Case 1:18-cv-01363-CFC Document 79-7 Filed 03/22/19 Page 1 of 15 PageID #:
`9508
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`Advanced Drug Delivery Reviews 58 (2006) 671 – 685
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`www.elsevier.com/locate/addr
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`Antibody productionB
`
`John R. Birch *, Andrew J. Racher
`
`Lonza Biologics plc, 228 Bath Road, Slough, Berkshire, SL1 4DX, UK
`
`Received 16 November 2005; accepted 6 May 2006
`Available online 22 May 2006
`
`Abstract
`
`The clinical and commercial success of monoclonal antibodies has led to the need for very large-scale production in
`mammalian cell culture. This has resulted in rapid expansion of global manufacturing capacity [1], an increase in size of
`reactors (up to 20,000 L) and a greatly increased effort to improve process efficiency with concomitant manufacturing cost
`reduction. This has been particularly successful in the upstream part of the process where productivity of cell cultures has
`improved 100 fold in the last 15 years. This success has resulted from improvements in expression technology and from
`process optimisation, especially the development of fed-batch cultures. In addition to improving process/cost efficiencies, a
`second key area has been reducing the time taken to develop processes and produce the first material required for clinical
`testing and proof-of-principle. Cell line creation is often the slowest step in this stage of process development. This article
`will review the technologies currently used to make monoclonal antibodies with particular emphasis on mammalian cell
`culture. Likely future trends are also discussed.
`D 2006 Elsevier B.V. All rights reserved.
`
`Keywords: Fed-batch culture; CHO; NS0; Gene expression systems; Downstream processing; Fermentation; Cell line selection
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`Contents
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`1.
`Introduction .
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`2.
`Expression systems .
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`2.1.
`Expression vectors .
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`2.1.1. DHFR expression systems .
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`2.1.2. Glutamine Synthetase (GS) Expression System .
`Increasing specific production rate by improving transcription .
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`2.2.
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`B This review is part of the Advanced Drug Delivery Reviews theme issue on bEngineered Antibody TherapeuticsQ, Vol. 58/5–6, 2006.
`* Corresponding author. Tel.: +44 1753 716576; fax: +44 1753 716595.
`E-mail address: john.birch@lonza.com (J.R. Birch).
`
`0169-409X/$ - see front matter D 2006 Elsevier B.V. All rights reserved.
`doi:10.1016/j.addr.2005.12.006
`
`

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`3. Cell lines .
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`3.1. CHO .
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`3.2. Murine lymphoid cell lines.
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`3.3. Hybridomas .
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`3.4. Other cell lines .
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`Screening of cell lines .
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`4.
`Transient and other expression systems for production of development material.
`5.
`6. Cell engineering to increase productivity or modify product characteristics .
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`7. Reactor systems used for large-scale antibody production .
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`7.1.
`Steps in a fed-batch process .
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`7.2. Culture media .
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`7.3. Optimisation of culture conditions .
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`7.4.
`Impact of process improvements on product .
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`8. Alternative production systems .
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`9. Downstream processing of antibodies .
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`10. Conclusions.
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`References .
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`1. Introduction
`
`there are 18 monoclonal antibodies
`Currently,
`approved for therapeutic use [2]. The majority (15)
`of these antibodies are produced by recombinant
`DNA technology although three are murine anti-
`bodies made in hybridomas. The recombinant anti-
`bodies are produced in mammalian cell expression
`systems using Chinese hamster ovary (CHO) or
`murine lymphoid cell lines (e.g., NS0, Sp2/0-Ag14).
`Most products in clinical trial are whole antibodies
`made in mammalian cell systems but some are
`antibody fragments, which can be made in micro-
`organisms such as E. coli. For example, CIMZIAk
`[2] is a pegylated FabV fragment made in a microbial
`system and is currently in phase III trials. Reichert et
`al. [2] report that, of the 15 antibodies they identified
`in phase III trials, six were single chain or Fab
`fragments. In this article, we review the technologies
`used to manufacture antibodies focusing particularly
`on the current status of mammalian cell culture and
`approaches taken in process development. There are
`two crucial issues, which have to be faced in process
`development. The first is to minimise the time taken
`to provide material
`for clinical studies and the
`second is to develop a process which can deliver
`sufficient drug substance to meet market demands at
`an acceptable price per dose.
`The industry continues to look at new technologies
`and process development strategies that will reduce
`
`timelines. The resulting processes must be easily
`scaleable, robust and meet quality and safety criteria.
`One approach to shortening the timelines is the use of
`platform technologies for cell culture processes, for
`example using standard media, feeds and growth
`conditions. Cell
`line construction and selection is
`often a critical path activity and needs to be
`completed rapidly without compromising quality
`criteria. Ideally, one would like to rapidly create a
`highly productive cell
`line that could be used for
`long-term manufacture obviating the need to create an
`improved second generation cell line at a later stage
`of development.
`Productivity of mammalian cell processes has
`improved dramatically in recent years [3] and modern
`cell culture processes can achieve antibody concen-
`trations exceeding 5 g/L [4,5]. This has resulted from
`improvements in expression technology and from
`process optimisation, particularly of the upstream, cell
`culture stage. Most current processes are based on fed-
`batch culture and the development of
`feeds in
`particular has made a significant contribution to
`increased antibody yields. Highly productive cell
`lines result from using a host cell line that has the
`desired characteristics, an appropriate expression
`system, and a good transfection and selection proto-
`col. A number of expression systems with the
`potential
`to produce cell
`lines with high specific
`production rates ( Qp) are available. The challenge is
`to create cell lines that not only have high Qp but also
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`673
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`have the growth characteristics that will lead to high
`productivity in the manufacturing process. This can be
`achieved by selecting cell lines with the combination
`of a high Qp, an ability to achieve high space–time
`yields of viable biomass and consequent high volu-
`metric production rate in a screen designed to mimic
`the final production process. The resulting cell lines
`are also selected for stability, growth in suspension
`culture using a chemically defined, animal compo-
`nent-free (CDACF) medium and the ability to make
`the desired post-translational modifications.
`Purification strategies are based on chromatograph-
`ic procedures usually including a protein A affinity
`step. With increasing upstream concentrations, signif-
`icant attention is now being paid to improving
`downstream efficiency.
`
`2. Expression systems
`
`The ability of a cell line to achieve high volumetric
`productivities results from a combination of character-
`istics. Efficient transcription of the antibody genes is
`achieved by using an appropriately designed expres-
`sion vector. Secondly, one requires a cell line capable
`of efficiently translating antibody mRNAs, assembling
`and modifying the antibody at high rates with minimal
`accumulation of incorrectly processed polypeptides,
`and having sufficient secretory capacity for secreting
`the resulting assembled antibody. It is probable that
`with the current generation of cell lines, productivity is
`not limited by transcription. It has been reported that,
`for a panel of antibody-producing GS-NS0 cell lines
`with Qp values varying between 0.05 and 0.95 pg/(100
`pg cell protein h), there was no correlation between Qp
`and mRNA levels [6]. These data suggest that events
`downstream of transcription are the limiting factors.
`Thirdly, the cell line must be capable of achieving high
`viable cell concentration within an acceptable time. An
`additional criterion is that the cell line must produce
`antibody with the desired product quality character-
`istics, such as glycosylation.
`
`2.1. Expression vectors
`
`The expression vector systems most frequently
`used for the production of therapeutic monoclonal
`antibodies are the Glutamine Synthetase (GS) Gene
`
`Expression System (Lonza Biologics; [5]) and those
`based on dihydrofolate reductase (DHFR) genes. A
`selection of expression vectors developed for the
`expression of immunoglobulin genes is shown in
`Table 1. To achieve high levels of gene expression,
`GS and DHFR vectors usually have strong promoters
`to drive expression of
`the antibody genes. The
`promoters are typically of viral origin (e.g., human
`cytomegalovirus) or they are derived from genes that
`are highly expressed in a mammalian cell [10,11].
`Historically, the vectors have also included favourable
`RNA processing signals such as polyA tail, 5V and 3V
`untranslated region, presence of an intron to encour-
`age export from the nucleus and a splice site to
`remove this intron. Coding sequences may also be
`optimised to remove, for example, cryptic splice sites
`or cryptic polyA tails, or sequences that
`lead to
`unfavourable folding of
`the mRNA. To increase
`mRNA processing and improve secretion, codon
`usage can be optimised for the target cell type, GC
`content increased and signal sequences used to target
`the heavy and light chain polypeptides to the correct
`part of the secretory pathway.
`
`2.1.1. DHFR expression systems
`DHFR expression systems use the folate analogue
`methotrexate (MTX)
`to inhibit
`the function of
`DHFR, an essential metabolic enzyme. Transfection
`with an expression vector containing a DHFR gene
`prevents MTX poisoning of transfected cells. The
`antibiotic resistance gene frequently used in DHFR
`expression vectors acts as the selectable marker: the
`primary function of the DHFR gene is then to facilitate
`vector amplification. The DHFR gene is usually under
`the control of a weak promoter, such as one from
`SV40. The use of a weak promoter to regulate DHFR
`
`Table 1
`Expression vector systems for use with expression of immunoglo-
`bulin genes
`
`Expression vectors and selectable markers
`
`References
`
`GS vectors
`DHFR intron vector/hygromycin
`Neomycin
`DHFR bicistronic vector
`CHEF/DHFR
`DHFR/neomycin
`
`www.lonza.com, [5]
`[7]
`[8]
`[9]
`[10,11]
`[12]
`
`Where resistance to an antibiotic is used to select transfectants, the
`target antibiotic is shown.
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`gene expression should reduce promoter interference
`(due to read-through from the upstream promoter
`inhibiting expression from the downstream promoter)
`thus increasing expression of the immunoglobulin
`genes.
`
`2.1.2. Glutamine Synthetase (GS) Expression System
`GS synthesises glutamine from glutamate and
`ammonium. Since glutamine is an essential amino
`acid, transfection of cells that lack endogenous GS with
`the GS vector confers the ability to grow in glutamine-
`free media. GS expression vectors contain the GS gene
`downstream of a SV40 promoter, which offers similar
`benefits to those seen when a weak promoter is used to
`drive DHFR gene expression.
`
`2.2. Increasing specific production rate by improving
`transcription
`
`limiting antibody
`Transcription is probably not
`secretion in the current generation of cell lines. This is
`because they were constructed using expression
`vector systems developed to give high mRNA levels.
`Several options exist to increase transcription. In early
`expression systems,
`this was generally by gene
`amplification. Gene amplification is usually achieved
`by constructing the expression vector so that the genes
`of interest are linked to an amplifiable gene (e.g.,
`thymidine kinase, adenosine deaminase, GS or
`DHFR). Tansfected cells are then exposed to increas-
`ing levels of a specific enzyme inhibitor at concen-
`trations substantially higher
`than those used for
`selection of transfectants. If the drug inhibits an
`enzyme (e.g., GS or DHFR) essential for the survival
`of the cell, only cells that overproduce this enzyme
`will survive. The overproduction of
`the enzyme
`commonly results from increased levels of its partic-
`ular mRNA, resulting from either an increase in gene
`copy number
`(i.e., amplification), or
`from more
`efficient transcription [13]. Often more DNA (up to
`1000 kb) than just
`the target gene is amplified.
`Therefore, when the transfected genes are amplified,
`other tightly linked sequences, including the immu-
`noglobulin genes, on the vector are co-amplified. The
`high copy numbers of the expression vector seen upon
`amplification, especially with the DHFR expression
`it can also have a
`system, may increase Qp but
`detrimental effect on other cellular properties. Ampli-
`
`fication of the transgenes will frequently result in poor
`growth performance of the resulting cell population
`and may alter cellular metabolism. Amplification and
`the resulting variation in copy number can also alter
`the inherent stability of expression and often requires
`the continued presence of the selective agent. If the
`selective agent is required in the production bioreac-
`tor,
`it will be necessary to demonstrate that
`the
`purification process removes this compound from
`the bulk drug substance.
`The GS system [5] and some variants of the DHFR
`one [12] do not rely upon amplification to achieve high
`productivities. Instead, these systems rely upon inser-
`tion of the antibody construct into a transcriptionally
`active region to achieve high productivities, selecting
`against insertion into the heterochromatin.
`One approach is to use site specific recombination
`of the gene(s) of interest into a known transcription-
`ally active locus. Expression vectors can be con-
`structed that contain a specific targeting sequence that
`will direct
`the vector to integrate by homologous
`recombination into a particular active site. Such a
`sequence has been identified in the immunoglobulin
`locus of the murine myeloma cell
`line NS0 [14].
`Vectors containing this sequence are targeted to the
`immunoglobulin locus in more than 75% of high
`producing NS0 cell lines.
`A corollary of
`this approach is to take the
`sequences flanking the transcriptionally active locus
`and incorporate them into the expression vector.
`Thus, the vector should create a favourable environ-
`ment for expression independent of its integration
`site into the genome. Vectors incorporating ubiqui-
`tous chromatin opening elements [15], matrix attach-
`ment regions and anti-repressor sequences [16,17] or
`the flanking sequences of
`the Chinese hamster
`elongation factor-1a gene [10,11] have been shown
`to increase and maintain transgene expression and
`are being actively evaluated with immunoglobulin
`genes.
`An alternative approach is to transfect the cells
`with a conventional expression vector (i.e., randomly
`integrate the expression vector into the genome) but
`then bias the selection method so that only trans-
`fectants where the vector integrated into a transcrip-
`tionally active site are selected. This can be done by
`using a selection system that only allows transfectants
`producing sufficient levels of the selectable marker
`
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`gene product to proliferate. Expression systems using
`a selectable marker gene with either the weak SV40
`promoter [5] or an impaired Kozak sequence upstream
`of the marker gene [12] are included in this class of
`selection system. Linkage of the antibody construct to
`the selectable marker gene results in the over-
`production of antibody as both genes are integrated
`into a transcriptionally active locus. The choice of
`selection conditions is extremely important for the
`success of this approach [5].
`
`3. Cell lines
`
`The key issues affecting the choice of a cell line for
`use in a manufacturing process are: the capability to
`produce high antibody concentrations in the chosen
`production system, the ability to consistently produce
`a product of uniform characteristics, and the speed
`with which a high yielding cell line can be obtained.
`The availability of a suitable expression system and the
`importance of post-translational modifications of the
`recombinant antibodies may also affect this choice.
`There are 18 therapeutic antibodies currently
`licensed for use of which 10 are manufactured in
`Chinese hamster ovary (CHO) cell lines and 8 are
`made in murine lymphoid cells (including NS0 and
`Sp2/0-Ag 14). These parental cell lines are also the
`ones most commonly used for antibodies currently in
`clinical
`trials. In addition, murine hybridomas and
`other cell lines such as the human cell line PER.C6 are
`used.
`
`3.1. CHO
`
`CHO cells are widely used to produce recombinant
`antibodies using both the DHFR and GS expression
`systems. The most commonly used CHO strains with
`DHFR expression vectors are DUKX-B11 and DG44,
`which both lack dhfr. The GS system uses the CHO-
`K1 strain, or a derivative of the CHO-K1, CHOK1SV.
`Although both CHO-K1 and CHOK1SV [18] express
`functional GS enzyme, inclusion of the GS inhibitor
`methionine sulphoximine (MSX)
`in the medium
`allows use of the GS expression vectors. Endogenous
`GS in CHO cells is inhibited by 3 AM MSX, which is
`a cytotoxic concentration. By selecting GS-CHO
`transfectants in the presence of 50 AM MSX, only
`
`lines that have stably incorporated the
`those cell
`expression vector into a transcriptionally active locus
`will form transfectant colonies. These cells produce
`enough GS enzyme to titrate out the MSX whilst
`leaving sufficient functional enzyme to meet
`the
`cellular demand for glutamine. Recombinant CHO
`cell lines show efficient post-translational processing
`of complex proteins, while the glycosylation patterns
`of native and CHO-derived recombinant proteins are
`similar.
`The preferred culture format for large-scale (sub-
`stantially greater than 10 L) is single cell suspension,
`ideally using chemically defined, animal component-
`free (CDACF) media. Wild type CHO strains have
`adherent cell morphology and require serum supple-
`mentation for growth. Adaptation of recombinant
`CHO cell lines from adherent to suspension culture
`formats and adaptation to CDACF media can take up
`to 9 months, which is not compatible with short
`development timelines. The industry trend has been to
`pre-adapt the parental CHO cell line to suspension
`culture in CDACF media, reducing timelines by about
`6 months [18,19].
`
`3.2. Murine lymphoid cell lines
`
`The host cell lines NS0 and Sp2/0-Ag14 are widely
`used for antibody production. Both cell lines were
`derived from a plasmacytoma cell
`line originating
`from a BALB/c mouse. The starting cell
`line
`underwent numerous rounds of cloning, and in the
`case of Sp2/0-Ag14, fusion with spleen cells from
`another BALB/c mouse,
`to generate these two
`parental cell lines [20,21]. Both the cell lines lack
`the ability to synthesise and secrete immunoglobulin
`proteins. The parental cell type of the two cell lines is
`a differentiated B cell, which is inherently capable of
`high levels of immunoglobulin production. These two
`characteristics favour
`their use for manufacturing
`antibodies. The genotype of NS0 cells makes them
`particular suited for use with the GS expression
`system. Unlike other cell types, NS0 cells are obligate
`glutamine auxotrophs: glutamine independence can be
`conferred upon NS0 cells following transfection with
`a functional GS gene. In the case of Sp2/0-Ag14,
`mutants that no longer
`require glutamine occur
`spontaneously with relatively high frequency. This is
`not observed with NS0 cells [22].
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`3.3. Hybridomas
`
`In addition to murine antibodies, it is now possible
`to make human antibodies using murine hybridoma
`technology. However, unlike the original hybridomas
`[23],
`the spleen cells are taken from a transgenic
`mouse which has the murine immunoglobulin locus
`replaced by the human genes.
`
`3.4. Other cell lines
`
`line
`types of cell
`There are reports of other
`being used to produce monoclonal antibodies
`including the hamster
`line BHK21 [24] and the
`human PER.C6 cell line [8]. The PER.C6 cell line
`is derived from human embryonic retinal cells by
`transfection with the adenovirus E1 region followed
`by selection for
`transfectants with an immortal
`phenotype. Analysis of
`the glycosylation profiles
`IgG1 antibody revealed no high mannose or
`of
`hybrid structures; all were biantennary with core
`fucose. Galactosylation was similar to human serum
`IgG1.
`
`4. Screening of cell lines
`
`The function of the expression vectors described
`in the previous sections is to generate cell lines with
`high Qp values. Transfectants with high Qp are rare
`events and this is the reason to use expression
`technologies that provide stringent selection and/or
`an increased frequency of high producers. However,
`a transfectant with a high Qp does not necessarily
`result
`in a cell
`line that performs well
`in the
`production process. Hence, a sufficient number of
`cell
`lines need to be generated to allow for the
`attrition in numbers when screening for other desired
`characteristics.
`The issue is, therefore, how can the hit rate for
`finding highly productive cell lines be increased? The
`simplest approach is to screen more transfectants, but
`how many? Simulations run by one of us (AJR,
`unpublished) suggest that several thousand should be
`screened, even after being enriched with a stringent
`selection system, to be confident of getting multiple
`transfectants with the desired productivity character-
`istics [5].
`
`Conventional methods for the screening of cell
`lines are labour intensive, which limits the number of
`cell lines that can be screened. Increasingly robotics is
`being used to automate the liquid handling and cell
`transfer stages. This does not address the need to
`screen large numbers of
`transfectants to identify
`sufficient high producers to screen against
`the
`additional growth criteria that contribute to high
`productivity in a manufacturing process. The number
`can be reduced by using a fluorescence-activated cell
`sorting (FACS) technique to identify cells secreting
`high levels of antibody and sort them away from the
`lower producers. One such method, based on the
`capture of the secreted antibody by a capture matrix
`and its detection by a labelled probe, has been
`described by Holmes and Al-Rubeai [25]. The cells
`can be sorted into large populations (bbulk sortingQ),
`from which cell lines can be isolated by conventional
`cloning methods, or by single cell sorting using
`FACS. Strictly, these approaches enrich for cells with
`a high Qp since the secreted monoclonal antibody is
`captured close to the cell surface and secretion occurs
`over a short time period. Qp is not the only phenotypic
`characteristic contributing to productivity and cell
`lines need to be screened for other characteristics.
`Typically, several criteria are used to select
`the
`line including a high Qp, growth
`production cell
`characteristics such as the magnitude of the time
`integral of the viable cell concentration and maximum
`cell concentration, antibody concentration at harvest,
`cell line stability and product quality. An important
`feature of any screening scheme is that it incorporates
`a technique that
`is a predictive model of
`the
`manufacturing process. This screening step is often
`carried out in shake flask cultures, which may include
`the feeding techniques used in the final process.
`
`5. Transient and other expression systems for
`production of development material
`
`In order to produce quantities of material for
`process development studies, rapid expression tech-
`nologies are frequently used that allow the generation
`of milligrams to grams of material in advance of a
`stable manufacturing cell
`line becoming available.
`These methods include the use of uncloned trans-
`fectant pools and the application of transient expres-
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`sion technology. Large-scale (up to 100 L) transient
`expression systems are being developed to meet this
`demand [26].
`
`6. Cell engineering to increase productivity or
`modify product characteristics
`
`High monoclonal antibody concentrations are the
`result of high Qp values and space–time yield of viable
`cells. As discussed above, Qp is probably limited by
`events downstream of transcription, only some of
`which can be addressed by vector engineering. An
`alternative approach is to modify the translational or
`secretory pathways where antibody production is
`considered limited at folding and assembly reactions.
`Dinnis and James [27] recently reviewed ways of
`increasing Qp through cell line engineering. These
`authors proposed that, since foldases and chaperones
`exist as large multi-protein complexes, global expan-
`sion of all components of the secretory pathway is
`required for generic improvement of antibody secretion
`rather than over-expression of selected proteins. This
`would be similar to the events occurring during the
`differentiation of B cells into plasma cells, where the
`unfolded protein response (UPR), an important intra-
`cellular signalling pathway, is induced. Protein expres-
`sion in differentiating B cells is coordinated by
`components of the UPR to achieve maximum antibody
`production. It has been proposed that there may be
`benefit
`in over-expression of proteins known or
`suspected of having a key role in modulating signalling
`pathways, e.g., BLIMP-1, or initiation of ER expansion
`(XBP-1, ATF6) [27]. An alternative approach to over-
`expression of specific genes is to use randomised zinc
`finger protein-transcription factor (ZFP-TF) libraries
`[28]. Theoretically, the ZFP-TF libraries can modulate
`the expression of any gene, so that a specific phenotype
`can potentially be created without a detailed knowledge
`of the molecular basis of the phenotype.
`High space–time yields of viable biomass are
`achieved by using a cell line capable of growing to a
`very high viable cell concentration and then maintain-
`ing it for extended periods. The maintenance of high
`viability for such cultures requires minimisation of the
`death rate. The major cause of cell death in animal cell
`cultures is by apoptosis pathways. Since apoptosis can
`be induced by various chronic insults and is mediated
`
`by a number of pathways, numerous strategies have
`been developed to limit cell death [5,29]. Nutrient
`limitation can induce apoptosis, so one strategy for
`limiting apoptosis is to prevent nutrient limitation.
`Although operating the culture in fed-batch mode can
`delay the onset and reduce the extent of apoptosis, the
`cells will still eventually die by apoptosis. Alternative-
`ly, resistance to apoptosis can be engineered into the
`cell lines. As activation of the apoptotic pathways is
`lethal
`to the cell,
`the pathways must be tightly
`regulated. The best understood regulatory mechanism
`involves the Bcl-2 family of proteins. The anti-
`apoptotic properties of Bcl-2 family members have
`been used to protect industrially important cell lines
`from insults typically experienced during cell culture
`operations. However, the results are contradictory with
`respect
`to productivity and there are few reports
`describing the behaviour of cell lines engineered to
`have increased apoptosis resistance in modern antibody
`manufacturing processes.
`In addition to changing characteristics related to
`productivity, there are also examples where it has been
`advantageous to alter the cellTs ability to carry out
`particular post-translational steps such as glycosyla-
`tion. This has been driven by increasing awareness of
`the role of glycosylation in effector functions such as
`antibody-dependent cellular cytotoxicity (ADCC).
`ADCC is believed to play a role in the function of
`some therapeutic monoclonal antibodies, with various
`studies showing that oligosaccharide engineering may
`optimise ADCC. The degree of galactosylation and
`fucosylation and the proportion of bisecting GlcNAc
`residues have all been implicated in modulating
`effector functions. Oligosaccharide engineering has
`thus become an important research area for increasing
`antibody potency. Yamane-Ohnuki et al. [30] created a
`FUT8 double knockout of the CHO DG44 host that
`lacks a-1, 6-fucosyltransferase activity and cannot
`synthesise fucosylated antibodies. The ADCC of the
`resulting antibody was increased 100-fold compared
`to the fucosylated form. In a different approach, over-
`expression of N-acetylglucosaminyltransferase III in
`CHO increased the proportion of bisecting GlcNAc
`residues,
`increasing the ADCC substantially com-
`pared to the parental molecule [31,32].
`Increasingly, our ability to isolate useful variant
`cells or to engineer them will come from a better
`understanding of
`the biology which defines the
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`desired phenotype. The bomicsQ tools which have
`become available in recent years will play a major part
`in providing this knowledge base and we are already
`seeing examples of the power of these methods.
`Recently, Smales et al. [6] compared the proteomes of
`GS-NS0 cell lines with varying monoclonal antibody
`production rates and were able to demonstrate
`changes in abundance of several proteins associated
`with changes in productivity.
`
`7. Reactor systems used for large-scale antibody
`production
`
`A consequence of the rapidly growing demand for
`monoclonal antibodies has been a dramatic increase in
`capacity in the industry [1] and an increase in the scale
`of reactors used for production. Two types of culture
`system are used for large-scale manufacture, fed-batch
`and continuous perfusion culture [33]. The principles
`of these types of reactor are shown in Figs. 1 and 2.
`Fed-batch processes are by far the most common and
`are now operated at scales up to 20000 L working
`volume. Several authors [33,34] have reviewed the
`types of cell culture reactor systems and processes in
`industrial use.
`In fed-batch culture, small volumes (in our case less
`than 10% of the reactor volume) of key nutrients are fed
`to the culture during the fermentation process to
`
`Fig. 1. Schematic representation of batch and fed-batch culture
`systems. The fed-batch system is supplied with a concentrated
`nutrient solution: no spent culture medium is removed. In a batch
`system, no additions of nutrient solutions are made.
`
`