figure 3 shows data thereof.

Figure 1. Schematic presentation of the liposome production unit, consisting of vessel 1 (drug solu-
tion) vessel 2 (dilution vessel), and vessel 3 for the lipid ethanol solution. The black cross in the mid-
dle of the sketch is the injection module.
 GMP Production of Liposomes 313
Downloaded By: [St George's Medical School Library] At: 13:01 3 October 2007

Figure 2. Different sized liposome batches in 50 nm, 170 nm, and 350 nm size. Sizing is performed
by varying the process specific parameters like lipid concentration, injection pressure, injection
whole diameter, and buffer flow rate.

Figure 3. Reproducibility data of liposome production batches. Encapsulation rates are or passive
entrapment of rh-Cu/Zn-SOD.

Compared to other technologies like the film method which is normally followed by
size reduction through high-pressure homogenization, ultrasonication, or extrusion,
no mechanical forces are needed to generate homogeneous and narrow distributed lipo-
somes. This is an important issue for the generation of long-term stable liposomes. Data
presented in Fig. 4 show long-term stable aqueous suspensions of liposomal caroverine
hydrochloride.
Another important advantage of this method is the suitability for the entrapment of
many different drug substances such as large hydrophilic proteins by passive encapsula-
tion, small amphiphilic drugs by an one-step remote loading technique or membrane asso-
ciation of antigens for vaccination approaches. In the following Material and Methods
section, several different products are presented demonstrating the versatility of this prep-
aration/production system.
 314 A. Wagner et al.
Downloaded By: [St George's Medical School Library] At: 13:01 3 October 2007

Figure 4. Long-term stability data of a caroverine-loaded liposome suspension at 3°C–5°C. The

lines show the amount of remote loaded Caroverine after gelfiltration for free drug removal deter-
mined by hplc Size distribution graphs were determined at day 0 and after 101 weeks.

Experimental Methods
Liposomal drug and vaccine formulations of recombinant human superoxide dismutase
(rh-Cu/Zn-SOD), caroverine hydrochloride, or viral membrane proteins were produced by
the cross-flow injection technique using the multiple injection modes. As described ear-
lier, all reagents such as the buffer solution, the protein solution, and the lipid-ethanol
solution and nitrogen for the injection process are transferred into the sterilized contain-
ment by filtration through 0.2 μm filters. The aqueous phases and the lipid/ethanol mix-
tures are tempered at 20°C–55°C. The injection pressure amounted to 4–6 bar. The
aqueous phase is pumped from vessel 2 to vessel 3 passing the cross-flow injection mod-
ule where the ethanol/lipid solution is injected which is immediately diluted in the stirred
buffer solution in vessel 3. The final ethanol concentration in the resulting liposome sus-
pension amounted to 7.5%.

Passive Entrapment of rh-Cu/Zn-SOD

The liposomes are produced with the multiple injection mode. The 33 kDa and hydro-
philic rh-Cu/Zn-SOD, which is produced in E.coli, is passively entrapped in liposomes
consisting of DPPC, cholesterol, and stearylamine. An additional formulation for direct
application to the lung via nebulization has been established whereby stearylamine is
replaced by egg-phosphatidyl-glycerol. The protein solution was pumped from vessel 2 to
vessel 3 passing the cross-flow injection module where the ethanol/ lipid solution is
injected into the protein solution which is immediately diluted in the stirred buffer solu-
tion in vessel B (Wagner et al., 2002). This process variation allows very high lipid
 GMP Production of Liposomes 315
Downloaded By: [St George's Medical School Library] At: 13:01 3 October 2007

concentrations per aqueous phase volume, based on the fact that up to 50% ethanol per
protein solution can be injected followed by immediate dilution to an ethanol content
below 7%.

Remote Loading of Caroverine Hydrochloride

Caroverine hydrochloride, a marketed drug for the treatment of tinnitus, was entrapped
with a one-step remote loading technique. Remote loading of amphiphillic drugs using a
citric acid sodium carbonate gradient has been described earlier by Redelmeier et al.
(Mayer et al., 1986). In our approach caroverine hydrochloride is dissolved in citric acid
pH 3.5, which is pumped from vessel 2 to vessel 3 passing the cross-flow injection mod-
ule, where the ethanol/lipid solution is injected. The unentrapped citric acid is immedi-
ately neutralized with sodium carbonate (pH 9.0) in vessel 3. By this, liposome formation
and generation of the pH gradient is performed within one step. Liposomes consisted of
DPPC:cholesterol in a molar ratio of 55:45 with a concentration of 10 μmol/mL.

Membrane Entrapment of Viral Antigens

For the membrane association of antigens to be used as a vaccine, we combined the etha-
nol injection method with the detergent dilution method. Herein, recombinant HIV-1
membrane protein gp41 or native proteins from lysed virus are dissolved in a micellar
solution using PBS/ß-OG or PBS/Chaps with a detergent concentration well above the
critical micellar concentration (CMC). Liposomes are composed of EPC, EPG, EPE, and
cholesterol mimicking the viral membranes. Again the solution from vessel 2 is pumped to
vessel 3 accompanied by ethanol/lipid injection. Immediately after injection, the solution
is diluted in vessel 3 resulting in a detergent concentration reduction below the CMC.
Thereby vesicles are formed. For the development of this method several experiments
were undertaken to determine the optimal dilution ratio that stable liposomes were formed
(Wagner et al., 2006).
Liposomes of all experiments were analyzed with respect to size and size distribution
by photon correlation spectroscopy using a Malvern Nano ZS. The amount of liposomaly
entrapped rh-Cu/Zn-SOD was determined by ELISA and activity was tested in a microti-
ter enzyme activity assay. The amount of remote-loaded caroverine hydrochloride
together with cholesterol was determined by RP-HPLC as shown in Fig. 5. The membrane
associated antigens were analyzed by electrophoresis and western blot with respect to
encapsulation efficiency, protein configuration, and stability. In addition, antigen antibody

Figure 5. Typical rp-hplc chromatogram of caroverine-hydrochloride (1) and cholesterol (2).

 316 A. Wagner et al.
Downloaded By: [St George's Medical School Library] At: 13:01 3 October 2007

binding studies were done by FACS analysis using the in-house developed hmAb 2F5 and
4E10 which bind specifically to gp41.
All products were additionally tested for the absence of microbial contaminants in test
procedures described in the European Pharmacopoeia.

Results and Discussion

Recombinant human Cu/Zn superoxide dismutase was liposomally entrapped by the mul-
tiple injection procedure as described elsewhere (Wagner et al., 2002a, 2002b; Wagner,
Vorauer-Uhl, Katinger, 2002). Until now liposome batches up to 200 were produced.
Throughout all production batches, 25–30% of the present protein was entrapped into
liposomes, only feasible with the injection of high lipid/ethanol volumes in the drug solu-
tion with subsequent dilution for ethanol concentration reduction. Once the parameters
were established, defined and reproducible liposome suspensions were produced. As
shown in Fig. 3, z-average means of about 180 nm and pDI of 0.15–0.18 were reproduc-
ible in all production batches. This radical scavenging enzyme in our liposomal formula-
tions has been used in several clinical and preclinical studies. For instance, the liposomal
rh-Cu/Zn-SOD (Lipoxysan™) has passed a phase 3 clinical trial for the topical treatment
of induratio penis plastica (IPP) with very encouraging results (Claus et al., 2005). Pain,
the main parameter observed in this study, was reduced in more than 90% of the patients.
Additional parameters such as plaque size and hardness were also improved in 30–50% of
the treated patients.
Furthermore, animal studies were performed to prove the concept of improved wound
healing after burning by topical application of liposomal rh-Cu/Zn-SOD, showing satisfy-
ing results compared to control groups (Vorauer-Uhl et al., 2001, 2002). These data
encouraged us to start with a limited number of case studies for treating chronic ulcers in a
compassionate use program. These experiments showed in some patients improved wound
healing and closure of chronic ulcers within weeks.
An additional animal study is currently in progress whereby rh-Cu/Zn-SOD
containing liposomes are nebulized by a new device, the Pari E-Flow (Wagner et al.,
2006), for the treatment of lipopolysaccheride-induced acute respiratory distress
syndrome.
Based on the side-effects of parenteral administered tinnitin (trade name of caroverine
hydrochloride) this drug was entrapped for the local treatment of tinnitus via drops direct
at the transtympanic membrane. With the above described technique, a pH gradient was
created which induced the active loading of the amphiphillic drug. By loading into 150 nm
vesicles loading rates of 250–300 nmol drug/μmol lipid could be realized depending on
the pH-difference inside and outside the liposomes, demonstrated in Fig. 6. With this
method up to 95% of the admitted drug can be entrapped within one production step. The
big advantage of this method is that liposome formation and pH-gradient formation is per-
formed within one step and no additional buffer exchange is necessary for the remote
loading of the drug. This mild one step active loading method generated very stable lipo-
somes, where the drug was retained within the bilayers for more than 2 years stored in sus-
pension at 3°C–8°C as shown in Fig. 4.
Currently, additional drug substances are under investigation, which are also actively
loaded by this one-step remote loading method. For instance liposomal galantamine
hydrobromide is currently in a phase 2 trial for the topical treatment of peripheral
neuropathy and a liposomal formulation of sildenafil citrate is under development for a
possible local treatment of male and female sexual dysfunction.
 GMP Production of Liposomes 317
Downloaded By: [St George's Medical School Library] At: 13:01 3 October 2007

350
300
nmol drug / µmol l 250
200
150
100
50
0
pH inside 4,5 pH inside 4,0 pH inside 3,5

Figure 6. Increasing amount of remote-loaded caroverine hydrochloride by decreasing the pH

inside the liposomes.

In the field of HIV the Institute of Applied Microbiology and Polymun Scientific
have been working on the development of monoclonal antibodies. Currently, three of the
five worldwide known monoclonal antibodies with neutralizing activity are owned by
Polymun Scientific. Working in the field of liposomology, we combined the HIV
knowhow with liposome knowhow to establish possible vaccine candidates (Lenz et al.,
2005).
For the virosomal vaccine project against HIV recombinant the HIV-1 membrane
protein gp41 is entrapped within the bilayer. As shown in the electrophoreses in Fig. 7,
nearly all admitted gp41 is liposomaly bound. In the filtrate lane hardly any gp41 can be
detected. These liposomes were generated by the combined ethanol injection detergent
dilution technique established for this purposes. The liposomal associated antigen ana-
lyzed in antibody binding studies using mAb 2F5 and 4E10 and trimeric structure was
controlled by chemical cross-linking studies (Wagner et al., 2006). In addition, this mate-
rial was administered intraperitoneally to mice. After three immunizations mouse sera
were investigated by ELISA and significant IgG titers were found (Fig. 8a). Furthermore,
these antisera demonstrated neutralizing activity as shown in Fig. 8b.

1 2 3 4 5 6 7 8 9 10 11 12
1: gp41 chrtm5 liposomes
2: gp41 chrtm5 liposomes after filt.
3: gp41 chrtm5 liposomes after filt.
4: filtrate
5: protein marker
6: ---------
7: gp41 ctm liposomes
8: gp41 ctm liposomes after filt.
9: gp41 ctm liposomes after filt.
10: filtrate
11: free gp 41 ctm
12: protein marker

Figure 7. Electrophorese of two different liposomal gp41 batches (lane 1–4 construct 1; lane 7–11
construct 2) for determination of the amount of liposomally associated pg41 (lanes 2, 3, and 8, 9).
 318 A. Wagner et al.
Downloaded By: [St George's Medical School Library] At: 13:01 3 October 2007

Figure 8. 1: IgG titer after three immunizations in balb c mice determined by ELISA a and neutral-
ization data determined by a synctium inhibition assay.

Based on these results we started working with native proteins from lysed virus and
used them for association with liposomes. This project is currently under way and spon-
sored by funds of the European Union (EC-Contract/Project number: 012183).
For this approach we grow the virus in mammalian cell culture, and then lyse the
virus by incubation with a suitable detergent. After destroying the RNA by Benzonase
treatment the reverse transcriptase is removed. Subsequently, liposomes are generated in
the micellar aqueous phase containing different HIV specific proteins.
Until now stable encapsulation rates of intact membrane proteins could be observed
and ongoing preclinical in vivo studies show promising results.
In addition, other studies are in progress where synthetic palmitoylated peptides are
liposomaly associated by the same method. These liposomes are used in mouse studies for
the treatment of Alzheimer´s disease (Nicolau et al., 2002).
Subsequently, the potential of this technique has been demonstrated by the entrap-
ment of several product classes. For characterization, size and size distribution, protein
encapsulation efficiency, biological activity of the protein, membrane stability, and micro-
bial contamination were determined and showed satisfying results.

Conclusions
In conclusion, liposome manufacture with the cross-flow technique is suitable for the pro-
duction of clinically relevant material. The main advantage of this technique is the feasibility
of manufacturing batches of 5–10 mL which is directly scalable to several liters.
This variant avoids cost-intensive scale-up and permits early prognosis about product
quality, thus liposomes can be screened more quickly and more efficiently. This is of par-
ticular importance when it comes to cost-intensive drugs that are to be manufactured by
novel biotechnological procedures.

References
Allison, A. G., Gregoriadis, G. (1974). Liposomes as immunological adjuvants. Nature Nov
15;252(5480):252.
Lenz, O., Dittmar, M. T., Wagner, A., Ferko, B., Vorauer-Uhl, K., Stiegler, G., Weissenhorn, W.
(2005). Trimeric membrane anchored gp41 inhibits HIV membrane fusion. J. Biol. Chem.
280(6):4095–4101.
Mayer, L. D., Bally, M. B., Cullis, P. R. (1986). Uptake of adriamycin into large unilamellar vesicles
in response to a pH gradient. Biochimica Biophyica Acta 9;857(1):123–126.
 GMP Production of Liposomes 319
Downloaded By: [St George's Medical School Library] At: 13:01 3 October 2007

Nicolau, C., Greferath, R., Balaban, T. S., Lazarte, J. E., Hopkins, R. J. (2002). A liposome-based
therapeutic vaccine against ß-amyloid plaques on the pancreas of transgenic NORBA mice.
PNAS 99 (4):2332–2337.
Riedl, C. R., Sternig, P., Gallé, G., Langmann, F., Vcelar, B., Vorauer, K., Wagner, A., Katinger, H.,
Pflüger, H. (2005). Liposomal recombinant human superoxide dismutase for the treatment of
Peyronie’s disease: A randomized placebo-controlled double-blind prospective clinical study.
European Urology 48(4):656–661.
Vorauer-Uhl, K., Fürnschlief, E., Wagner, A., Ferko, B., Katinger, H. (2001). Topically applied
liposome encapsulated superoxide dismutase reduces postburn wound size and edema formation.
European Journal of Pharmaceutical Sciences, 14/1, 63–67.
Vorauer-Uhl, K., Fürnschlief, E., Wagner, A., Ferko, B., Katinger, H. (2002). Reepithelization of
experimental scalds effected by topically applied superoxide dismutase: controlled animal
studies. Wound Repair and Regeneration 10(6):366 – 371.
Wagner, A., Vorauer-Uhl, K., Katinger, H. (2002). Liposome produced in a pilot scale: production,
purification and efficiency aspects. European Journal of Pharmaceutics and Biopharmaceutics
54:213–219.
Wagner, A., Vorauer-Uhl, K., Katinger, H. (2006). Nebulization of liposomal rh-Cu/Zn–SOD with a
novel vibrating membrane nebulizer. Accepted for publication in Journal of Liposome research.
Wagner, A., Vorauer-Uhl, K., Kreismayr, G., Katinger, H. (2002a). The crossflow injection tech-
nique – an improvement of the ethanol injection method. Journal of Liposome Research
12(3):259–270.
Wagner, A., Vorauer-Uhl, K., Kreismayr, G., Katinger, H. (2002b). Enhanced protein loading into
liposomes by the multiple injection technique. Journal of Liposome Research 12(3):271–283.
Wagner, A., Quendler, H., Stiegler, G., Vorauer-Uhl, K., Katinger, H. (2006). One step membrane
incorporation of viral antigens as a Vaccine Candidate against HIV.

View publication stats