Downstream processing of recombinant proteins from
transgenic feedstock
Zivko L Nikolov and Susan L Woodard
The search for inexpensive production systems capable
of producing large quantities of recombinant protein has
resulted in the development of new technology platforms
based on transgenic plants and animals. Over the past
decade, these transgenic systems have been used to
produce several products and potential therapeutic
proteins. Improvements continue to be made, not only
in how the proteins are expressed but also in how the end
products are obtained. As improvements in expression are
realized, cost-saving measures will increasingly focus on
downstream processing.
Addresses
Department of Biological and Agricultural Engineering, Texas A&M
University, MS 2117, College Station, Texas 77843, USA

e-mail: znikolov@tamu.edu
Current Opinion in Biotechnology 2004, 15:479–486
This review comes from a themed issue on
Biochemical engineering
Edited by Manuel Carrondo and John G Aunins
Available online 11th September 2004
0958-1669/$ – see front matter
# 2004 Elsevier Ltd. All rights reserved.
DOI 10.1016/j.copbio.2004.08.006
Introduction
The potential of ‘molecular pharming’ — using trans-
genic plants or animals as bioreactors to produce ther-
apeutic proteins — has been amply demonstrated with
several plant- and animal-derived recombinant protein
products currently in clinical trials. The status of these
two general transgenic platforms has been recently
reviewed, with emphasis given primarily to the upstream
production and processing of pharmaceutical proteins
[1–6]. This review provides a brief overview of recent
advances in transgenic production systems and focuses
on methods developed for the recovery and purification
of transgenically produced proteins over the past two
years.
Advances in animal production systems
A wide variety of animal species, including cows, goats,
rabbits, sheep and chickens, have been used as an alter-
native source of properly processed recombinant proteins.
In addition to animal variety, one has the option to select
the expression tissue and transgenic protein can be har-
vested from body fluids such as milk, egg, blood, urine
www.sciencedirect.com
and seminal fluid [5,7,8,9]. Mammary gland is currently
the preferred bioreactor, because of the large volumes of
milk produced by lactating animals and expression levels
of transgenic protein in excess of 1 g/L. More than 20
potential products, such as human monoclonal antibodies,
a-glucosidase, human C1 inhibitor, tissue plasminogen
activator, antithrombin III, human lactoferrin, procolla-
gen, protein C and other therapeutics, have been
expressed in transgenic animals and are currently in
various stages of clinical trials [4].
The recent successful expression and purification of
several therapeutic proteins in transgenic egg white
[8,10] indicates that avian transgenic systems offer yet
another scalable system for recombinant protein produc-
tion. A potential advantage of eggs is the existence of an
established regulatory pathway for the clinical approval of
vaccines produced in eggs. The current expression levels
of therapeutics in transgenic eggs are around 0.1 mg per
egg [10]; thus, at least a 100-fold increase in expression
would be needed before this system could become eco-
nomically viable (Z Nikolov, unpublished).
Numerous examples (see Table 1) attest to the capacity
of transgenic tissues, and mammary gland in particular, to
synthesize and secrete heterologous proteins. Yet, the
commercialization of transgenic animals as bioreactors
has been unexpectedly slow. The technical challenges
that have hampered commercialization efforts include
the efficiency of producing transgenic animals, hetero-
geneous post-translational modifications, the ability to
predict and control the specificity of expression, and
challenges in downstream processing [1,5,11].
Recent developments that are helping to improve the
efficiency of host production include the replacement of
microinjection with nuclear transfer technology, the use
of transfected somatic cells as nuclear donors, and the use
of sperm-mediated gene transfer and totipotent (having
unlimited capacity) embryonic stem cells to produce
chimeric animals [6,8,12]. A promising technology that
might further increase the efficiency of generating trans-
genic animals uses artificial chromosomes as transgenic
vectors [13,14]. Transgene expression is continuously
being improved through the use of a combination of
factors and/or elements such as gene insulators, chromatin
openers, matrix-attached regions, enhancers and introns
[5]. Post-translational modification is an issue that has to
be dealt with on a product-by-product basis, because
differences in glycosylation between animals and humans
might not necessarily affect biological activity.
Current Opinion in Biotechnology 2004, 15:479–486
480 Biochemical engineering

Table 1

Downstream processing studies with transgenic animals.

Transgenic system Recombinant protein Purification steps employed Comments Reference

Ewe milk Human fibrinogen Clarification - defatting by centrifugation >65% yield [21]
Purification - cation exchange >60% pure
Cow milk Human lactoferrin Clarification (NR) Yield and purity not reported [23]
Purification
- cation exchange
- viral inactivation
- cation exchange
- diafiltration and liquid formulation
Sheep milk a1-PI Clarification 44% yield [17]
- defatting by centrifugation >99% pure
- casein precipitation with PEG
Purification
- PEG precipitation of a1-PI
- cation exchange
- anion exchange
- protein G affinity
- immobilized Ni affinity
- hydrophobic interaction
(phenyl-sepharose)
Goat milk Human IgG Clarification Modeled microfiltration of defatted [7]
- defatting by centrifugation transgenic milk
- casein removal by microfiltration
Porcine semen Follicle-stimulating Clarification No other details reported [9]
hormone - membrane filtration (UF/DF)
Purification
- several chromatographic steps
Egg white Interferon a-2b Purification Proof-of-concept study [10]
- cation exchange chromatography Yield and purity not reported
- RP-HPLC

DF, diafiltration; IgG, immunoglobulin G; NR, not reported; PEG, polyethylene glycol; a1-PI, a1 proteinase inhibitor; RP-HPLC, reverse
phase high-performance liquid chromatography; UF, ultrafiltration.

From a commercialization standpoint, the possible con-
tamination of transgenic products with animal viruses and
prions is a major concern that is being addressed through
the implementation of measures to ensure the health of
transgenic herds [15,16]. The removal of pathogens is
currently carried out during downstream processing of
non-transgenic animal-derived therapeutics and mamma-
lian cell culture, and the same approach will be applied
to products originating from transgenic fluids. To fully
exploit the low production cost potential of transgenic
animal systems, the development of efficient downstream
processing technologies is the remaining technical chal-
lenge to be addressed.
clarification of milk [7]. Other techniques for casein
removal, such as the use of salt, acid and polyethylene
glycol precipitation, have been reviewed by McCreath
[21]. The principal disadvantage of casein precipitation is
the potential loss of target protein trapped in the protein
precipitate [19].
The processing of transgenic eggs starts with egg white
fractionation from egg yolk followed by the reduction of
egg white viscosity by homogenization or ovomucin
precipitation using dilution with acidified water [10,22].
Clarified egg white can then be treated in a similar
manner to clarified milk.

Downstream processing of proteins Details of the processing and purification of recombinant

produced in animal bioreactors
Figure 1 illustrates the similarity of the purification
procedures for transgenic proteins from milk or egg white,
which differ only in the initial processing steps. The
removal of fat globules and casein micelles from milk
is deemed critical for reducing fouling in the subsequent
purification steps [7,17,18–21]. Several methods for
defatting and casein removal from transgenic milk have
been explored. Centrifugation of fat globules followed by
membrane filtration of casein micelles or membrane
filtration alone appear to be preferred methods for the
proteins expressed in transgenic animals are scarce in the
literature (summarized in Table 1). Most downstream
processes were developed by industry and published in
patents. One such report by Lee and Antonsen [17]
describes the purification of a-1 proteinase inhibitor
(a1-PI) from transgenic sheep milk expressing the protein
at 10–12 g/L. Stringent product specifications of >99.99%
purity necessitated the development of a ten-step pur-
ification procedure to remove whey protein from the final
product. By contrast, human lactoferrin expressed at
2–3 g/L in transgenic cow milk was purified by two cation

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 Processing proteins from transgenic feedstock Nikolov and Woodard 481
Figure 1
Transgenic animal systems
Milk
Acid
Fat globules,
Clarification casein micelles,
soluble lipids
Pre-purification Milk proteins,
(Capture step) salts, lactose
Purification (HIC, Trace proteins
affinity, IEx, etc.) Process-related
contaminants
Viral inactivation
and removal
Bulk product
Downstream processing of recombinant proteins from transgenic animal systems. The stages of downstream processing of transgenic milk
are shown on the left and those of eggs appear on the right. Note that viral inactivation is typically executed early in the process, but is shown
at the end for simplicity. HIC, hydrophobic interaction chromatography; IEx, ion exchange.
exchange chromatography steps [23]. Although develop-
ment of downstream processing for recombinant proteins
from transgenic eggs lags behind that of milk, Rapp et al.
[10] purified interferon a-2b from egg white by cation
exchange and reverse phase chromatography to charac-
terize the glycosylation pattern of the chicken-derived
protein. Milk, being more complex (containing casein
micelles, milk fat and lactose), will probably require more
downstream processing steps than egg white (no lipids) to
achieve the same degree of purification.
Advances in plant production systems
Over 100 recombinant proteins expressed in more than
20 plant species have been reported in the past 20 years
[2,3,24,25]. Although the expression of a variety of
complex proteins has been sufficiently demonstrated,
reports on purification development and the commercial
viability of developed products have been missing. In
addition to intellectual property and proprietary technol-
ogy concerns, other probable reasons for this void include
lower than optimal expression, containment issues and
suboptimal purification processes.
Plant-based systems share similar general technical chal-
lenges to those of transgenic animals: consistent and high
expression levels, speed to market, expression specificity,
appropriate post-translational modifications and, to a les-
ser extent, downstream processing. A new challenge
www.sciencedirect.com
Egg white
Precipitation
Clarification Ovomucin
Pre-purification Egg white
(Capture step) proteins
Purification (HIC, Trace proteins
affinity, IEx, etc.) Process-related
contaminants
Viral inactivation
and removal
Bulk product
Current Opinion in Biotechnology
specific to plants, imposed largely by the food and phar-
maceutical industries, is the environmental containment
of plant production systems. In response to this prefer-
ence, aquatic production systems are being developed to
support the growth and production of recombinant pro-
teins in bioreactors or contained environments. These
new systems include unicellular green algae [26], moss
Physcomitrella paten suspension culture [27], the rhizose-
cretion of hairy roots into hydroponic medium [28] and
seed germination (sprouting) in airlift bioreactors (http://
www.unicrop.fi), as well as aquatic plants such as Lemna
[29].
With the addition of aquatic systems, the choice of plant-
based protein production systems has increased making
it convenient to divide them into two broad categories:
terrestrial (soil-supported crops) and aquatic systems.
Thus, plant systems offer more production options than
animal systems. With this variety comes a greater varia-
bility of bioprocessing strategies, handling and purifica-
tion design requirements. Owing to space limitations,
only basic features and advances of the various plant
systems will be summarized emphasizing considerations
that are important for optimal downstream processing.
For recent advances in expression strategies, glycosyla-
tion, applications and suitability of each plant group for a
specific application or product category, refer to reviews
by Ma et al. [3], Horn et al. [30] and Gomord et al. [25].
Current Opinion in Biotechnology 2004, 15:479–486
482 Biochemical engineering

Table 2

Downstream processing studies with transgenic plants.

Transgenic system Recombinant protein Purification steps employed using Comments Reference
plant extracts and concentrates
Corn Aprotinin Clarification by 3 mm filtration 49% yield [47]
Purification 79% pure
- trypsin affinity chromatography
- immobilized copper affinity
chromatography
Corn Trypsin Clarification 25% yield [48]
- centrifugation >98% pure
Purification
- trypsin inhibitor affinity chromatography
- cation exchange
Canola GUS Clarification Compared expanded-bed [41]
and fixed-bed
- centrifugation >65% yield
- 0.22 mm filtration (packed-bed only) >30-fold purification
Purification
- expanded-bed and fixed-bed anion
exchange chromatography
Potato tubers Monoclonal antibody (IgG) Clarification 10% yield [49]
- centrifugation (2) Major protein bands (59 kDa and
Purification 28 kDa) on SDS–PAGE
- protein G affinity (2)
Tobacco Thermostable xylanase Clarification (NR) >85% yield [35]
(chloroplast) Purification Major band on SDS–PAGE
- heat precipitation (70 8C)
- anion-exchange chromatography
(Q-sepharose)
Tobacco GUS-polyhistidine-g- Clarification (NR) Fusion yield after Ni affinity 75% [50]
(chloroplast) interferon fusion Purity: NR
Purification
- anion exchange
- immobilized Ni affinity
- cation exchange
Tobacco Antimicrobial peptide– Clarification Yield and purity not reported [51]
CBD fusion
- two centrifugation steps
Purification
- size exclusion
- chitin affinity chromatography
Tobacco Monoclonal antibody (IgG) Clarification <60% yield [42]
- basket centrifugation >90% purity
- tubular centrifugation
Purification
- expanded-bed protein A affinity
Tobacco GUS–CaM Clarification 85% yield [52]
- two centrifugation steps 20-fold purification
Purification Purity: NR
- ammonium sulfate precipitation
- dialysis
- concentration
- phenothiazine affinity chromatography
Tobacco Trichosanthin Clarification Purification details not given [53]
- centrifugation Yield and purity not given
Purification
- dialysis
- sizing
- anion exchange
- UF concentration
- cation exchange
Lemna Interferon Clarification (not required) Purification details not given [29]
Purification Yield and purity not reported
- UF concentration (50)
- mAb affinity chromatography

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 Processing proteins from transgenic feedstock Nikolov and Woodard 483

Table 2 continued
Transgenic system Recombinant protein Purification steps employed using Comments Reference
plant extracts and concentrates
Pea GUS-polyhistidine fusion Clarification Evaluated potential of [54]
different methods
- centrifugation 260-fold purification
- 0.22 mm filtration Threefold Fourfold
Purification 1.5-fold
- immobilized cobalt affinity
- anion exchange chromatography
- isoelectric precipitation
- polyelectolyte precipitation
Rice Human lactoferrin Clarification Yield not reported [55]
- three-step filtration Single band on SDS–PAGE
Purification
- cation exchange chromatography
Rice Human lysozyme Clarification 60% yield [56]
- three-step filtration Single band on SDS–PAGE
Purification
- cation exchange chromatography
- size exclusion

CaM, calmodulin; CBD, cellulose-binding domain; GUS, b-glucuronidase; mAb, monoclonal antibody; NR, not reported; SDS–PAGE,
sodium dodecyl sulphate–polyacrylamide gel electrophoresis.

Tobacco, alfalfa and potato leaves (leafy crops) are dis-
tinguished by a very high biomass yield per acre, but
these systems have issues regarding protein stability and
contaminants [31,32]. Extracts containing phenolics and
released proteases could reduce the choice of initial
downstream processing steps and flexibility by requiring
product-tailored step(s), such as the addition of protease
inhibitors, pH adjustment, thermal inactivation of
enzymes or the use of a fast initial processing step.
The desired and achieved high level of expression in
the vegetative organs could potentially interfere with
plant growth and development. The development of
transplastomic (transformed plastid) expression in
tobacco offers distinct advantages, such as very high
expression levels and minimal protein degradation; the
limitation of this approach is the lack of glycosylation
machinery in plastids [33–35].
Of terrestrial seed crops, most effort has been spent on
product development from transgenic corn, tobacco, rice,
safflower and alfalfa [30,36]. Other crops of potential
significance include barley, peas, potato tubers and
leaves, flax and other oilseeds [3,31]. Seed crops are
distinguished by providing a more stable environment
in the seed and extracts alike [24,30,31], but have lower
production volumes (kg/acre) than tobacco or alfalfa and
require a flowering cycle to produce seed. The contain-
ment concern is greatest for transgenic corn and to a lesser
extent for other seed crops that are self-pollinating (e.g.
rice, barley and safflower). Aquatic production systems
offer containment and the possibility to recover and
purify secreted protein product from a simple aqueous
medium, thus eliminating the need for tissue homogeni-
zation or grinding. The potential disadvantages include
very low expression levels in the medium and unproven
production technology on a larger scale. The scale up of
suspension culture bioreactors could well face the same
challenges experienced when scaling up mammalian cell
culture.
Downstream processing of proteins
produced in plants
From its inception, the prospect of utilizing plants as
factories for the production of recombinant proteins was
thought to hinge on the low cost of downstream proces-
sing [37]. And, although detailed cost analyses to date are
scarce [38,39], simulations based on small-scale data
suggest that a high percentage of the overall production
costs is incurred during downstream processing. There-
fore, research to develop and improve downstream pro-
cessing methods could greatly reduce the overall costs
associated with many products, and perhaps dictate
(along with expression levels) commercial feasibility.
Reports concerning the downstream processing of trans-
genic plant material in the past two years outnumber
those of transgenic animals (see Table 2). An extensive
discussion of various processing considerations for trans-
genic plant-derived proteins is given in a recent review
by Menkhaus et al. [24]. In the past ten years detailed
process analyses were conducted on corn, canola and
tobacco, while the downstream processing of other crops
such as alfalfa, wheat, rice and barley have been given
only limited evaluation. Aquatic systems are an emerging
technology and minimal information on downstream
processing is currently available.
Initial downstream processing steps applied to transgenic
plant-expressed proteins are rather diverse. Generally
speaking, the particular plant host as well as the final

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484 Biochemical engineering

Figure 2

(a) Terrestrial transgenic systems

Seed crops Leafy crops

Buffer and
Grinding Grinding
antioxidant

High ionic-
strength Solid-liquid Pressing Biomass
buffer extraction
Protein precipitate
Heat treatment Residual biomass
Clarification Biomass and clarification particles

Plant proteins, Plant proteins,

Pre-purification Pre-purification
sugars, phenolics, sugars, phenolics,
(Capture step) (Capture step)
phytic acid, salts salts

Purification Trace proteins Purification Trace proteins

(HIC, affinity, IEx, etc.) Process-related (HIC, affinity, IEx, etc.) Process-related
contaminants contaminants

Bulk product Bulk product

(b) Aquatic transgenic systems

Lemna, plant hairy roots, Microalgae
moss, sprouts

Harvesting Water

Biomass removal Biomass Cell rupture

Concentration Clarification Biomass

Pre-purification Bulk protein, Pre-purification Bulk protein,

(Capture step) salts (Capture step) salts

Purification Trace proteins Purification Trace proteins

(HIC, affinity, IEx, etc.) Process-related (HIC, affinity, IEx. etc.) Process-related
contaminants contaminants

Bulk product Bulk product

Current Opinion in Biotechnology

Downstream processing of recombinant proteins from transgenic plant systems. (a) Terrestrial systems. The stages of downstream processing
of seed crops are depicted on the left and those of leafy crops are shown on the right. (b) Aquatic systems. The stages of downstream
processing of aquatic culture systems where proteins are secreted are shown on the left and those for non-secreting aquatic culture systems,
such as microalgae, are shown on the right. HIC, hydrophobic interaction chromatography; IEx, ion exchange.

destination of the secreted recombinant biomolecule
(tissue or media) will influence the selection of unit
operations before purification. The handling of transgenic
plant raw material containing the recombinant protein
of interest from post-harvest to final formulation of the
product can be divided into three general stages, as
depicted schematically in Figure 2. For terrestrial crops
(Figure 2a) these consist of plant material pre-processing
(i.e. grinding and/or fractionation); protein extraction and
solid/liquid clarification; and protein purification [40].
Terrestrial seed crops typically require an extraction that
can be conducted concurrently with grinding (wet grind-
ing) or subsequent to particle size reduction using a

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 Processing proteins from transgenic feedstock Nikolov and Woodard 485
preferred dry grinding and fractionation method [24].
Terrestrial leafy crops are macerated in the presence of
antioxidants (such as ascorbic acid) to reduce phenol
oxidase activity. Clarification in most cases is performed
by centrifugation and, in some cases, may be followed by
polishing steps such as dead-end filtration or another
centrifugation step (Table 2). The integration of clarifica-
tion and adsorption steps has been studied by Bai and
Glatz [41] and Valdes et al. [42] using expanded bed
adsorption. Valdes et al. [42] found that clarified tobacco
extract fouled Protein A Sepharose when used in a fixed-
bed mode and to a lesser extent Protein A Streamline
resin (expanded bed resin). Similar observations were
made by others using transgenic canola [41] and corn
extracts [24]. Miller et al. [32] found two-phase parti-
tioning in the presence of non-ionic detergent useful in
removing both chlorophyll and phenolics from potato leaf
extracts containing recombinant protein.
The initial downstream processing stages of aquatic sys-
tems would be similar to those utilized in submerged
culture fermentation, starting with biomass removal/
clarification and followed by concentration and purifica-
tion (Figure 2b) [29]. The development of an efficient
and inexpensive method for rupturing/breaking algal cell
walls is a technical challenge facing microalgae systems (P
Heifetz, personal communication).
The extent of processing and purification of plant-derived
products will depend on the intended use. These range
from processed and/or sterilized edible tissue, which
requires no purification steps [43,44], to highly purified
protein product for parenterally administered therapeu-
tics in compliance with FDA guidelines [45,46].
Conclusions
Molecular pharming using transgenic plant and animal
systems provides low-cost alternatives to traditional pro-
tein production systems. Refinements made in utilizing
these platforms are resulting in commercial products.
Increasingly, improvements in the recovery and purifica-
tion of products will be sought to maximize cost-savings
and to provide the potentially large volumes required to
meet the market demand for these products.
References and recommended reading
Papers of particular interest, published within the annual period of
review, have been highlighted as:
 of special interest
 of outstanding interest
1. Larrick JW, Thomas DW: Producing proteins in transgenic
plants and animals. Curr Opin Biotechnol 2001, 12:411-418.
2. Twyman RM, Stoger E, Schillberg S, Christou P, Fischer R:
Molecular farming in plants: host systems and expression
technology. Trends Biotechnol 2003, 21:570-578.
3. Ma K-CJ, Drake PMW, Christou P: The production of
recombinant pharmaceutical proteins in plants.
Nat Rev Genet 2003, 4:794-805.
www.sciencedirect.com
4. Das RC: Production of therapeutic proteins from transgenic
animals. Biobusiness 2001, Feb:60-64.
5. Houdebine LM: Transgenic animal bioreactors. Transgenic Res
2000, 9:305-320.
6. Houdebine LM: Antibody manufacture in transgenic animals
and comparisons with other systems. Curr Opin Biotechnol
2002, 13:625-629.
7. Baruah GL, Couto D, Belfort G: A predictive aggregate transport
 model for microfiltration of combined macromolecular
solutions and poly-disperse suspensions: testing model with
transgenic goat milk. Biotechnol Prog 2003, 19:1533-1540.
This paper discusses the role of submicron fat globules and casein
micelles as putative foulants in microfiltration and proposes a model
for predicting permeate flux during cross flow microfiltration of raw milk.
8. Ivarie R: Avian transgenesis: progress towards the promise.
Trends Biotechnol 2003, 21:14-18.
9. Birse D, Lacroix D, Dyck M, Pothier F, Sirard M-A:
Biotherapeutics from transgenic porcine sources:
bioprocessing approaches and challenges. Bioprocessing J
2004, 3:37-44.
10. Rapp JC, Harvey AJ, Speksnijder GL, Hu W, Ivarie R: Biologically
active human interferon a-2b produced in the egg white of
transgenic hens. Transgenic Res 2003, 12:569-575.
11. Lubon H, Palmer C: Transgenic animal bioreactors – where we
are. Transgenic Res 2000, 9:301-304.
12. Clark J, Whitelaw B: A future of transgenic livestock.
Nat Rev Genet 2003, 4:825-833.
13. Kuroiwa Y, Kasinathan P, Choi YJ, Naeem R, Tomizuka K,
Sullivan EJ, Knott JG, Duteau A, Goldsby RA, Osborne BA et al.:
Cloned transchromosomic calves producing human
immunoglobulin. Nat Biotechnol 2002, 20:889-894.
14. Robl JM, Kasinathan P, Sullivan E, Kuroiwa Y, Tomizuka K,
Ishida I: Artificial chromosome vectors and expression of
complex proteins in transgenic animals. Theriogenology 2003,
59:107-113.
15. Montgomery SA: Transgenic animals: walking bioreactors.
 BioProcess Int 2004, 2 (Supplement 2):40-51.
This overview contains useful information on commercial considerations
for producing recombinant proteins in transgenic animals.
16. Kues WA, Niemann H: The contribution of farm animals to
human health. Trends Biotechnol 2004, 22:286-294.
17. Lee VW, Antonsen KP: Purification of a-1 proteinase inhibitor.
 US Patent 2003, 6,525,176 B1.
A detailed description of a1-PI purification, essentially free of whey
protein. Good discussion of individual purification steps and considera-
tions used in selecting resin functionalities and process parameters.
18. Pollock DP, Kutzko JP, Birck-Wilson E, Williams JL, Echelard Y,
Meade HM: Transgenic milk as a method for the production
of recombinant antibodies. J Immunol Methods 1999,
231:147-157.
19. Morcol T, He Q, Bell SJD: Model for removal of caseins from
milk of transgenic animals. Biotechnol Prog 2001, 17:577-582.
20. Wilkins TD, Velander W: Isolation of recombinant proteins from
milk. J Cell Biochem 1992, 49:333-338.
21. McCreath G: Purification of fibrinogen from milk by use of
cation exchange chromatography. US Patent 2002, 2002/
0025932 A1.
22. Ransohoff TC, Owen MJ: Immunoglobulin purification. US
Patent 2003, 2003/0176660.
23. van Berkel PH, Welling MM, Geerts M, van Veen HA,
Ravensbergen B, Salaheddine M, Pauwels EK, Pieper F,
Nuijens JH, Nibbering PH: Large scale production of
recombinant human lactoferrin in the milk of transgenic cows.
Nat Biotechnol 2002, 20:484-487.
24. Menkhaus TJ, Bai Y, Zhang C-M, Nikolov ZL, Glatz CE:
 Considerations for the recovery of recombinant proteins from
plants. Biotechnol Progr 2004, 20:1001-1014.
Current Opinion in Biotechnology 2004, 15:479–486
486 Biochemical engineering
A comprehensive review of transgenic crops and their composition, as
well as an evaluation of recombinant protein recovery and purification
methods.
25. Gomord V, Sourrouille C, Fitchette A-C, Bardor M, Pagny S,
Lerouge P, Faye L: Production and glycosylation of plant-made
pharmaceuticals: the antibody as a challenge. Plant Biotechnol
J 2004, 2:83-100.
26. Franklin SE, Mayfield SP: Prospects for molecular farming in the
green alga Chlamydomonas. Curr Opin Plant Biol 2004,
7:159-165.
27. Decker EL, Reski R: The moss bioreactor. Curr Opin Plant Biol
2004, 7:166-170.
28. Gaume A, Komarnytsky S, Borisjuk N, Raskin I: Rhizosecretion of
recombinant proteins from plant hairy roots. Plant Cell Rep
2003, 21:1188-1193.
29. Gasdaska JR, Spencer D, Dickey L: Advantages of therapeutic
protein production in the aquatic plant Lemna. Bioprocessing J
2003, 2:49-56.
30. Horn ME, Woodard SL, Howard JA: Plant molecular farming:
systems and products. Plant Cell Rep 2004, 22:711-720.
31. Fischer R, Stoger E, Schillberg S, Christou P, Twyman RM:
Plant-based production of biopharmaceuticals. Curr Opin
Plant Biol 2004, 7:152-158.
32. Miller KD, Gao J, Hooker BS: Initial clarification by aqueous
 two-phase partitioning of leaf extracts from Solanum
tuberosum plants expressing recombinant therapeutic
proteins. Bioprocessing J 2004, 3:47-51.
A non-traditional application of aqueous two-phase partitioning technol-
ogy for removing chlorophyll and phenolics from leaf tissue extracts.
Chlorophyll and phenolics were extracted in the PEG phase and target
proteins remained in the aqueous phase.
33. Fernandez-San Millan A, Mingo-Castel A, Miller M, Daniell H:
A chloroplast transgenic approach to hyper-express and
purify human serum albumin, a protein highly susceptible
to proteolytic degradation. Plant Biotechnol J 2003, 1:71-79.
34. Tregoning JS, Nixon P, Kuroda H, Svab Z, Clare S, Bowe F,
Fairweather N, Ytterberg J, van Wijk KJ, Dougan G et al.:
Expression of tetanus toxin fragment C in tobacco
chloroplasts. Nucleic Acids Res 2003, 31:1174-1179.
35. Leelavathi L, Gupta N, Maiti S, Ghosh A, Reddy VS:
Overproduction of an alkali- and thermostable xylanase
in tobacco chloroplasts and efficient recovery of the
enzyme. Mol Breeding 2003, 11:59-67.
36. Huang N: High-level protein expression system uses
self-pollinating crops as hosts. BioProcess Int 2004, 2:54-59.
37. Whitelam GC: The production of recombinant proteins in
plants. J Sci Food Agric 1995, 68:1-9.
38. Mison D, Curling J: The industrial production costs of
recombinant therapeutic proteins expressed in transgenic
corn. BioPharm 2000, 13:48-54.
39. Evangelista RL, Kusnadi AR, Howard JA, Nikolov ZL: Process and
economic evaluation of the extraction and purification of
recombinant b-glucuronidase from transgenic corn.
Biotechnol Prog 1998, 14:607-614.
40. Kusnadi AR, Nikolov ZL, Howard JA: Production of recombinant
proteins in transgenic plants: practical considerations.
Biotechnol Bioeng 1997, 56:473-484.
41. Bai Y, Glatz CE: Capture of a recombinant protein from
 unclarified canola extract using streamline expanded bed
anion exchange. Biotechnol Bioeng 2003, 81:855-864.
A thorough analysis of key variables that affected expanded-bed recov-
ery of recombinant b-glucuronidase from transgenic canola extract
Current Opinion in Biotechnology 2004, 15:479–486
by anion-exchange chromatography. Packed-bed and expanded-bed
operation modes were compared. Experimental observations explained
by equilibrium and mass transfer correlations.
42. Valdes R, Reyes B, Alvarez T, Garcia J, Montero JA, Figueroa A,
 Gomez L, Padilla S, Geada D, Abrahantes MC et al.: Hepatitis B
surface antigen immunopurification using a plant-derived
specific antibody produced in large scale. Biochem Biophys
Res Commun 2003, 310:742-747.
This study describes the difficulties experienced in removing plant pig-
ments and phenolics from a monoclonal antibody pool after Protein A
expanded-bed adsorption. Attempts to remove the yellow color from a
purified antibody pool by ion exchange, gel filtration, ultrafiltration and
precipitation were unsuccessful. Protein A resin in expanded-bed mode
performed poorly when used without the removal of residual suspended
particles from centrifuged tobacco extract.
43. Streatfield SJ, Howard JA: Plant-based vaccines.
Int J Parasitol 2003, 33:479-493.
44. Johnson E: Edible plant vaccines. Nat Biotechnol 1996,
14:1532-1533.
45. Stein KE, Webber KO: The regulation of biologic products
derived from bioengineered plants. Curr Opin Biotechnol 2001,
12:308-311.
46. FDA: Guidance for industry. Drugs, biologics, and medical
devices derived from bioengineered plants for use in humans
and animals. URL: http://www.fda.gov/cber/gdlns/bioplant.pdf
September 2002.
47. Azzoni AR, Kusnadi AR, Miranda EA, Nikolov ZL: Recombinant
aprotinin produced in transgenic corn seed: extraction and
purification studies. Biotechnol Bioeng 2002, 80:268-276.
48. Woodard SL, Mayor JM, Bailey MR, Barker DK, Love RT,
Lane JR, Delaney DE, McComas-Wagner JM, Mallubhotla HD,
Hood EE et al.: Maize (Zea mays)-derived bovine trypsin:
characterization of the first large-scale, commercial
protein product from transgenic plants. Biotechnol
Appl Biochem 2003, 38:123-130.
49. De Wilde S, Peeters K, Jacobs A, Peck I, Depicker A: Expression
of antibodies and Fb fragments in transgenic potato plants: a
case study for bulk production in crop plants. Mol Breed 2002,
9:271-282.
50. Leelavathi L, Reddy VS: Chloroplast expression of His-tagged
GUS-fusions: a general strategy to overproduce and
overproduction of an alkali- and thermostable xylanase in
tobacco chloroplasts and efficient recovery of the enzyme.
Mol Breeding 2003, 11:59-67.
51. Morassutti C, De Amicis F, Skerlavaj B, Zanetti M, Marchetti S:
Production of a recombinant antimicrobial peptide in
transgenic plants using a modified VMA intein expression
system. FEBS Lett 2002, 519:141-146.
52. Desai UA, Sur G, Daunert S, Babbitt R, Li Q: Expression and
affinity purification of recombinant proteins from plants.
Protein Expr Purif 2002, 25:195-202.
53. Krishnan R, McDonald KA, Dandekar AM, Jackman AP, Falk B:
Expression of recombinant trichosanthin, a ribosome-
inactivating protein, in transgenic tobacco. J Biotechnol 2002,
97:69-88.
54. Menkhaus TJ, Pate C, Krech A, Glatz CE: Recombinant protein
purification from pea. Biotechnol Bioeng 2004, 86:108-114.
55. Nandi S, Suzuki YA, Huang J, Yalda D, Pham P, Wu L, Bartley G,
Huang N, Lonnerdal B: Expression of human lactoferrin in
transgenic rice grain for the application in infant formula.
Plant Sci 2002, 163:713-722.
56. Huang J, Nandi S, Wu L, Yalda D, Bartley G, Rodriguez R,
Lonnerdal B, Huang N: Expression of natural antimicrobial
lysozyme in rice grains. Mol Breed 2002, 10:83-94.
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