BIOT_0206 03.02.2006 11:41 Uhr Seite 164
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`Biotechnology
`Journal
`
`Review
`
`DOI 10.1002/biot.200500051
`
`Biotechnol. J. 2006, 1, 164–186
`
`Manufacturing of recombinant therapeutic proteins in
`microbial systems
`
`Klaus Graumann and Andreas Premstaller
`
`Novartis Biopharmaceutical Operations, Sandoz GmbH, Kundl, Austria
`
`Recombinant therapeutic proteins have gained enormous importance for clinical applications. The
`first recombinant products have been produced in E. coli more than 20 years ago. Although with
`the advent of antibody-based therapeutics mammalian expression systems have experienced a
`major boost, microbial expression systems continue to be widely used in industry. Their intrinsic
`advantages, such as rapid growth, high yields and ease of manipulation, make them the premier
`choice for expression of non-glycosylated peptides and proteins. Innovative product classes such
`as antibody fragments or alternative binding molecules will further expand the use of microbial
`systems. Even more, novel, engineered production hosts and integrated technology platforms
`hold enormous potential for future applications. This review summarizes current applications and
`trends for development, production and analytical characterization of recombinant therapeutic
`proteins in microbial systems.
`
`Received 21 December 2005
`Accepted 11 January 2006
`
`Keywords: Microbial expression systems · Fermentation · Downstream processing · In-process control · Product characterization
`
`1 Introduction
`
`Since the very early days of recombinant DNA technolo-
`gy, recombinant bacterial expression systems, above all
`Escherichia coli, have played a major role. The very first
`modern biopharmaceuticals such as recombinant human
`insulin or recombinant human growth hormone entered
`the market in the early 1980s and thereby dramatically im-
`proved the availability, quality and safety aspects regard-
`ing these products which were only made available by tis-
`sue extraction before. To date, a large number of protein
`
`Correspondance: Dr. Klaus Graumann, Novartis Biopharmaceutical
`Operations, Sandoz GmbH, Biochemiestrasse 10, 6250 Kundl, Austria
`E-mail: klaus.graumann@sandoz.com
`Fax: +43 (0) 5338 200 461
`
`Abbreviations: AEX, anion exchange chromatography; AOX, alcohol oxi-
`dase; C, carbon; CEX, cation ion exchange chromatography; DSP, down-
`stream processing; EBA, expanded bed adsorption; ER, endoplasmatic
`reticulum; FDA, Food and Drug Administration; HCP, host cell protein;
`HIC, hydrophobic interaction chromatography; IB, inclusion bodies; IMAC,
`immobilized metal ion affinity chromatography; IEX, ion exchange; PAT,
`process analytical tool; PEI, polyethyleneimine; RPC. reverse-phase chro-
`matography; scFv, single-chain variable fragment; SEC, size exclusion chro-
`matography; USP, upstream processing
`
`products have been developed and are being produced for
`the market using different microbial expression systems,
`and many more are currently under development.
`With the availability of several microbial genomes and
`their better understanding, enormous potential to engi-
`neer traditional hosts, to remove bottlenecks and open
`doors to new technical applications has been created. To-
`gether with the inherent benefits of microbial cells such
`as rapid growth, ease of manipulation and efficient ex-
`pression of target genes, engineered hosts and new prod-
`uct classes will further increase the value of microbial sys-
`tems for the biotech industry in the future. In this review,
`several aspects relevant for development and manufac-
`turing of biopharmaceuticals will be discussed and an
`overview of future trends will be given.
`
`1.1 Selection of microbial expression systems
`
`In the industry, the selection of the host cell will be strong-
`ly influenced by the type and use of the product, as well
`as economic or intellectual property issues. A number of
`new, sophisticated host-vector combinations have be-
`come available in recent years. However, their use in com-
`mercial applications lags behind. E. coli K12 derivatives
`are most widely used, often with protease-deficiencies
`
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`and other mutations. Recently, B strains, e.g. BL21,
`gained in popularity due to their efficiency in recombi-
`nant protein expression. More recent developments make
`the use of E. coli even more attractive due to product se-
`cretion to the extracellular space (for references see be-
`low). Like eukaryotes, yeasts perform posttranslational
`modifications and possess efficient secretion machiner-
`ies. Although Pichia pastoris and Saccharomyces cere-
`visiae are the most widely used strains, other yeasts with
`attractive properties for heterologous protein production
`have been developed (for references see below).
`
`2 E. coli expression systems and pathways
`
`Different E. coli B and K12 strains are the main work hors-
`es for expressing non-glycosylated peptides and proteins
`at research level and in the industry. E. coli is genetically
`well characterized and efficient expression of recombi-
`nant product to more than 50% of total cell mass has been
`reported [6–9]. The selection and design of the expression
`plasmids influence synthesis rates, plasmid copy number,
`the segregational plasmid stability and therefore produc-
`tivity and regulatory issues. Recently, also chromosomal
`
`Table 1. Overviewa) of microbially derived peptides and proteins for therapeutic use. Many of these molecules exist in different formulations and/or as dif-
`ferent analogs
`
`Moleculeb)
`
`Produced in
`
`Remarks
`
`Tissue Plasminogen activator
`(r-tPA) mutant
`r Hirudin
`rh Insulin and analogs
`rh Insulin and analogs
`r Human Growth Hormone (rhGH)
`
`Pegylated rhGH analogue
`h Parathyroid Hormone
`(rPTH and analogs)
`r salmon Calcitonin
`rh Glucagon
`rh G-CSF, pegylated rh G-CSF
`rh GM-CSF
`(Pegylated) rh Interferon α−2a
`or Interferon α−2bPlough
`Interferon alfacon-1
`r Interferon β-1b
`r Interferon γ-1b
`
`rh IL-1receptor antagonist
`r IL-2
`r IL-2-diphtheria toxin fusion
`r IL-11
`r HBsAg
`r OspA
`r Urate oxidase
`rh B-type natriuretic peptide
`rh TNFα
`rh PDGF
`r Pertussis toxin
`r Cholera toxin B subunit
`Asparaginase
`
`E. coli
`
`Inclusion body, affinity purification
`
`S. cerevisiae
`S. cerevisiae
`E. coli
`E. coli
`
`E. coli
`E. coli
`
`E. coli
`S. cerevisiae
`E. coli
`S. cerevisiae
`E. coli
`
`E. coli
`E. coli
`E. coli
`
`E. coli
`E. coli
`E. coli
`E. coli
`S. cerevisiae
`E. coli
`S. cerevisiae
`E. coli
`E. coli
`S. cerevisiae
`E. coli
`E. coli
`E. coli
`
`Secretion
`Secretion
`Inclusion body
`Inclusion body or periplamic
`
`Random pegylation
`Inclusion body
`
`Secretion, in vitro amidation
`
`Inclusion body, N-terminal pegylation
`Secretion, glycosylated
`Random pegylation
`
`Inclusion bodies
`Inclusion bodies
`Inclusion bodies
`
`Stand alone or combination vaccine
`Lipoprotein
`Secretion
`Inclusion bodies
`
`Part of combination vaccine
`Part of combination vaccine
`
`Companies
`
`Roche
`
`Aventis, Novartis
`Novo Nordisk
`Eli Lilly, Aventis
`Genentech, Eli Lilly, Pfizer,
`Schwartz Pharma, Novo Nordisk,
`and others
`Pfizer
`Eli Lilly
`
`Unigene
`Novo Nordisk
`Amgen
`Berlex / Schering AG
`Hoffmann-LaRoche, Schering
`
`Valeant
`Schering AG, Chiron
`Genentec
`Intermune
`Amgen
`Chiron
`Seragen / Ligand
`Genetics Institute
`GlaxoSmithKline, Aventis, Merck
`SmithKline Beecham
`Sanofi-Synthelabo
`Scios/Johnson & Johnson
`Boehringer Ingelheim
`Ortho-McNeil, Janssen-Cilag
`Chiron
`SBL Vaccine
`Merck
`
`a) [1–5]
`b) r, recombinant; h, human; rh, recombinant human; rhG-CSF, recombinant human granulocyte-colony stimulating factor; rHBsAg, recombinant Hepatitis B antigen;
`IL, interleukin; TNFα, tumor necrosis factor alpha; rPDGF, recombinant platelet-derived growth factor
`
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`integration of expression cassettes has been proposed for
`E. coli [10], which trade off segregational plasmid stabili-
`ty issues with potentially lower productivity. In general,
`constitutive and inducible systems have been described
`for E. coli while the latter dominate. The yield and quality
`of the heterologous product mainly depends on gene
`dosage, the efficiency of transcription, mRNA stability
`and the efficiency, as well as fidelity of translation (see
`[6–9, 11] for review).
`Choice and design of the plasmid play an essential role
`in balancing production yield and metabolic burden [12,
`13]. For industrial applications, selective pressure by an-
`tibiotics is mainly maintained in pre-cultures, main cul-
`tures are usually grown without selective pressure.
`The ideal expression vector combines medium to high
`copy numbers with tight regulation of gene expression to
`achieve rapid cell growth to high densities before the in-
`duction phase [9, 11]. Furthermore, promoter strength
`should be tunable and low priced non-toxic inducers
`should be available for large scale production. Common
`promoters are lac, tac, trc for induction with lactose or the
`lactose analog IPTG, [14, 15], araBAD under the control of
`L-arabinose or λpR/pL for induction by temperature shifts
`[16–20]. For expression of protein complexes or multimer-
`ic proteins such as antibodies and fragments thereof,
`polycistronic or two-cistronic vectors have been devel-
`oped. To balance intrinsic differences in translation, the
`translational initiation regions (TIRs) and thereby mRNA
`affinity for ribosomes may be adapted [21].
`A common problem with large scale protein produc-
`tion is the metabolic overload and stress, since plasmid
`maintenance and heterologous protein expression bur-
`dens upon the host cell. Especially in high cell density
`fermentations, mixing problems and resulting nutrient,
`pO2 and pCO2 gradients may even enhance cellular
`stress and favor culture heterogeneity. Growth and ener-
`gy status of the host cells may be compromised and ex-
`pression yields suboptimal. The development and design
`of the fermentation process and the fermenter itself play
`a key role for achieving productivity and robustness at
`scale. Glucose feed and dissolved oxygen concentrations
`determine the formation of acetate which may be toxic
`for the cells [22]. Apart from host/vector specific factors,
`plasmid retention may be dependent on fermentation pa-
`
`rameters and nutritional status [23–25]. Besides vector
`and fermentation process design, several additional
`strategies to enhance yields have been developed. E. coli
`strains usually carry mutations in or deletions of several
`proteases, to reduce product proteolysis [26, 27]. At the
`promoter level, several strategies for tuning the tran-
`scription machinery have been described [28]. T7 RNA
`polymerase-based systems have the advantage of com-
`plete repression in the non-induced state. However, the
`strength of the promoter and the efficiency of the T7 poly-
`merase overload the cellular machinery quickly. Con-
`trolled inductor feeding may help controlling the amount
`of derepressed promoters and thereby the activity of the
`RNA polymerase [15].
`Stabilization of mRNA transcripts represents another
`level of intervention, by altering mRNA conformation or
`mutations of nucleases (see [11] for review). To overcome
`limitations in translation, codon optimization is the stan-
`dard method employed. Additionally, overexpression of
`rare tRNAs has been proposed [29–31] and respective
`host/vector systems are commercially available. If rate
`limiting, the availability of tRNAs may influence the
`speed of translation, folding and thereby the intracellular
`solubility of a heterologous protein.
`
`2.1 Cytoplasmic expression
`
`Many mammalian proteins expressed in E. coli tend to ag-
`gregate and form inclusion bodies (IB). IB formation is
`mainly due to limited solubility and the lack of appropri-
`ate chaperone systems in the prokaryotic host [32–34].
`Many industrial processes make use of these protein ag-
`gregates although this strategy requires solubilization
`and renaturation of the product. Examples of products de-
`rived from IB and currently in development or on the mar-
`ket are interferons, interleukins, growth hormones or an-
`tibody fragments (see Table 1). The widespread use of IB
`technology can be explained by several advantages pro-
`vided. Besides high expression yields, IBs can easily be
`isolated by host cell disruption and continuous centrifu-
`gation; both unit operations are well-established at large
`scale and ensure high volumetric throughput. Soluble
`host cell components such as proteins and nucleic acids
`as well as cell wall debris can be efficiently removed by
`
`Table 2. Pathways for E. coli expression: advantages, disadvantages and improvements
`
`Expression route
`
`Advantages
`
`cytoplasmic
`
`periplasmic
`
`secretory
`
`Inclusion bodies, removal of most
`contaminants
`Disulfide bridging, natural secretion
`signals
`Easy product harvest, pure product
`in the supernatant, reasonably
`efficient for peptides
`
`Limitations
`
`Refolding necessary
`
`Empirical, sometimes inefficient
`translocation
`Secretion machinery not fully
`understood, inefficient for large)
`(proteins
`
`Improvements
`
`Integrated fusion protein
`technologies [237]
`Co-expression of chaperones,
`tuning factors
`Co-expression of tuning factors
`
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`differential separation (combined wash and centrifuga-
`tion step). Therefore, IB paste or suspension contains rea-
`sonably pure protein aggregates, which can be stored
`frozen for at least several months, thus providing flexibil-
`ity for production plants due to uncoupling fermentation
`and purification processes.
`Whether a product remains soluble or aggregates in
`the cytoplasm depends on intrinsic properties of the pep-
`tide or protein sequence, but also on promoter strength
`and fermentation parameters such as temperature and
`growth rate (for review see [35]). Choosing an appropriate
`fusion partner may influence the solubility and proteolyt-
`ic stability. The fusion protein or peptide tag may also be
`useful for isolation and purification (see below).
`Soluble expression strategies in the bacterial cyto-
`plasm have disadvantages like increased proteolytic
`degradation, N-terminal methionylation and more labori-
`ous product isolation and purification out of a highly com-
`plex mixture of host cell components. On the other hand,
`soluble, properly folded protein can be obtained by adapt-
`ing process conditions [36] or overexpressing chaperones
`[37]. Furthermore, strains mutated in redox system-relat-
`ed genes provide the potential for disulfide bridging in the
`cytoplasm [38].
`
`2.2 Authentic amino termini
`
`Intracellular expression in E. coli frequently yields me-
`thionylated polypeptides. In addition, acylation or acety-
`lation of the N-terminal methionine residue may further
`contribute to the pool of product-related impurities. Only
`if the second residue is small, uncharged and non-desta-
`bilizing, endogenous deacylase and methionyl aminopep-
`tidases will co-translationally cleave off the Nα acyl group
`and the N-terminal methionine, respectively [39–42]. If
`authentic amino acid sequences are desired from E. coli
`expression, several strategies may be followed: i) genetic
`fusion to signal sequences directing the product to the
`periplasmic or extracellular space, cleavage of the signal
`peptide during translocation yields authentic amino ter-
`mini; ii) fusion of the target sequence to a partner mole-
`cule which is chemically or enzymatically cleaved off dur-
`ing isolation and purification; iii) co-expression of
`aminopeptidases which cleave off the N-terminal methio-
`nine residue in vivo [43].
`
`2.3 Secretion
`
`To express correctly folded protein within E. coli, secre-
`tion to the periplasmic space represents an efficient al-
`ternative in many cases. The efficient expression and se-
`cretion of cytokines [44], of whole antibodies [21] or anti-
`body fragments [45–49] has been described recently. The
`detailed study of bacterial translocation pathways pro-
`vides several targets for optimization. One strategy de-
`scribed by several authors is the co-expression of tuning
`
`factors or chaperones to improve folding in the cytoplasm
`or periplasm [50–55].
`Protein secretion by E. coli into the extracellular space
`has been described by several authors [6, 56, 57]. Ray et
`al. [58] developed an efficient production process for
`glycine-extended recombinant salmon calcitonin. They
`co-expressed secretion
`factors to enhance peptide
`translocation efficiency. Fernandez and De Lorenzo [59]
`successfully secreted a fully oxidized single-chain vari-
`able fragment (scFv) to the extra-cellular space via the
`type I secretion pathway. Increasing knowledge on bac-
`terial transport mechanisms [60, 61] formed the basis for
`these developments and may hold additional potential for
`improvements. Humphreys et al. [62] performed a sys-
`tematic study on signal sequences from different species.
`To enhance the efficiency of protein folding in the bac-
`terial periplasm, the co-expression of chaperones and ox-
`idoreductases has been proposed [63, 64].
`
`2.4 Yeast expression systems
`
`As eukaryotes, yeasts offer several advantages over
`prokaryotic expression hosts. While yeast cultures usual-
`ly also grow fast and hold the potential to express heterol-
`ogous proteins efficiently, they perform posttranslational
`modifications while secreting proteins to the culture
`medium. A major advantage of efficient secretion is the
`easier isolation and purification of the product. On its way
`through the endoplasmatic reticulum (ER) and Golgi ap-
`paratus, N- and O-linked glycan structures may be at-
`tached to the polypeptide and processed [65]. However,
`these glycans may be introduced at positions that are
`non-glycosylated in the natural host and their composi-
`tions may differ significantly from human glycosylation
`patterns mainly through their content in mannose
`residues. The yeast Golgi apparatus does not perform
`trimming reactions of N-glycans like the mammalian Gol-
`gi. Instead long mannose chains (hyperglycosylation) may
`be added to the core glycan thus contributing to the het-
`erogeneity of secreted heterologous products, altering
`their properties and raising concerns about their potential
`immunogenicity [66]. On the other hand, due to their abil-
`ity to perform post-translational modifications, yeasts are
`very attractive hosts and several products in development
`or already on the market are being expressed in different
`yeast systems (see Table 3).
`A number of plasmid vectors have been developed for
`yeasts, where selection with auxotrophic markers, elimi-
`nating the need for antibiotics, is most popular [67]. Most-
`ly, these vectors are designed as E. coli shuttle vectors to
`ease manipulation and handling.
`Saccharomyces cerevisiae has been used in food
`biotechnology for ages and the wealth of physiological
`and genetic knowledge expanded its use to modern
`biotechnology. Being a workhorse for biochemists, it is
`also an important organism for industrial applications.
`
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`Table 3. Examples of proteins and protein complexes expressed success-
`fully in different yeasts
`
`Moleculea)
`
`Yeast strain
`
`Remarks
`
`r Glucagon
`Urate oxidase
`rPDGF
`CB2 cannabinoid receptor
`Insulin precursor
`
`Dengue virus type 2
`envelope protein
`hIL-1b, hG-CSF, hGH
`Erythrocyte binding
`antigen (EBA-F2)
`h caseinomacropeptide
`
`Hirudin
`Interferon α-2b
`Hepatitis B vaccine
`Hepatitis B vaccine
`Hirudin
`
`S. cerevisiae
`S. cerevisiae
`S. cerevisiae
`P. pastoris
`S. cerevisiae
`
`P. pastoris
`
`S. cerevisiae
`P. pastoris
`
`S. cerevisiae,
`P. pastoris
`S. cerevisiae
`H. polymorpha
`S. cerevisiae
`H. polymorpha
`H. polymorpha
`
`see Table 1
`see Table 1
`see Table 1
`[291]
`e.g. [78],
`see Table 1
`[292]
`
`[293,294]
`[295]
`
`[76]
`
`[75]
`[77]
`see Table 1
`[81]
`[296], Table 1
`
`a) rPDGF, recombinant platelet-derived growth factor; hIL-1b, human Inter-
`leukin 1b; hG-CSF, human granulocyte-colony stimulating factor;
`hGH, human growth hormone
`
`Several strategies have been developed to optimize cell
`yield while keeping ethanol levels low [68–70]. Due to its
`limited respiratory capacity [71], glucose feed has to be
`tightly controlled. Frequently, complex nitrogen sources
`lacking certain amino acids are being used and inducible
`promoters (e.g. gal) are being preferred over constitutive
`ones. Galactose has been widely employed as an inducer.
`Due to its excellent growth efficiency and high prod-
`uct titers, Pichia pastoris has gained popularity in recent
`years (see [66] for review). Other than for S. cerevisiae, P.
`pastoris expression vectors are integrated into the
`genome, sometimes in multiple copies, thus eliminating
`potential plasmid retention issues. Pichia can be grown to
`extremely high cell densities since it strongly favors con-
`version into cell mass (respirative) over ethanol produc-
`tion (fermentative). The medium is usually fully defined
`using glycerol or methanol as carbon (C) and ammonium
`salts as nitrogen (N) sources, respectively. Frequently,
`methanol is used to induce the highly efficient alcohol ox-
`idase (AOX1) promoter. If needed, explosion proof facili-
`ties may increase investment cost significantly. Several
`alternative promoters are available, some of which are
`constitutive and eliminate the need for methanol induc-
`tion [72]. Also host strains with mutations regarding
`methanol utilization (AOX genes) have been created,
`which may benefit heterologous expression and reduce
`
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`
`the need for methanol feed [67]. Other approaches to re-
`duce methanol handling are mixed feeding during induc-
`tion [73] or optimized feeding profiles [74].
`Intracellular and extracellular expression of target pro-
`teins has been described for S. cerevisiae [75, 76] and P.
`pastoris [67]. Dependent on the product, efficient secre-
`tion may be achieved using yeast or product-specific se-
`cretion signals (e.g. α-mating factor, acid phosphatase,
`invertase). Problems with correct processing of the leader
`sequence have been described for different yeasts [67, 77]
`and the use of synthetic leader sequences to improve se-
`cretion have been reported [78]. Additionally, proteolytic
`degradation of secreted products may be influenced by
`fermentation conditions [79]. Protease deficient strains
`may improve the yield of correctly processed product [80],
`often with some drawbacks regarding growth rate and vi-
`ability [67].
`
`2.5 Alternative microbial expression systems
`
`Hansenula polymorpha has been used for development
`and manufacturing of several biopharmaceuticals [77–82]
`(see also Table 3). Being a facultative methylotroph like
`Pichia, it may be cultivated using methanol, glycerol or
`glucose as carbon sources. The expression plasmids are
`usually integrated into the host genome. Several cy-
`tokines, vaccines and coagulation factors have been suc-
`cessfully expressed in this host and have entered the mar-
`ket.
`Kjaerulff and Jensen [83] expressed GFP using differ-
`ent secretion signals in Schizosaccharomyces pombe and
`propose this host for heterologous protein expression
`since compared with Saccharomyces species, glycosyla-
`tion and other post-translational modifications are more
`similar to mammalian cells.
`A number of vectors, genetic markers, transformation
`methods, etc. for alternative yeasts have been reviewed
`by Wang et al. [84]. There are about 800 different yeast
`species with varying metabolic features [85] and we sus-
`pect that there will be substantial progress regarding the
`use of yeast strains for biotechnological purposes in the
`future. Most notably, yeast engineering approaches are
`underway to achieve human-like glycosylation in yeast
`[86–88].
`Also, alternative bacterial hosts are being investigat-
`ed for their potential in recombinant processes [89–95].
`Recently, Zhang et al. [96] developed an E. coli expression
`system that could make proteins with designed, homog-
`enous glycosylation accessible at large scale [97].
`Filamentous fungi [98] such as Aspergillus niger have
`been used for production of enzymes. Recently, also the
`successful expression of whole antibodies has been de-
`scribed [99].
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`2.6 Fermentation processes
`
`In the industry, recombinant microorganisms are usually
`cultivated at a several cubic meter scale and fed-batch op-
`erations have made high cell density fermentations possi-
`ble, thus increasing the productivity of bioprocesses sig-
`nificantly. A robust and efficient aerobic fermentation
`process requires intense process development and tight
`control of process parameters such as carbon source con-
`centration, temperature, pH and pO2 [100]. Frequently glu-
`cose or glycerol serve as carbon sources, while complex ni-
`trogen sources such as yeast extract, plant derived hy-
`drolysates or peptones are usually used. Complex media
`components may be critical since they provide a source for
`process variability [101]. Methylotrophic yeasts such as
`Pichia are cultivated on fully defined media and protein ex-
`pression is often induced with methanol. In general nutri-
`ent feeds, pH and pO2 levels have to be tightly controlled
`to optimize conditions for cell growth before and product
`formation during induction [102]. Limiting the availability
`of the C source controls cell growth and concomitant het-
`erologous gene expression. In many cases, segregational
`plasmid stability is also higher under limiting conditions
`[103, 104]. Metabolic and engineering approaches have
`been proposed to overcome the overload of the cellular ma-
`chinery and prolong production phases. Inductor feed con-
`trol represents a strategy to limit heterologous protein ex-
`pression and to extend the production period of a culture
`[15, 105, 106]. Also, alterations of the promoter itself have
`been described [107]. Besides heterologous protein ex-
`pression, also environmental factors in high cell density
`fermentations contribute to stress reactions [108, 109].
`Norleucine incorporation instead of methionine has been
`reported as a massive problem for IL-2 expression [110,
`111], feeding strategies have been developed to keep this
`misincorporation under control [112].
`A number of chemometric approaches using different
`on-line monitoring tools have been proposed to gain more
`insight into cellular processes during process develop-
`ment or production. Acquisition of fluorescence spectra
`combined with modeling of parameters of interest
`[113–115] may represent a valuable tool for fermentation
`development and optimization. Reischer et al. [116] re-
`ported the development of a monitoring host cell system
`for analyzing stress induced by heterologous protein ex-
`pression.
`Transcriptional and translational analysis during re-
`combinant fed-batch fermentations reveal overexpres-
`sion of stress genes (chaperones) and down regulation of
`ribosomal proteins [117–120]. The use of real-time PCR
`and/or microarray technology to assess alterations in
`gene expression patterns with fermentation parameter
`changes significantly expands the analytical toolbox
`available [121–123].
`The complexity of fermentation processes is further
`demonstrated by reports on metabolic oscillations in E.
`
`coli fermentations at 10 L and 1000 L scale [124]. The au-
`thors have linked this specific case to the amino acid me-
`tabolism of a W3110 strain. Holistic systems biology ap-
`proaches using bioinformatics tools to combine data sets
`derived from different analytical methods will certainly
`aid in understanding better the complexity of bioprocess-
`es. Furthermore, this knowledge should lead to targeted
`host/vector engineering and better bioprocess control
`and, ultimately, performance.
`
`3 Downstream processing (DSP)
`
`3.1 Primary separation
`
`After harvest of fermentation broth or cell culture super-
`natant, cells have to be disrupted, extracted or simply re-
`moved as a first step of product isolation. At industrial
`scale, disruption of bacterial or yeast cells is usually per-
`formed by high-pressure homogenization. Continuous
`centrifugation and/or filtration methods efficiently sepa-
`rate solids from liquid. In case of product aggregation in
`form of inclusion bodies, differential separation combin-
`ing concentration and inclusion body wash represents a
`highly effective first separation method [125]. In case a
`soluble product has to be isolated from the cytosol, meas-
`ures have to be taken against proteolysis in the cell ho-
`mogenate. Protease inhibitors are rarely used for biophar-
`maceutical processes due to toxicity concerns and cost.
`Frequently, lowering the temperature, shortening hold
`times to a minimum or addition of complexing agents,
`e.g. EDTA, are methods of choice at large scale. If prod-
`ucts are expressed in the bacterial periplasm, osmotic
`shock provides an elegant method for extraction while
`keeping cells largely intact [126]. Therefore, the clear ex-
`tract contains by far less host cell contaminants compared
`to cleared cell homogenate. As an alternative to cell ho-
`mogenization, extraction of intracellular proteins from
`whole cells has been reported [127]. For clarification and
`protein purification purposes, a wide variety of filters for
`protein processing is meanwhile available. Combinations
`of different types and grades or charged surfaces often en-
`hance throughput per square meter of filter area. Intact
`cell removal to gain clear supernatant is either performed
`by centrifugation or microfiltration. For processing large
`volumes, combinations of continuous centrifugation and
`filtration are usually more economical and therefore well
`established in large scale processing.
`To remove excess nucleic acids from cell ho-
`mogenates or periplasmic extracts, precipitation with
`polycationic agents such as polyethyleneimine (PEI) or
`other agents [128, 129] may be performed before captur-
`ing the product on chromatographic resins. However, the
`capacity of the further DSP sequence to quantitatively re-
`move PEI from the therapeutic protein has to be demon-
`strated. Also, precipitation by addition of salts or acidic
`
`© 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
`
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`Journal
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`Biotechnol. J. 2006, 1, 164–186
`
`pH may be efficient ways to significantly decrease the
`load of contaminants such as host cell proteins into the
`purification sequence. Recently, suitable materials for
`affinity precipitation of therapeutic protein have been de-
`veloped [130]. By combining the virtues of selective bind-
`ing and precipitation, their application could open the
`door to highly efficient purification steps.
`Most frequently, product capture from crude or clari-
`fied load material is performed using ion exchange chro-
`matography methods. Ion exchange resins combine rea-
`sonable capacity and selectivity, binding and elution con-
`ditions are easily adapted by varying buffer salt, conduc-
`tivity and pH [131]. Hydrophobic interaction (HIC) resins
`may also be useful for product capture (e.g. [132]), espe-
`cially in cases where the starting material contains high
`salt concentrations. Affinity [133] and pseudo-affinity
`methods [134, 135] represent another class of resins wide-
`ly employed for capture purposes. Despite their high cost,
`Protein A resins currently represent the most frequently
`used tools for monoclonal antibody purification [136]. Sev-
`eral Protein A resins are available commercially, they
`combine excellent purification factors, due to selective
`IgG binding, with good capacities. Despite ligand leakage
`and limited tolerance towards caustic agents, repeated
`use of Protein A resins for way above 200 cycles has been
`reported [137, 138]. For several enzymes, serum proteins
`or cytokines, dye affinity chromatography is used at in-
`dustrial scale (see [139] for review). Cibacron blue or Pro-
`cion red are two examples of synthetic dyes used, and
`products with different base matrices are available. Met-
`al chelate or immobilized metal ion affinity chromatogra-
`phy (IMAC) [135] is widely used at lab scale for purifica-
`tion of histidine-tagged proteins. For industrial process
`development, only a few applications have been de-
`scribed which make use of the excellent selectivity based
`on histidine tags [140] or intrinsic histidine residues [135].
`Heavy metal leaching and contamination of the thera-
`peutic product are of concern; their use is usually con-
`fined to Cu++ or Zn++ ions. The removal of heavy metal
`ions from the prod