Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reexamination Certificate
1998-11-16
2002-06-25
Guzu, David (Department: 1636)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S069100, C435S320100, C435S252330, C435S252300, C436S548000, C536S023100, C536S023530
Reexamination Certificate
active
06410270
ABSTRACT:
The invention relates to a fed-batch fermentation process which uses special
E. coli
host/vector systems for the efficient formation of recombinant proteins, in particular recombinant antibody molecules, in particular antibody fragments such as miniantibodies.
Under the conditions according to the invention, the
E. coli
cells can grow up to very high cell densities at a maximum specific growth rate. After recombinant formation of the product has been switched on, it is only the formed product which has a limiting effect on growth; substrates or metabolic by-products do not limit growth. In this way, and in conjunction with the novel expression vectors which are specially adapted for this purpose and which exhibit high stability, it is possible to achieve high space-time yields of recombinant proteins, which proteins exhibit high biological activity, in particular in the case of antibody fragments.
The culture of
E. coli
cells to high cell densities is an essential prerequisite for efficient recombinant protein formation. The following cultures are state of the art for this purpose: following unlimited growth (&mgr;=&mgr;
max
) in the batch phase, a carbon source (glucose or glycerol) is customarily metered in, in the subsequent fed-batch phase, in a limited manner such that the formation of growth-inhibiting by-products, for example acetate, is avoided, with the consequence that the growth can be continued in a manner which is only substrate-limited (&mgr;<&mgr;
max
) until high cell densities are reached (e.g. Riesenberg et al., 1991, J. Biotechnol., vol. 20, 17-28; Strandberg et al., 1994, FEMS Microbiol. Rev., vol. 14, 53-56; Korz et al., 1995, J. Biotechnol. 39, 59-65; EP-B-0 511 226). Growth at a reduced growth rate naturally results in long fermentation times and consequently also in lower space-time yields. Owing to the immediate consumption, the concentration of the carbon source in the culture solution in these fermentations is virtually zero. The substrate-limited conditions are not altered after the recombinant product formation has been switched on.
Fed-batch cultures which use
E. coli
are also known in which the carbon source is added discontinuously at relatively large time intervals and then in relatively large quantities, with a rise in the pO
2
value usually being used as an indicator of substrate exhaustion for the purpose of initiating the subsequent dose of the carbon source (e.g. Eppstein et al., 1989, Biotechnol. 7, 1178-1181). This procedure means frequent switching from relatively long-term substrate excess conditions to substrate limiting conditions and consequently implies metabolic imbalances.
In that which follows, fed-batch cultures are dealt with in which the cells can grow at maximum specific growth rate (&mgr;=&mgr;max) in the fed-batch phase. Fed-batch cultures in which relatively large quantities of carbon source are added to the culture at relatively large time intervals, in accordance with off-line determinations, for the purpose of avoiding substrate limitations are experimentally elaborate and suffer from the disadvantage that the concentration of the carbon source is constantly changing during the whole of the fermentation (e.g. Pack et al., 1993, Biotechnol., vol. 11, 1271-1277, Hahm et al., 1994, Appl. Microbiol. Biotechnol. 42, 100-107).
Fed-batch cultures have also been described in which the concentration of the carbon source is measured on-line and is regulated so that limitations are avoided, although these cultures— in particular in the high cell density region— suffer from the disadvantages which are described below. An autoclavable glucose biosensor for use of [sic] microbial fermentations in stirred tank fermenters has recently been described (M. R. Phelps et al., 1995, Biotechnol. Bioeng., vol. 46, 514-524). It was employed for
E. coli
cultures. This in-situ sensor provides, with a time delay of approximately 2 minutes, the current value in the culture solution. The signal supplied by the glucose sensor is dependent, inter alia, on the pH and pO
2.
The sensor has not been tested in the high cell density region (X>80 g/l). It is known from experience that growths on in-situ probes when
E. coli
is used can lead to additional erroneous values at very high cell densities. In addition, it is not possible to recalibrate the sensor exactly during an ongoing fermentation. Instead of being based on measurements using an in-situ sensor, other processes are based, for example, on determining the carbon sources using on-line flow injection analysers (FIA) or on-line HPLC in a culture solution which is removed semi-continuously from the fermenter and rendered cell-free by being subjected to filtration or microcentrifugation (Kleman et al., 1991, Appl. Environ. Microbiol. 57, 910-917 and 918-923; Turner et al., 1994, Biotechnol. Bioeng. 44, 819-829). Prediction and feedback control algorithms have decreased the fluctuations in the glucose concentration during growth up to X=65 g/l (Kleman et al., 1991, Appl. Environ. Microbiol., vol. 57, 910-917). In the region of very high cell densities (from approx. 80 g/l to 150 g/l ), it becomes increasingly more difficult and more time consuming to separate the cells and the nutrient solution, so that the time delay in determining the current glucose value in the fermenter also increases in a biomass-dependent manner and makes it more difficult, or impossible, to maintain the glucose level constant. By contrast, the glucose concentration is measured with a time delay which is constant and brief using an appliance which does without this cell separation (Pfaff et al., 1995, pp. 6-11, in: Proceedings of the 6th International Conference on Computer Appl. in Biotechnol. Garmisch-Partenkirchen, FRG). According to the method of Pfaff et al., an FIA possessing an enzymic-amperometric glucose sensor is employed in the immediate vicinity of the sampling site after the culture has been diluted with a growth inhibitor.
During aerobic culture,
E. coli
cells which are not obliged by the dosage regime to grow in a sub-strate-limited manner customarily form the metabolic by-product acetate to an increased extent (Riesenberg 1991, Curr. Opinion Biotechnol., vol. 2, 380-384), which acetate accumulates in the nutrient solution and has a growth-inhibitory effect when present in relatively large quantities (Pan et al. 1987, Biotechnol. Lett., vol. 2, 89-94). For this reason, it has only previously been possible to effect fed-batch cultures to high cell densities using special
E. coli
strains whose accumulation of acetate has been reduced by means of specific genetic alterations, while tolerating other disadvantages which are associated with this. The descendants of
E. coli
K12 include phosphotransacetylase-negative mutants (Bauer et al., 1990, Appl. Environ. Microbiol., vol. 56, 1296-1302; Hahm et al., 1994, Appl. Microbiol. Biotechnol., vol. 42, 100-107) whose growth is, however, strongly reduced in glucose/mineral salt media. Phelps and collaborators (see above) used the
E. coli
strain TOPP5 as the host for non-substrate-limited culture up to a biomass of X=85 g/l. However, this
E. coli
strain, which evidently does not accumulate acetate in a pronounced manner, is not a K12 strain.
E. coli
TOPP5 forms haemolysin and is consequently a pathogenic strain which is not suitable, for reasons of safety, for use as a host for forming recombinant DNA products in the industrial sector. A reduction in acetate accumulation by means of specifically reorienting the intermediary metabolism was achieved by transforming
E. coli
cells with a plasmid containing a gene encoding acetolactate synthase (ALS) (San et al., 1994, in: Ann.N.Y.Acad.Sci., vol. 721, 257-267). However, this procedure suffers from the disadvantage that instabilities usually occur under high cell density conditions when an ALS-encoding plasmid is used in combination with a second plasmid carrying the “production” gene. The efficiency of recombinant product formations is frequently decreased by pl
Horn Uwe
Knüpeer Uwe
Krebber Anke
Kujau Marian
Matzku Siegfried
Guzu David
Leffers, Jr. Gerald G.
Merck Patent Gesellschaft
Millen White Zelano & Branigan P.C.
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