Optimizing Cell and Culture Environment Production of Recombinant Proteins
By J E N N I E
Introduction Cloning a gene is only the first step in the study of genetic regulation and gene function. Further studies usually require that the gene be expressed in vitro, frequently in a mammalian cell culture system. The cell culture and expression systems chosen will have a significant impact on the amount and, frequently, the biochemical characteristics of the protein produced from the cloned gene. While the exact parameters necessary for successful expression of a given protein in mammalian cells cannot yet be predicted with great accuracy, several factors have been shown to contribute to optimizing expression. This article outlines some of the critical elements in producing acceptable expression levels in minimum time, especially where the techniques for handling cell lines expressing recombinant proteins might differ from normal cell culture practice.
Choice of Cell Line, Plasmid, and Transfection M e t h o d The choice of cell line, plasmid construction, and transfection method are interdependent and beyond the scope of this article. However, in all cases, cells which are cultured in conditions which lead to optimum growth will yield higher transfection frequencies and be easier to select, clone, and grow in order to obtain optimal expression of the desired recombinant protein. The choice of a cell line which can be grown in serum-free medium may later prove to be a significant advantage in detecting the recombinant protein at low levels of expression where the only available assays are not specific for the protein of interest or do not work in the presence of serum. The use of serum-free medium may also provide an advantage in purifying secreted proteins from the medium. Different cell types expressing the same protein and the same cell type expressing different proteins should be treated as separate cell lines, and each one will frequently have distinctly different properties. However, the optimal medium and growth supplements will be most closely related in cells derived from the same parent cell line. This is important in selecting the conditions for comparing recombinant proteins produced from two separate transfection events (e.g., expressed in two different cell lines). It is METHODS IN ENZYMOLOGY, VOL. 185
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EXPgESSION IN MAMMALIAN CELLS
frequently more meaningful to compare two lines each grown in conditions optimal for that line, rather than in identical conditions. It should be remembered that substances such as methotrexate or antibiotics, which are used to select for successfully transfected cells or to amplify genes, are toxic to cells and frequently mutagenic. In addition, the recombinant product being expressed may be detrimental to cell growth and viability at the high concentrations desired. Therefore, cells transfected with recombinant genes are frequently required to grow in adverse conditions. For this reason, it is best to optimize the culture conditions to the fullest extent possible, first for the parent line to be used for transfection and then for the selected recombinant line.
Cloning The purpose of cloning is to ensure that all of the cells in the culture are descended from a single cell, i.e., are genetically identical. This prevents the rapid and unpredictable changes in culture phenotype which may occur in mixed cell populations when conditions change to favor one cell type over another (e.g., a nonproducer cell over one producing a recombinant product). Perhaps more importantly, it allows the separation of high producer from low or nonproducing cells after the initial transfection and selection events. It should be emphasized, however, that there can be considerable change with time even in cloned populations. These changes can be genetic, and therefore irreversible, or only a phenotypic response to changing or marginal culture conditions which can be controlled or reversed. There are two methods of cloning which will be discussed here: (1) cloning by limiting dilution; and (2) cloning with the use of cloning rings. Cloning in soft agar is also frequently used, but works best with highly transformed cells and is somewhat more cumbersome than the other two methods described. Clones can also be picked from plates using sterile cotton swabs. The cells are then transferred by dipping the swab in fresh medium in another plate. This method is rapid but is more likely to result in the cross-contamination of clones than the two methods mentioned above unless the colonies are very widely spaced on the plate from which the clones are picked. Cloning by limiting dilution can be used with suspension or attached cells and should be used with suspension cells or with cells which are very mobile when attached. Conditioning of the medium is sometimes necessary to get good growth at the very low cell densities used in cloning. This is especially true when
CELL CULTURE OPTIMIZATION
cloning amplified recombinant cells which may be growing under adverse conditions or in low-serum or serum-free conditions. To condition medium, the parent line used for transfection should be grown to high density, the medium changed, and this "conditioned" medium collected 24-48 hr later, before the medium components have been exhausted. This medium is then filtered through a 0. l g m filter to remove any cells which may be floating in the medium and then it is resterilized. The conditioned medium mixed l : l (v/v) with fresh medium is used when plating the cells for cloning. Cells should be diluted so that approximately half of the wells of a 96-well multiwell plate contain cells when 50 #l/well of medium is added (10 cells/ml if the cells have a 100% plating efficiency). The cloning ring method allows one to inspect visually the colonies to be selected more easily and thus to select cells with desired, or different, morphologies. It can also be easier to use with cells with very low plating efficiencies. Cloning rings are usually used when cloning between amplification steps, as the cells are frequently already growing in widely dispersed colonies at the end of the selection. The plates should be examined microscopically and selected colonies marked on the plate. After placement of the cloning rings they can be checked visually to ensure that only one colony has been included. The following points refer to both methods. 1. When plating cells for cloning it is essential that they be in a dispersed single-cell suspension. Visually inspect to see that most cells in the suspension are separate before plating. 2. Check colonies visually early during growth and mark wells or colonies which arise from a single cell. If two cells are in a well, or a colony starts from two cells it should be discarded. 3. When using any of the above methods, the cloning should be repeated 2 - 3 times in succession to ensure that one has a true clone. 4. To obtain the maximum plating efficiency when cloning, the medium used should be the normal maintenance medium without methotrexate or other selective agent during the low density growth prior to cloning and during the first subsequent passage. Afterward the selective agent can be returned to the medium. This is less stressful on the cells and allows a higher plating efficiency. An exception to this may be cloning from plates in which only a few colonies have survived after amplification or selection. 5. When clones are picked, they should be passaged first into one well of a 24-well dish, then to 35-mm and 100-mm plates. This maintains a relatively high cell density throughout the growth of the clone. Conditioned medium may also be used in the first passage after cloning, if necessary.
EXPRESSION IN MAMMALIAN CELLS
Pools For some applications, such as transient expression and expression of stably integrated genes in some cell types, cloning may not be necessary. This saves a good deal of time initially. However, the pool may not be as stable to prolonged culture as a clone would be. The pool is also likely to be a mixture of cells with widely differing expression levels, including cells not producing the desired recombinant protein, and therefore will not produce at as high a level as a clone selected for high production. Adequate numbers of vials of the pool should be frozen down as soon as possible so that new cells can be thawed periodically (every few weeks).
Fluorescence-Activated Cell Sorting If there is a way of fluorescence tagging living cells a fluorescence-activated cell sorter (FACS) may be used to select for, and clone, cells producing high levels of the desired recombinant protein. Conditioned medium, as described above for cloning by limiting dilution, may improve cloning efficiency. The FACS may also be used to assess the homogeneity of the population and the need for cloning from the original pool. If the selected pool is heterogeneous for expression of the recombinant protein, it is best to obtain a clonal population. It should be emphasized that FACS sorting is an excellent method for obtaining cells which produce high amounts of cell-associated protein (e.g., cytoplasmic, membrane-bound), but cellular levels of secreted proteins may not correlate well with specific productivity.
Amplification If large quantities of the recombinant protein are desired for purification and further studies, amplification of the transfected gene may be accomplished using one of several amplifiable systems, such as dihydrofolate reductase/methotrexate,~ ornithine decarboxylase/dihydrofluoromethylornithine,2 or glutamine synthase/methionine sulfoxamine. 3 These systems frequently require the use of selective media which are deficient in some essential nutrients and selective agents which are toxic to R. Kaufman and P. A. Sharp, J. Mol. Biol. 159, 601 (1982). 2 T. R. Chang and L. McConologue, Mol. Cell. Biol. 8, 764 (1988). 3 C. R. Bebbington and C. C. G. Hentschel, in "DNA Cloning, Volume III: A Practical Approach" (D. M. Glover, ed.), p. 163. IRL Press, Oxford, 1987.
CELL CULTURE OPTIMIZATION
normal cells. In these instances, it is again especially important to use cell culture media that are optimal for the growth of the cell used to express the protein to be amplified. Amplification of the desired gene will frequently result in poor growth performance of the resulting cell population. At some point this poor growth may offset the gains of the increased specific productivity of the cells. This may be counteracted to some extent by selecting for cells with good growth characteristics, as well as high specific productivity when picking clones. In its simplest form, this would mean assaying for protein production after a short (24 hr) and a more extended (e.g., 1 week) period in culture. The initial value would primarily reflect the amount of product produced per cell, while the extended production of protein will be a reflection of both specific productivity and viable cell days in culture (which in turn reflects both growth rate and viability), as well as other parameters such as protein stability.
Choice of Culture M e t h o d The choice of culture method will depend on the characteristics of the recombinant protein, the type of studies to be carded out in the transfection system, the amount of recombinant protein desired, and the properties of the recombinant cell line obtained. Thinking through these factors carefully initially can save a good deal of time later in obtaining a workable experimental or production system. If the purpose of the transfection is to demonstrate that the transfected gene can be expressed and/or to study the genetic regulation of that expression, very little of the actual protein will be required and any one of a number of established cell lines can be used for expression. The choice might then be dictated by the efficiency of transfection and expression or the characteristics of the cell line itself (e.g., use of a liver cell line to study regulation of expression of a secreted protein). If large amounts of protein will be required for future studies, then some type of culture system should be used which can be scaled up to produce large amounts of cells or culture fluid. Roller bottle production is the most straightforward, but is labor intensive and limited in the degree to which it can be scaled up. The next choice might be growth in spinners, or fermentors. This requires a cell line which naturally grows in suspension or has been adapted to do so (see below). The fermentor systems have been scaled up to thousands of liters. 4 4 W. R. Arathoon and J. R. Birch, Science 232, 1390 (1986).
EXPRESSION IN MAMMALIAN CELLS
Suspension Adaptation The purpose of suspension adaptation is to obtain a cell line which will grow as single cells unattached to a substrate. These cells are then capable of being easily scaled up for production in spinners or fermentors using existing technologies. This is usually the fastest and least expensive method of obtaining relatively large amounts of recombinant protein for further study. There are several approaches to obtaining this end: (1) alter the medium so that the ability of cells to attach is eliminated or diminished, (2) select for cells which will not attach in the standard medium conditions, or (3) select for cells which will grow in suspension in the standard media when the surfaces available have been treated to prevent attachment but will attach to standard tissue-culture-treated surfaces in standard serumcontaining medium. The first approach has the disadvantage that the media devised to promote cell detachment (generally with much reduced magnesium and calcium, e.g., Joklik's medium) are frequently suboptimal for supporting high titers of desired proteins. The second approach is adequate for production cell lines but is more difficult than the third and the resulting cell lines less flexible in use. The third approach is generally (but by no means always) reasonably rapid, and results in a line that can be grown in an attached state for further manipulation such as cloning or transfection. The approach outlined below is designed to suspension adapt cells in this third sense with as little alteration in other cell properties as possible. The one exception to this rule is that we have in several cases chosen to suspension adapt in a reduced-serum, hormone-supplemented medium in order to obtain a line which will grow continuously in these conditions. In at least one case, this strategy also improved our ability to suspension adapt the cells and obtain a stable phenotype after transfection. There are other ways to suspension adapt cells but we have been most successful with this one. In cases where maintaining selective pressure on the cells during suspension adaptation is desired, this may be done, but it may make the adaptation to suspension more lengthy or more difficult. Cultures can most easily be adapted to suspension and then the selective pressure reintroduced after the cells are growing well in suspension, or the cells recloned and clones selected which are high producers and grow well in suspension. After the suspension adaptation described below, cells may be grown as attached cells for cloning, and cloned populations grown up and reintroduced into suspension with little or no difficulty.
CELL CULTURE OPTIMIZATION
Protocol Medium. The medium used should be the medium selected for the optimal growth of the desired cell line (see below). Supplement the medium with 1 - 50/0(v/v) fetal bovine serum and insulin (5/tg/ml) or the ITS supplement (which can be obtained from Collaborative Research, Bedford, MA). Determine the minimum amount of serum necessary for optimal growth in the medium to be used by obtaining a serum dose-response curve in the presence and absence of the insulin or ITS supplement. Further supplementation with a polyol such as F-685 at 0.1% (w/v) and an organic buffer such as HEPES ( 10- 20 raM) will help protect the cells from mechanical damage and provide additional buffering capacity during the suspension growth. The presence of polyols can be critical for success in suspension adapting some cell lines (see Fig. 1). Spinners. Use well siliconized spinners. Solutions for silicone coating glassware can be obtained from Dow Chemical (Dowcoat). We prefer using 250-ml spinners with 50- to 100-ml volumes of culture medium. Spinners are run at a rate sufficient to keep cells and small aggregates in suspension (50-I00 rpm) while minimizing damage to the cells. Set up two spinners in parallel, subculturing on different days. If the cells accumulate around the spinner shaft and on the sides of the spinner at the medium surface the spinner is not properly siliconized. If this occurs, cells tend to form clumps which break off and allow other cells to attach to them, thus reducing the likelihood that they will grow as single cells. Culture. Trypsinize starting culture and set up the spinners at 2 - 5 × 105 cells/ml. Check cells daily. Sterile sodium bicarbonate solution may be added if the pH drops below 6.8. After the first day or so, the caps on the spinners should be loosened to allow for increased oxygen exchange. On day 3 or 4, the cells should be counted and passaged. Initially, cells should be centrifuged and fresh medium added to bring the cell number back to between 2 and 5 × 105/ml. As the cells start growing to densities over 106/ml they may be passaged by dilution of the suspended cells with fresh medium. The cell density should be such as to allow at least a 1 : 5 split. If cells clump excessively, large clumps should be allowed to settle before subculture and not passaged. After 1 - 2 months, the cells should be capable of logrithmic growth in suspension and reach densities of > 106 cells/ml when inoculated at densities of 5 - 10 )< 104 cells/ml. Cell viability should remain at > 90% throughout the growth period. At this point, the cells are termed "suspension s A. Mizrahi, J. Clin. Microbiol. 2, 11 (1975).
EXPRESSION I N M A M M A L I A N
t~ 105 k~ --
,o, I 0
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li / .
." I f
/ .... I
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Day FIG. I. Suspension adaptation of a CHO-derived recombinant cell line. (A) Cells were grown in spinners in medium which did not contain a polyol. The cells never grew to high densities. (B) The same cells were grown in medium with the addition of 0.1% F-68. The cells grew well by the second week and were fully adapted to suspension growth by 6 weeks in culture.
adapted" even though they will still attach to tissue culture plastic in serum-containing medium and may exhibit some clumping in suspension under some conditions. In Fig. 1 the cell counts and passage times (arrows) are shown for the course of adapting a C H O recombinant clone. The cells were considered suspension adapted after 5 weeks. The amount of time required for this
CELL CULTURE OPTIMIZATION
degree of adaptation may vary widely from one cell line to another or from one clone to another. Where suspension growth is essential, it is wise to start adapting several clones and eliminate those that do not adapt well. Growth to High Cell Densities High-density growth may be desired to obtain high titers of secreted recombinant proteins. If this is desirable, cells should be screened for the ability to grow to high densities during the cloning stage. The extended screen described above will tend to identify cells which can grow to high densities. Selection of a medium designed for high-density cell growth (e.g., Dulbecco's modified Eagle's medium or FI2/DME) rather than the use of medium designed for clonal growth (e.g., Ham's nutrient mixtures) is essential. Selection of Optimal M e d i u m The optimization of culture media, especially for serum-free or lowserum growth, has been carefully outlined elsewhere. 6-8 The optimization of each individual component of the medium is the only way to ensure that one is using the best medium for a given cell line. However, it is relatively easy, and much quicker, to screen a number of commercially available media as a good first approximation. For best results, these media formulations should be purchased as dry powdered mixtures, prepared in the laboratory using highly purified water, and stored for no more than 1 month at 6 °. Serum, F-68, and HEPES may be added at the time of medium preparation but other additions, such as insulin and other hormones, should be prepared as separate stocks and added at the time of use. These media should be screened in the presence of the minimal amount of serum required, as optimizing the nutrient mixture frequently results in a decreased serum and/or hormone requirement. Media designed for the growth of cells frequently used for expression of recombinant proteins such as CHO (e.g., Hand Biologicals, Alameda, CA, CHO medium) in lowserum levels (e.g., 0.1 -2%) are becoming more widely available. 6 R. G. Ham, in "Methods for Preparation of Media, Supplements, and Substrata for Serum-Free Animal Cell Culture" (D. W. Barnes, D. A. Sirbasku, and G. H. Sato, eds.), p. 3. Alan R. Liss, New York, 1984. 7C. Waymouth, in "Methods for Preparation of Media, Supplements, and Substrata for Serum-Free Animal Cell Culture" (D. W. Barnes, D. A. Sirbasku, and G. H. Sato, eds.), p. 23. Alan R. Liss, New York, 1984. 8j. p. Mather and M. Tsao, in "Large Scale Mammalian Cell Culture Technology"(A. R. Lubiniecki, ed.), in press. Marcel Decker, New York, 1990.
EXPRESSION IN MAMMALIAN CELLS
Serum-Free Medium A great deal has been written about the development of serum-free media. The use of serum-free media is advantageous in instances where experimental control over the culture environment is required. Thus, if one wishes to study the hormonal regulation ofgene expression, it is best to do these studies in a defined, serum-free, hormone-supplemented medium where all of the culture variables can be studied independently. Serum-free or low-serum media can also provide a significant advantage when a secreted protein is to be purified from the culture medium. Elimination of the serum results in the desired protein constituting a significant percentage of the protein in the conditioned medium. The most straightforward way to achieve this end is to transfect the gene of choice into a cell line for which a defined medium is already available. A number of these culture systems have been described in detail in the last decade?- ~ This same medium should then be sufficient to grow the recombinant cell line, particularly if clones picked for study are tested for production of the protein in the defined medium. Any clones unable to grow in this medium can be eliminated initially. Persistent inability to grow transfected cells in the medium that the parent line grew in probably reflects an inhibitory action of the recombinant protein on the cells or medium components. Selection for Desired Characteristics The use of mammalian cells to express recombinant proteins, especially for the large-scale production of these proteins, has tremendous potential for engineering the cells as hosts in ways that go beyond transfecting the gene coding for the desired product. The methods employed can be divided into four categories: (1) selection of cells with the desired characteristic, (2) introducing the desired characteristic through mutation, (3) fusion of two cells each of which exhibits a desired trait followed by selection for these traits, or (4) introduction of new genes whose expression results in the desired phenotype. The most critical step for the success of any of these approaches is setting up the conditions which will eventually be used to select for the desired phenotype. Examples of these three approaches are 9 G. H. Sato and R. Ross, "Hormones and Cell Culture," Books A and B. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1979. ~oD. W. Barnes, D. A. Sirbasku, and G. H. Sato, "Cell Culture Methods for Molecular and Cell Biology," Vols. 1-4. Alan R. Liss, New York, 1984. H G. H. Sato, A. B. Pardee, and D. A. Sirbasku, "Growth of Cells in Hormonally Defined Media," Books A and B. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1982.
ANALYSIS OF SYNTHESIS, PROCESSING, AND SECRETION
the suspension adaptation of cells as outlined above, the selection of a dhfrmutant for use in gene amplification, and the creation of hybridomas producing monoclonals of desired specificities. Each of these techniques is extremely powerful and not yet used to its full potential. The future of recombinant gene expression should see the optimization of the characteristics of the cells themselves as well as the media and cell culture systems.
 A n a l y s i s o f S y n t h e s i s , P r o c e s s i n g , a n d S e c r e t i o n o f P r o t e i n s E x p r e s s e d in M a m m a l i a n C e l l s B y ANDREW J. D O R N E R a n d R A N D A L J. K A U F M A N
Introduction The secretion of biologically active protein from mammalian cells is the final step in a complex pathway of posttranslational modifications performed in the endoplasmic reticulum (ER) and Golgi complex (GC). Many of the steps of the secretory pathway in mammalian cells have been reviewed (see Fig. 1).~,2Proteins destined for the exocytic pathway are first cotranslationally translocated into the lumen of the ER. During translocation, an amino-terminal leader peptide is, in most cases, proteolytically removed and a high-mannose oligosaccharide core is enzymatically transferred to asparagine residues located in the sequence Asn-X-Ser/Thr, where X can be any amino acid except proline. In the ER the initial steps of carbohydrate processing occur. Terminal glucose residues are rapidly removed by glucosidases I and II and at least one a-1,2-1inked mannose residue is removed by an a-l,2-mannosidase in the ER. Acylation with long-chain fatty acids may also occur in the ER, usually by addition of palmitate or myristate to cysteine or serine residues. Protein is transported to the GC where further modifications occur. Transit out of the ER has been identified as a potential rate-limiting step in secretion, a During traversal of the GC a series of reactions separated spatially and temporally involve the removal of mannose residues by mannosidases I and II and addition of N-acetylglucosamine, fucose, galactose, and sialic acid residues by specific transferases to modify high-mannose carbohydrate to complex forms (Fig. 1). In addition to N-linked glycosylation, proteins can also have carbohydrate attached to serine and threonine residues. The initial step in this O-linked glycosylation is the direct transfer of N-acetylR. Kornfeld and S. Kornfeld, Annu. Rev. Biochem. 54, 631 (1985). 2 A. M. Tartakoff, Int. Rev. Cytol. 85, 221 (1983). 3 H. F. Lodish, N. Kong, M. Snider and G. J. Strous, Nature (London) 304, 80 (1983).
METHODS IN ENZYMOLOGY,VOL. 185
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