Commercial Biotechnology: An International Analysis (Part 5 of 35)

PART IThe Technologies

Chapter 3

The Technologies

Contents

PageIntroduction . . . . . . . . . . . . . . , . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Recombinant DNA Technology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Structure and Function of DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Preparing Recombinant DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Recombinant DNA Technology in Industrial Processes . . . . . . . . . . . . . . . . . . . . . . . . . . .

Monoclinal Antibody Technology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Preparing Monoclonal Antibodies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Monoclinal Antibodies and Recombinant DNA Technology . . . . . . . . . . . . . . . . . . . . . . .Large-Scale Production of Monoclinal Antibodies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Industrial Uses for Monoclinal Antibodies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Conclusion. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Bioprocess Technology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Bioprocess Essentials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Processing Modes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Raw Materials. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Biocatalyst . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Bioprocess Monitoring and Associated Instrumentation . . . . . . . . . . . . . . . . . . ~ . . . . . . .Separation and Purification of Products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Culture of Higher Eukaryotic Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Priorities for Future Research . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Chapter 3 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . ~ . . . . . . . . . . . . . . . . . . . . . . . . . . .

333333363738404242434344464751515254555657

Tables

Table No. Pagel. Volume and Value of Biotechnology Products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 442. Situations Potentially Requiring Large-Scale Eukaryotic Cell Culture . . . . . . . . . . . . . . . 553.Comparison of Microbial and Mammalian Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57

Figures

Figure No. Page3.The Structure of DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 344.The Replication of DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 355.Mechanism of Gene Expression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 356. Recombinant DNA: The Technique of Recombining Genes From One Species

With Those of Another . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 367. Structure of an Antibody Molecule . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 398. Preparation of Monoclinal Antibodies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 409. Steps in Bioprocessing. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46

Introduction —

Chapter 3

The Technologies

This chapter reviews the scientific bases for thetechnologies discussed in this assessment. Themost publicized and broadly applicable of thesetechnologies is recombinant DNA (rDNA) tech-nology, which includes gene cloning, and isexplained first. The second technology discussedis monoclinal antibody (MAb), or hybridoma,technology. This technology, used to preparecomplex molecules known as MAbs which can beused to recognize or bind a large variety ofmolecules, has an expanding number of applica-tions. The last technology discussed, bioprocess

technology, allows the scaling-up of a biologicalproduction process so that large quantities of aproduct can be made. Bioprocess technology is, inmany respects, the most difficult and least under-stood of the technologies, so it receives a moreintensive discussion in this chapter. Because ofthe lack in the United States of broadly applicableknowledge in bioprocess engineering, the sectionon bioprocess technology also ends with prioritiesfor future research, giving a focus to whereFederal research funds might best be spent.

Recombinant DNA technology

The development of rDNA technology-the join-ing of DNA from different organisms for a specificpurpose—has allowed a greatly increased under-standing of the genetic and molecular basis of life.This technology has also led to the founding ofmany industrial ventures that are addressing theproduction of numerous compounds rangingfrom pharmaceuticals to commodity chemicals.This section introduces some aspects of the scien-tific basis of rDNA technology, discusses methodsthat are used to construct rDNA, and notes sev-eral additional features of the commercial use ofrDNA technology.

Structure and function of DNA

Throughout the spectrum of life, the traitscharacteristic of a given species are maintainedand passed on to future generations, preservedsimply and elegantly by the information systemcontained within DNA. DNA can be thought ofas a library that contains the complete plan foran organism. If the plan were for a human, thelibrary would contain 3,000 volumes of 1,000pages each. Each page would represent one gene,or a unit of heredity, and be specified by 1,000letters. As shown in figure 3, DNA, a double-

Photo credit: Science Photo Llbraty and Porton/LH International

The DNA of the bacterium Escherichia co/i

stranded, helical molecule, is composed, in part,of four nucleotide bases—adenine (A), cwytosine (C),guanine (G), and thymine (T)-which are the let-ters of the chemical language. A gene is anordered sequence of these letters, and each genecontains the information for the composition ofa particular protein and the necessary signals forthe production of that protein.

33

—

34 ● Commercial Biotechnology: An International Analysis

The mechanism by which DNA replicates is in-herent in the structure of DNA itself. As can beseen from figure 3, the nucleotide bases arepaired to form the rungs of the twisted DNA lad-der. This pairing is absolutely specific: A alwayspairs with T and C always pairs with G. The pair-ing is accurate, but not very strong. Thus, in celldivision, the DNA can “unzip” down the middle,leaving a series of unpaired bases on each chain.Each free chain can serve as a template for mak-ing a complementary chain, resulting in two iden-tical DNA molecules, each a precise copy of theoriginal molecule. Figure 4 illustrates the replica-tion of DNA.

The DNA present in every cell of every livingorganism has the capacity to direct the functionsof that cell, Gene expression, shown in figure 5,is the mechanism whereby the genetic directionsin any particular cell are decoded and processedinto the final functioning product, usually a pro-tein. In the first step, called transcription, the DNAdouble helix is locally unzipped near the gene of

interest, and an intermediate product, messengerRNA (mRNA), a single-stranded, linear sequence ofnucleotide bases chemically very similar to DNA,is synthesized. The transcription process dictatesthe synthesis of mRNA that is complementary tothe section of unzipped DNA in a manner thatis somewhat similar to the replication of DNA. Inthe second step of gene expression, translation,the mRNA, after release from the DNA, becomesassociated with the protein-synthesizing machin-ery of the cell, and the sequence of nucleotidebases in the mRNA is decoded and translated intoa protein. The protein goes on to perform its par-ticular function, and when the protein is nolonger needed, the protein and the mRNA codingfor that protein are degraded. This mechanismallows a cell to “fine tune” the quantity of its pro-teins while keeping its DNA in a very stable andintact form.

Proteins perform most of the necessary func-tions of a cell. By far the most diverse group ofproteins is the enzymes, which are the proteins

t Figure 3.–The Structure of DNA

Base pairs

lgar-phosphateckbone

A schematic diagram of the DNA double helix. A thr~dimensionai representation of the DNA doubie helix.

The DNA molecule is a doubie helix composed of two chains. The sugar-phosphate backbones twist around the out-side, with the paired bases on the inside serving to hold the chains together.

SOURCE: Office of Technology Assessment.

Ch. 3—The Technologies ● 3 5

Figure 4.–The Replication of DNA

When DNA replicates, the original strands unwind and serveas templates for the building of new complementary strands.The daughter molecules are exact copies of the parent,

with each having one of the parent strands

that catalyze biological reactions. Another groupis the structural proteins, which are found, forinstance, in cell membranes. Other proteins haveregulatory functions; these include some hor-mones. Still others have highly specialized func-tions (hemoglobin, for example, carries oxygenfrom the lungs to the rest of the tissues).

The code by which genetic information is trans-lated into proteins is the same for all organisms.Thus, because all organisms contain DNA and all

Figure 5.—Mechanism of Gene Expression

I Transcription

mRNA released andtransported to protein-synthesizing machinery

Translation

+

Protein

~ ~ ~ ~ ~


organisms interpret that DNA in the same man-ner, all organisms, in essence, are related. It isthis concept that forms the basis for the industrialuse of DNA. In nearly every instance, a produc-tion process using rDNA technology depends onthe expression of DNA from one species inanother species. Only a universal genetic codewould allow DNA to be used in this manner.

Despite the existence of a universal geneticcode, regulatory signals indicating starts and stopsof genes are known to vary among species. Thus,a gene removed from one organism and placed

36 . Commercial Biotechnology: An International Analysis

in another will code for the same protein as it didin its native system, but its synthesis needs to beinduced by the proper host signal. one of thegreat challenges of rDNA technology is to con-struct DNA molecules with signals that optimal-ly control the expression of the gene in the newhost .

Preparing recombinant DNA

The amount of DNA present in each cell of ahuman (or most higher animals) is approximately3 billion base pairs (2), and an average gene isabout 1,000 base pairs, or about one millionth ofthe DNA. It is extremely difficult to study one genein a million. Therefore, powerful tools have beendeveloped to isolate genes of interest, place themin a foreign, simpler system, and replicate themmany times to give a large amount of a singlegene. The isolation of genes from higherorganisms and their recombination in simple cellshas already yielded a wealth of information, in-cluding insight into how genes determine thedifferences between different types of cells, howgene expression is regulated, and how genes mayhave evolved. For industrial uses, however, notonly must the gene be cloned (reproduced), butthat gene must also be expressed (the proteinbased on the gene be produced).

The basic technique of preparing rDNA isshown in figure 6. Preparations of restriction en-zymes (enzymes that are made in certain bacteriaand cut DNA at specific sites) are used to cutdonor DNA (usually from a higher organism) intofragments, one of which contains the gene of in-terest, The resulting DNA fragments are then in-serted into a DNA “vector,” which is most oftena plasmid. ” Each plasmid vector will contain a dif-ferent donor DNA fragment. These rDNA plas-mids are introduced into host cells in a processcalled ‘(transformation.” Once inside the host cells,the rDNA plasmids replicate many times, thusproviding many copies of each donor DNA frag-ment. of the many bacteria transformed by plas -mids containing donor DNA, only a few will con-tain the DNA fragment of interest. The desired

“A plasmid is a circular, double-stranded piece of DNA whichreplicates in cells apart from the chromosome.

Figure 6.-Recombinant DNA: The Technique ofRecombining Genes From One Species

With Those of Another .

+ +

Donor DNA

SOURCE Off Ice of Technology Assessment


Photo credit: Science Photo Service and Po170rVLH International

Molecular biologist in laboratory

gene is located among the vast number of bac-teria containing plasmids with a suitable probe. *Vectors other than plasmids can be used for clon-ing DNA. One method uses the DNA of viruses,and another uses cosmids, artificially constructedhybrids of plasmids and viruses. Another methoduses transposable elements, fragments of DNAthat can insert themselves into the host cell’s chro-mosomes.

All rDNA methods require the following:

●

●

●

a suitable vector that is taken up by the host,is capable of autonomous replication, and,during the process, replicates the segment ofdonor DNA faithfully;an adequate selection system for distin-guishing among cells that have, or have not,received rDNA; andan appropriate probe for detecting the par-ticular DNA sequence in question.

The most difficult part of the cloning processis isolating an appropriate probe. The genes thatwere first cloned were those that, in certain cells,produced large quantities of relatively puremRNA. Since the mRNA was complementary tothe gene of interest, the mRNA could be used asa probe. This method severely limited the numberof genes that could be cloned, however, because

● ,4 probe is a sequence of DNA that has the same sequence as

the desired gene and has been prepared in such a waj’ that it can

be identified after it base pairs with that gene,

most genes do not produce large amounts ofmRNA. More recently, a different technique hasbeen used that allows a much greater diversityof genes to be cloned. If the amino acid sequenceof a protein is known, then, working backwardsthrough the gene expression scheme, the nucleo -tide base sequence can be determined. Becauseof the advent of automated DNA synthesizers, aportion of DNA can be synthesized that is com-plementary to the gene. This piece of DNA canthen be used as a probe. Thus, if enough of a par-ticular protein can be isolated and sequenced, itscorresponding gene can be cloned.

At present, rDNA is grown principally in simplemicro-organisms such as bacteria and yeast.Yeasts, in addition to bacteria, are being used ashosts for rDNA cloning because they more closelyresemble cells of higher organisms. Yeasts per-form functions similar to those of higher euka-ryotic cells. These functions include adding sugargroups to some proteins. For the function of manyproteins, these sugar groups are essential. Recent-ly, scientists have learned how to introduce novelgenetic material into higher plants and animals.The special techniques that pertain to cloningDNA in plants are discussed in Chapter 6: Agri-culture.

Recombinant DNA technology inindustrial processes

The commercial use of rDNA technology hasseveral features in addition to those just discussed.In order to produce a product or improve a proc-ess, the cloned gene must be expressed to givea functional product. Since the signaIs thatregulate gene expression vary from species tospecies, achieving the expression of a gene in aforeign cell may be difficult. The commercialdevelopment of biotechnology is highly depend-ent on the ability to achieve gene expression, forit is proteins (or their metabolizes) that either arethe marketable products themselves or establishthe cellular environment necessary for perform-ing such practical tasks as degrading toxic wastesor increasing the efficiency of photosynthesis. Toa large extent, the problem of gene expressionhas been addressed through the manipulation ofthe adjacent vector DNA so that it contains thehost regulatory sequences. The cloned gene can

38 ● Commercial Biotechnology: An International Anaiysis

then be “switched on” by using host-regulatedcontrols (l). Moreover, it is possible to alterspecifically the regulatory sequences so that thegene is expressed at higher levels or so that itsexpression is more readily controllable in anindustrial situation (3).

The purification of a protein from an industrialbioprocess is greatly simplified if the protein issecreted from the cell into the growth medium.If the protein is secreted, it does not have to bepurified away from all the other cellular compo-nents. It is possible to attach additional regulatorysignals to the vector DNA that direct the cell tosecrete the protein and, thus, simplify its purifica-tion. The successful development of methods toenhance gene expression and product functionand secretion will undoubtedly enhance the com-mercial applicability of rDNA technology.

The computer-aided design of proteins isanother technology that will expand the use ofrDNA molecules industrially. In the past, enzymes

were modified by mutagenizing the host cell andthen selecting or screening for mutants that con-tained an altered enzyme. Now, through use ofthe techniques of X-ray crystallography, proteinsequencing, and computer modeling, the aminoacid sequence and three-dimensional structure ofthe protein can be determined and amino acidchanges that should bring about altered enzymeproperties can be selected. The DNA sequence ofthe cloned gene for an enzyme can then be modi-fied to incorporate the amino acid changes.Specific gene modification is made possible be-cause of technical advances resulting in rapid andinexpensive synthesis of small DNA segments thatcan be used to change specific base pairs in a DNAsequence. Near-term protein modification exper-iments could result in enzymes with increasedtemperature and pH stability. Longer term experi-ments could define the structure of active sitesof enzymes to be used for specific catalyticfunctions.

Monoclinal antibody technology

The production of antibodies in higher animalsis one aspect of a complex series of events calledthe immune response. Specialized cells called Blymphocytes, present in the spleen, lymph nodes,and blood, recognize substances foreign to thebody, or antigens, and respond by producing anti-bodies that specifically recognize and bind tothose antigens. Any given B lymphocyte canrecognize only one antigen. Thus, when a B lym-phocyte meets and recognizes an antigen for thefirst time, the B lymphocyte is stimulated andbecomes committed to producing a single type ofantibody for the duration of its life. The end resultof this aspect of an immune response is the anti-gen’s removal from the body.

Antibodies bind to antigens and carry out theirfunctions by virtue of the antibody’s unique struc-ture. All antibodies are comprised of four proteinchains in a precise orientation, as shown in figure7. One end of the antibody (the constant, or ef-fecter region) is nearly identical among antibodies.

This effecter region is associated with functionssuch as the secretion of antibodies from the B lym-phocyte and “signaling” to the immune systemafter the antibody binds with the target antigen.The other end of the antibody, the variable re-gion, contains the site that recognizes and bindsto a particular antigen, and the structure of thisend varies greatly from antibody to antibody toaccommodate a wide range of antigens.

Apart from their natural functions in theprotection of organisms via the immune response,antibodies have long been important tools forresearchers and clinicians, who use an antibody’sspecificity to identify particular molecules or cellsand to separate them from mixtures. Antibodiesalso have a major role in diagnosis of a widevariety of diseases. Antibodies that recognizeknown antigens are used to detect the presenceand level of drugs, bacterial and viral products,hormones, and even other antibodies in sensitiveassays of blood samples.

Ch. 3—The Technologies 39

Figure 7.—Structure of an Antibody Molecule ing and expensive, have not prevented the effec -

Effecterfunctions

Constantregion

SOURCE Off Ice of Technology Assessment.

The conventional method of producing anti-bodies for diagnostic, therapeutic, and investiga-tional purposes is to inject an antigen into alaboratory animal and, after evoking an immuneresponse, to collect antiserum (blood serum con-taining antibodies) from the animal. Although thismethod has been and continues to be widely used,there are several problems associated with con-ventional antibody technology, These include:

minor contamination of the injected antigenwith other molecules, so that the antiserumcollected from the animal contains a mixtureof antibodies against both the target antigenand the contaminating molecules;

● heterogeneous populations of antibodies withconcomitant differences in activity, affinityfor the antigen, and biological functions, es-pecially when a number of different animalsare used to prepare the antiserum; and

● the limited supply of quality antisera for anygiven purpose (10,28,32).

Since these difficulties are almost unavoidablein standard antibody preparations, the standardi-zation of immunoassay and the accumulation oflarge amounts of reference antisera have beendifficult. Such problems, although time-consum-

methods for continual production of largeamounts of pure antibodies has continued.

By what Cesar Milstein calls a “lucky circum-stance,” he and Georges Kohler began experiment-ing with the well-established technique of cellfusion in myeloma (antibody-producing tumor)cells adapted for cell culture. Milstein and Kohlerfused myeloma cells with antibody-producingspleen B lymphocytes from mice that had beenimmunized with sheep red blood cells (SRBCS),and they found that some of the resulting hybridcells, called hybridomas, secreted large amountsof homogeneous (monoclinal) antibodies directedagainst SRBCS (20)21). The myeloma parent cellconferred on the hybridoma the ability to growpermanently in cell culture and thus to supportalmost unlimited antibody production, while theB lymphocyte parent contributed the genescoding for the specific antibody against an SRBCantigen.

Photo credit: Scisnce Photo Service and PortodLH lnte?natlonal

Dr. Cesar Milstein, discoverer of monoclinal antibodies

40 ● Commercial Biotechnology: An lnternational Analysis

By using the method of hybridoma, or MAb,technology, it is now possible to “immortalize” in-dividual antibody-producing cells by fusion withtissue culture-adapted myeloma tumor cells in thelaboratory (4,5,8,13,22,25).

Preparing monoclinal antibodies

The method used to prepare MAbs is summa-rized in figure 8. The purified antigen of choiceis injected into a mouse, and a few weeks later,the spleen of the mouse is removed, The B lym-phocytes (antibody-producing cells) are isolatedfrom the spleen and fused with myeloma cells.The resulting cells are placed in a cell culturemedium that allows only the hybridomas to grow.

The many hybridomas that result are cloned, andeach clone is tested for the production of theantibody desired. A particular hybridoma cloneeither may be established in an in vitro culturesystem or may be injected into mice, where thehybridoma grows in abdominal cavity fluid(ascites) from which the antibodies are readilycollected.

This method allows the preparation of largequantities of highly specific MAbs against almostany available antigen. The antibodies producedby MAb technology are homogeneous, and theirproduction is predictable and repeatable, as com-pared to polyclonal antibodies produced with con-ventional immunological methods.

Figure 8.—Preparation of Monoclinal Antibodies

Mveloma cells

1 ar~ mixed andfused withB lymphocytes

Mouse myeloma(tumor) cellsare removedand placed intissue culture

Cells divide inliquid medium

The products of thisfusion are grown in aselective medium. Onlythose fusion productswhich are both “immor-tal” and contain genesfrom the antibody pro-ducing cells survive.These are called“hybridomas.”Hybridomas are clonedand the resulting cellsare screened for an-tibody production.Those few cells thatproduce the antibodiesbeing sought are grownin large quantities forproduction ofmonoclinal antibodies.

SOURCE: Off Ice of Technology Assessment, adapted from Y. Baakln, “In Search of the Magic Bullet,” Technology f7ev/ew, pp. 19-23.

Ch. 3—The Technologies ● 41

Photo credit: Sctence Photo Service and Por@n/LH international

Scanning electron micrograph of human hybridoma cells

Despite the great promise of MAbs, there areseveral persistent technical problems to beconsidered:

●

●

●

●

obtaining MAbs against certain weak antigens(antigens that do not produce a large immuneresponse) remains difficult (11,24);homogeneous antibodies cannot performsome functions such as forming a precipitatewith other antigen-antibody complexes, anecessary function for some diagnostic assays;low frequency of fusion is a continuing prob-1em in the preparation of hybridomas, as isthe stability of the hybridomas and antibodies(14); andsome MAbs are sensitive to small changes inpH, temperature, freezing and thawing, andcan be inactivated during purification.

Many of these problems are being alleviated orsolved as research with MAbs progresses.

Photo credit: Science Photo Library and PortmlLH International

One step in the isolation of hybridomas

Another problem being addressed is the devel-opment of hybridomas for specific species. Somesuitable myeloma cell lines exist for mice, rats,and humans (12,20,27), but a wider variety ofhuman cell lines and cell lines for other speciesare needed if wider applications of MAb tech-nology are to be made. Hybridomas are oftenmade with cells from two different species, butthese fusions regularly result in the preferentialloss of the spleen B lymphocyte chromosomes,resulting in an absence of antibody production(24). For therapeutic applications, it is desirableto treat people with human antibodies to avoidallergic reactions and other problems of antibodycross-reactivity. Thus, MAbs from a humanmyeloma/human spleen cell fusion are needed.Several investigators have reported the develop-ment of human myelomas that are suitable for

25-561 0 - 84 - 4


Photo credit: Science Photo L/brary and PortonlLH International

In vitro identification of specific cells using fluorescentlylabeled monoclinal antibodies

hybridoma preparation (9,17,23,27). Successfulfusions apparently result from using these celllines (6).

Monoclinal antibodies andrecombinant DNA technology

The combination of MAb technology and rDNAtechnology offers intriguing possibilities for fur-ther technological exploration. Recombinant DNAtechniques could be used to produce portions ofantibody molecules in bacteria to circumventsome of the problems (e.g., hybridoma instability)associated with MAb production in mice or tissueculture. Additionally, these MAbs would be freeof impurities, such as viruses, found in animalcells and possibly could be produced in largeamounts with a concurrent savings in cost.

The first cloning and expression of a completeantibody molecule in a bacterial system wasannounced recently by the U.S. firm Genentechand the City of Hope Medical Center and ResearchInstitute (19). The protein chains were expressedseparately in bacteria and reconstituted by theresearchers. The pharmaceutical applications ofbacterially produced antibody genes will belimited. Antibody molecules must be modified bythe cell to function in most diagnostic andtherapeutic applications. Bacteria do not performthe modifications necessary for proper function.However, it maybe possible to clone the antibodygenes in a cellular system such as yeast wherethe proper modifications can be made.

The production of MAbs in rDNA systems mayprove useful for making reagents used in indus-trial applications where only the antigen-bindingfunction may be necessary. With genes cloned forthe antigen-binding regions of the antibody, por-tions of MAbs may be produced more economical-ly in bacterial rDNA systems than in a large-scalemouse ascites or cell culture protocols.

Large-scale production ofmonoclinal antibodies

Although MAbs can be produced by severalmethods, manufacturers primarily use mouseascites to produce the modest amounts of MAbsneeded to service current diagnostic and researchmarkets. As applications for MAbs to humantherapy are developed, the need for larger quan-tities of MAbs (free from mousederived contami-nants that might cause allergic reactions) may .encourage a switch to the use of large-scale cellculture to produce MAbs. If MAbs are to be usedin industrial applications (e.g., in the purificationof proteins), production methods will be neededto produce even larger quantities of antibodies,In these cases, efficient cell culture or microbialbioprocess techniques will probably be necessaryto provide enough antibodies to fill these needs.

Improved, more controllable cell culturesystems will be needed for the production ofMAbs in the future. A crucial need for large-scalecell culture is either the isolation of hybridomacell lines that attach to surfaces or the use of tech-niques for immobilizing cells on a solid matrix.


Immobilized cells could be grown in large quanti-ties in culture; the MAbs secreted from the cellscould then be routinely collected from themedium. Immobilized cell methods may provevaluable for large-scale MAb production. Suchmethods are already used industrially, for exam-ple, in growing cells that produce polio virus forsubsequent vaccine production (26,31).

Damon Biotech Corp. (U. S.) has recently intro-duced the technique of microencapsulation toMAb technology (7). This method uses a porouscarbohydrate capsule to surround the hybridomacells and to retain the antibodies while allowingthe circulation of nutrients and metabolic wastes.After several days in culture, the encapsulatedcolonies are harvested and washed to remove thegrowth medium, the capsules are opened, and theantibodies are separated from the cells. Accord-ing to Damon Biotech, 40 to 50 percent (by weight)of the harvested medium is made up of MAbs.The company claims the microencapsulationmethod for producing MAbs is significantly lessexpensive than the ascites method, provides ahigh concentration of antibodies, and does notrequire the maintenance of animals (18).

Industrial uses for monoclinalantibodies

Because of their unique properties of homoge-neity, specificity, and affinity, MAbs can be usedeffectively in downstream purification systemsfor molecules, especially proteins, A MAb purifica-tion system relies on the binding of a targetmolecule to a MAb immobilized on a solid supportsuch as a bead, The beads are packed in a column,and a mixture containing the target molecule ispassed through the column. The MAb binds themolecule while the impurities wash through thecolumn. Then the binding is reversed, and thetarget molecule is released and collected from thecolumn.

Before MAb-based purification systems can beused in large-scale, several practical and technicalfactors must be optimized. These include cost,purification of the antibody itself, and elution ofthe desired product after purification by the anti-body. Elution requires the use of an antibody ofsomewhat lower affinity than one would use for

diagnostic or therapeutic applications so that thebinding can be reversed easily.

Various important proteins, including alpha-fetoprotein and leukocyte interferon, are nowpurified using MAbs (29,30). MAb purificationsystems may be used in the future to purify a vastnumber of compounds, particularly substancespresent in small amounts.

A simple extension of the procedure just de-scribed involves using MAbs to bind unique sur-face proteins and, with them, the cells to whichthey are attached. This permits separation of cellswith surface proteins of interest and is carriedout by passing the cells over a suitable matrix towhich the antibodies have been bound. In anotherprocedure, fluorescence-activated cell sorting,cells are mixed with fluorescently labeled MAbs,and the mixture is passed through a special instru-ment called a flow cytometer, which responds tothe fluorescent marker and sorts the cells intolabeled populations at rates of 50,000 cells perminute (15,16). So far, fluorescence-activated cellsorting has been used mostly for research pur-poses, but as the method is improved, it may beemployed in a range of clinical applications.

Conclusion

Many fields of biological research are beingaffected by MAb technology. Researchers nowuse MAbs to study problems in endocrinology,biochemistry, cell biology, physiology, parasi-tology, and many other fields, because the prod-ucts of MAb technology are easily standardizedand reproduced. Furthermore, many diagnostic,therapeutic, and industrial uses for MAbs arebecoming apparent, and, as outlined in subse-quent chapters of this report, several U.S. andforeign firms are developing these applications.Industrial purification applications of MAbs andthe widespread advantages of MAb technologyin preparing pure and easily standardized anti-bodies offer substantial benefits in industrial,research, and clinical laboratories. RecombinantDNA and MAb technologies can complement eachother, because rDNA technology can lead to theproduction of new compounds, and MAbs can aidin their identification and purification.

——


Bioprocess technology*

Bioprocesses are systems in which completeliving cells or their components (e.g., enzymes,chloroplasts, etc.) are used to effect desiredphysical or chemical changes.** Since the dawnof civilization, bioprocesses have been used to pro-duce alcoholic beverages and fermented foods.Until the 19th century, alcoholic fermentation andbaker’s yeast production were carried out in thehome or as local cottage industries. As industrial-ization occurred, both these bioprocesses movedinto factories.

Although other minor products made withbioprocesses were added over the years, bio-processes did not become significant in the overallspectrum of chemical technology in the UnitedStates until the introduction of commercialacetone and butanol production during and afterWorld War I. Somewhat later, large-scale micro-bial production of citric acid was introduced, andby the beginning of World War II, the U.S. bio-process industry was thriving, with solvent alco-hols and related low molecular weight compoundscomprising the bulk of bioprocess production,The rapid growth of the petrochemical industryduring World War H caused the displacement ofmicrobial production of industrial solvents,however, and by 1950, microbial production ofsuch solvents (including nonbeverage alcohol) hadvirtually disappeared in the United States.

This contraction of bioprocess manufacturingmight have been the death-knell for old biotech-nology had it not been for the introduction of,and the proliferation of markets for, antibioticsduring the 1940’s. The unique qualifications ofbiological processes for the synthesis of complexmolecules such as antibiotics rapidly becameapparent. Microbial production of a number of

● This section is based largely on a contract report prepared forthe Office of Technolo~ Assessment by Elmer Gaden, Universityof Virginia. The information in that report was extensively reviewedand added to by James Bailey, California Institute of Technology;Harvey Blanch, University of California, Berkeley; and CharlesCooney, Massachusetts Institute of Technology.

● *The term bioprocess is used here in preference to the morefamiliar term “fermentation” because it more correctly identifiesthe broad range of techniques discussed. A fermentation process,though often used to denote any biopmcess, strictly speaking refersonly to an anaerobic bioprocess.

vitamins and enzymes was initiated at about thistime, although only on a small scale. Thus, in thedecade from 1940 to 1950, there occurred a com-plete transformation of industrial bioprocesses.Production of high-volume, low-value-added in-dustrial chemicals (e.g., acetone, butanol) byanaerobic processes employing primarily yeastsand bacteria was largely replaced by more mod-est-scale production of high-value-added products(e.g., pharmaceuticals, vitamins, enzymes) madeby highly aerobic processes in a variety of lessfamiliar bacteria (e.g., the actinomycetes) andsome fungi (see table 1). These aerobic processesare generally quite vulnerable to contaminationby other micro-organisms and require muchcloser control of process conditions. Such aerobicprocesses continue to be used in industry today.

The advent of new biotechnology has sparkedrenewed interest in the industrial use of bioproc-esses. The discussion that follows examines thedependence of new biotechnology, includingrDNA and MAb technology, upon bioprocess tech-nologies. Two aspects of the interrelationship be-tween new genetic technologies and bioprocesstechnologies are emphasized:

● the engineering problems unique to genet-ically modified organisms, and

● the ways in which genetically modifiedorganisms or parts of organisms maybe usedto enhance the efficiency and usefulness ofbioprocesses.

In order to be viable in any specific industrialcontext, bioprocesses must offer advantages over

Table I.-Volume and Value of BiotechnologyProducts

Category ExamplesHigh volume, low value . . . Methane, ethanol, animal

feed, waste treatmentHigh volume, intermediatevalue . . . . . . . . . . . . . . . . . . . Amino and organic acids,

food products, polymersLow volume, high value . . . Pharmaceuticals, enzymes,

vitaminsSOURCE: Off Ice of Technology Assessment, adapted from A. T. Bull, G. HoIt,

and M. D. Lilly, B/otechno/ogy: International Trends and Perspectives(Paris: Organisation for Economic CoOperation and Development,19s2).


competing methods of production. In most cases,bioprocesses will be used industrially becausethey are the only practical way in which a desiredproduct can be formed. Biological processes maybe desirable:

. in the formation of complex molecular struc-tures such as antibiotics and proteins wherethere is no practical alternative,

. in the exclusive production of one specificform of an isomeric compound,

● because microa-ganisms may efficiently ex-ecute many sequential reactions, and

● because bioconversions may give high yields.

Examples of the categories of current uses ofbioprocesses are the folIowing:

production of cell matter ((’biomass” itself)(e.g., baker’s yeast, single-cell protein);production of celI components (e.g., enzymes,nucleic acids);production of metabolizes (chemical productsof metabolic activity), including both primarymetabolizes (e.g., ethanol, lactic acid) andsecondary metabolizes (e.g., antibiotics);catalysis of specific, single-substrate conver-sions (e.g., glucose to fructose, penicillin to6-aminopenicillanic acid); andcatalysis of multiple-substrate conversions(e.g., biological waste treatment).

Bioprocesses may offer the following advan-tages over conventional chemical processes:

●

●

●

●

●

●

●

●

milder reaction conditions (temperature,pressure, and pH);use of renewable (biomass) resources as rawmaterials for organic chemical manufacture,providing both the carbon skeletons and theenergy required for synthesis;less hazardous operation and reduced en-vironmental impact;greater specificity of catalytic reaction;less expensive or more readily available rawmaterials;less complex manufacturing facilities, requir-ing smaller capital investments;improved process efficiencies (e.g., higheryields, reduced energy consumption); andthe use of rDNA technology to develop newprocesses.

Some of the conceivable disadvantages ofbioprocesses, on the other hand, are thefollowing:

●

●

●

●

●

the generation of complex product mixturesrequiring extensive separation and purifica-tion, especially when using complex sub-strates as raw materials (e.g., lignocellulose);problems arising from the relatively diluteaqueous environments in which bioprocessesfunction [e.g., the problem of low reactantconcentrations and, hence, low reactionrates; * the need to provide and handle largevolumes of process water and to dispose ofequivalent volumes of high biological oxygendemand wastes; complex and frequentlyenergy intensive recovery methods forremoving small amounts of products fromlarge volumes of water);the susceptibility of most bioprocess systemsto contamination by foreign organisms, and,in some cases, the need to contain theprimary organism so as not to contaminatethe surroundings;an inherent variability of biological processesdue to such factors as genetic instability andraw material variability; andfor rDNA systems, the need to contain theorganisms and sterilize the waste streams, anenergy-intensive process.

Solutions to some of these problems throughthe use of biotechnology may make bioprocessesmore competitive with conventional chemical syn-theses. Genetic intervention may be used in someinstances to modify micro~rganisms so that theyproduce larger amounts of a product, grow inmore concentrated media, have enzymes withincreased specific activity, or grow at highertemperatures to help prevent contamination.Recombinant DNA technology may lead to thedevelopment of completely new products ormodification of important existing ones. In thepast, some potentially useful bioprocesses have

● It is often said that biochemical catalysis is many times more ef-fective than conventional chemical catalysis. ‘1’his contention is basedon the very high specific activities observed for individual enzymesin vitro. Such rates are seldom encountered under large-scale condi-tions. In general, bioprocesses are extremely slow in comparisonwith conventional chemical processes.


not been economical. Now, however, a combina-tion of improved engineering design and pro-cedures and rDNA technology may yield bioproc-esses that are more efficient than they have beenin the past and therefore more competitive.

Bioprocess essentials*

The steps in bioprocessing are presented sche-matically in figure 9. The substrate and nutrientsare prepared in a sterile medium and are put intothe process system with some form of biocata-lyst–free or immobilized cells or enzymes. Undercontrolled conditions, the substrate is convertedto the product and, when the desired degree ofconversion has been achieved, byproducts andwastes are separated.

Water is the dominant component of themedium for virtually all current bioprocesses.Even when micro-organisms are grown on solidmaterials, an unusual processing mode, thesubstrate must be dampened in order to permitmicrobial growth and enzyme action. Productsmust usually be purified from dilute, aqueoussolutions.

● The bioprocesses discussed here exclude uncontrolled environ-mental applications.

Figure 9.—Steps in Bioprocessing


Bioprocesses require a closely controlled en-vironment, and this necessity markedly influencestheir design. Biocatalyst generally exhibit greatsensitivity to changes in temperature, pH, andeven concentrations of certain nutrients or metalions. The success of a bioprocess depends on theextent to which these factors are controlled inthe medium where interaction between biocata-lyst and substrate takes place.

SUPPLY OF NUTRIENTS

In addition to establishing a suitable environ-ment, the medium must provide for the nutri-tional needs of living cells. A primary requirementis a source of carbon, In addition to supplying theenergy needed for metabolism and protein syn-thesis, carbon sources contribute structuralelements required for the formation of complexcompounds. Often, the carbon source may itselfbe the substrate for the catalyzed reaction, as inthe fermentation of sugar to ethanol. Sugars,starches, and triglycerides, and, to a lesser extent,petroleum fractions, serve as carbon sources.

Other important nutrients required by livingcells are nitrogen, phosphorus, and sometimes ox-ygen. Nitrogen and phosphorus are incorporatedinto structural and functional molecules of a celland may also become pafi of product molecules.Most of the microorganisms currently used byindustry are highly aerobic and require an ade-quate supply of oxygen, but others are strictlyanaerobic and must be protected from oxygen.A number of other nutrients, such as vitaminsand metal ions, though required only in very smallamounts, are nevertheless essential. Some of thesenutrients, especially metals, may appear in theproduct.

In order to make the substrate and nutrientsaccessible to the biocatalyst, the medium must bethoroughly mixed. Most bacteria and some yeastsused in bioprocesses commonly grow as individ-ual cells or as aggregates of a few cells suspendedin the medium, whereas fungi and actinomycetesgrow in long strands. As they grow, all these typesof cells increase the viscosity of the fluid in whichthey are growing in a batch process, making thefluid more difficult to mix, and thus more difficultfor nutrients to reach them.


Since most of the micro~rganisms currentlyused by industry perform their conversionsaerobically, they demand a constant supply ofoxygen. Oxygen’s low volubility in water repre-sents a significant stumbling block to efficientbioprocessing. Since oxygen is depleted duringconversion, the medium must be constantly aer-ated; the more viscous the medium, however, themore difficult it becomes to supply oxygen. Ap-proaches to maintaining an adequate oxygen sup-ply

●

●

●

include:

increasing reactor pressure to increase ox-ygen volubility,the use of oxygen-rich gas for aeration, andchanges in process design and operation.

PURE CULTURES AND STERILIZATION

Most of the products of bioprocesses areformed through the action of a single biocatalyst,either a microorganism or an enzyme. * If foreignorganisms contaminate the process system, theymay disrupt its operation in a variety of ways.They can directly inhibit or interfere with thebiocatalyst, whether it is a single enzyme or acomplete cell, and they may even destroy thebiocatalyst completely. Alternatively, contami-nating organisms may leave the catalyst un-affected, but modify or destroy the product.Foreign organisms can also generate undesirablesubstances that are difficult to separate from theprimary product. In the manufacture of pharma-ceutical products, the risk of toxic impurities isof particular concern.

To avoid or minimize contamination, most cur-rent bioprocess technologies employ pure culturetechniques. The medium and its container aresterilized, and a pure culture consisting of apopulation of a particular species is introduced.In order to avoid subsequent contamination, allmaterials entering the system, including the largeamounts of air required for aerobic processes, aresterilized. The apparatus must be designed andoperated so that opportunities for invasion byunwanted organisms are minimized.

*A significant exception to this generalization is the broad groupof biological waste treatment processes. These processes use mixedand varied populations of microorganisms developed naturally andadapted to the waste stream being treated.

Processing modes

Bioprocesses may, in principle, use any of theoperating modes employed by conventional chem-ical technology. These modes range from batchprocessing to continuous steady-state processing.

In batch processing, the reaction vessel is filledwith the medium containing the substrate andnutrients, the medium is sterilized, the biocata-lyst is added, and conversion takes place over aperiod ranging from a few hours to several days.During this period, nutrients, substrates, agentsfor pH control, and air are supplied to, and prod-uct gases are removed from, the reaction vessel.When conversion is complete, the reaction vesselis emptied, and the purification process begins.Turnover time between batches can account fora significant portion of total processing time.

In continuous steady-state processing, whichlies at the other end of the operational spectrumfrom batch processing, raw materials are suppliedto, and spent medium and product are withdrawnfrom, the reaction vessel continuously and atvolumetrically equal rates. Potential advantagesoffered by continuous processing over batchprocessing include significantly higher productivi-ty, greater ease of product recovery due to thelack of contaminating biocatalyst, and lower costdue to reuse of biocatalyst.

The simplest approach to the implementationof a continuous processing system is to modifya batch reactor so that fresh substrate andnutrients can continually be added while aproduct stream is removed. This simple arrange-ment has one serious drawback: the biocatalystleaves the reactor continuously with the outletstream and must be separated from the product.Several techniques, all of which involve fixing thebiocatalyst in some reamer) have been developedto avoid the biocatalyst’s escape with the reactionmixture and allow its repeated use. The develop-ment of techniques for the immobilization ofbiocatalyst has greatly expanded the possibilitiesfor continuous bioprocesses. Although still notwidely employed for large-scale bioprocesses, thebiocatalyst immobilization techniques now avail-able offer a diversity of new opportunities formore effective bioprocessing (see Box A.-Contin-uous Bioprocessing).

Ch. 3—The Technologies . 49

Figure BXA-1 .—Techniques for Immobilizing Enzymes and Whole Cells

Enzymes can be immobilized by adsorption or chemical bonding (a), by entrapment in a polymer matrix (b), or by microencapsulation (c).

CarrierSOURCE Adapted from E Gaden, “Production Methods in Industrial Microbiology)” Scientific American, September 1981, p. 182,

Lattice Membrane

Table BXA”l.—Characteristics of Immobilization Methods for Enzymes and Cells

Immobilization method

Physical Chemical Entrapment;Characteristic adsorption bonding encapsulation

Preparation . . . . . . . . . . . . . . . . Easy Difficult ModerateActivity . . . . . . . . . . . . . . . . . . . . Low High HighSpecificity . . . . . . . . . . . . . . . . . Unchanged Changeable UnchangedBinding force (retention) . . . . . Weak Strong StrongRegeneration . . . . . . . . . . . . ., . Possible Impossible Impossiblecost . . . . . . . . . . . . . . . . . . . . . . Low High Low

SOURCE Gaden, personal communication, 1983

● Biological. BiocataIwvst stability may be difficult to maintain for long periods of continuous operation.The phenomenon of “culture degeneration,” reported in many instances, deserves careful study.The results of such studies will surely be case-specific and may simply reflect inadequacies in thelmoivledge of nutrient requirements necessary to sustain long-term productivity. As the use of rDNAorganisms grows, this matter will require close attention because of concerns over the stability ofthese types of organisms.

(Operationaf. The primary technical factors acting to limit continuous bioprocessing in the past havebeen difficulties in maintaining sterile conditions and in handling biocatalytic suspensions, especiallythose of filamentous fungi or large cell clumps. The perplexing contamination problem has focusedimprovement efforts on the deficiencies of equipment (mainly pumps) for moving liquids and slurriesand on valves and transfer lines. Many specific difficulties have already been overcome in connectionwith batch operations, and improved equipment design and more rigorous operating proceduresmay result in successful continuous processes.

— —


Figure BXA.2.– Packed-Bed and Fluidized-Bed Reactors

Packed-bed reactorFresh medium

Fluidized-bed reactorSettling region

rOutlet

, . : ” ~ ” f stream

u Packed bed ui t

Spent medium and product Fresh mediumSOURCE: Adapted from E. Gaden, “ProductIon Methods in Industrial Microbiology,” Sckwrtlfk Arnerlcan, September 19S1, p. 19S.

Batch operation currently dominates specialtychemical and pharmaceutical bioprocesses and islikely to continue to do so in the near future, Inaddition to technical limitations on continuousprocessing (see box A), other considerations haveled manufacturers to choose the batch mode.Batch processing is often used, for example, be-cause it offers the operational flexibility neededwhen a large number of products are manufac-tured, each at fairly low production levels; eachprocess unit, more or less standard in design, caneasily and rapidly be switched from one product

line to another. Furthermore, a switch from batchto continuous processing is expensive, and, if acompany has unused batch equipment, it mayfind that a switch to continuous processing is noteconomically warranted in the near term.

Increased use of genetically manipulated bio-catalyst could affect the design and operation ofbioconversion units. Harvey Blanch points out(33):

. . . one of the difficulties which arises from theinsertion of foreign DNA into the organism is re -


version. This can be minimized by placing the cellin an environment in which cellular replicationis minimized, while cellular activity, such as theproduction of enzymes and products, is main-tained at high levels.

Achieving the dual objective of minimal growthand maximum conversion activity requires re-strictive nutrient supplies and high cell densities.Immobilized biocatalyst could be used to achievethese objectives.

Bioprocesses, unlike petroleum refining orpetrochemical operations which completely con-vert raw materials to products or consume themas process fuels, regularly produce large amountsof waste, mainly cell matter and residual nutri-ents. Bioprocesses also require large volumes ofclean water, discharge equivalent amounts ofdilute, high biological oxygen demand wastes, andproduce products in low concentrations. Onesolution to problems associated with bioprocess-ing might be the use of cleaner, more definedmedia, which produce fewer byproducts. An-other solution might be the use of more concen-trated media. The latter option is normally con-sidered in bioprocess development, but the micro-organisms now in use are limited in their toler-ance for high nutrient concentrations. Geneticmanipulation may provide micro-organisms thatare less sensitive to increased product concen-tration.

Raw materials

Current bioprocess technology uses an extreme-ly limited range of raw materials. Just a few agri-cultural commodities—starch, molasses, and veg-etable oil—are employed as raw materials in manyof the existing industrial bioprocesses, Industrychooses these feedstocks for several reasons.There are established markets for these materialsand, for primary products like starch, reasonablydefined quality standards and assay procedures.Several competing suppliers guarantee uniformquality and fairly stable prices. Bioprocess applica-tions constitute only a relatively small fraction ofthe market for agricultural commodities. Theneed for raw materials for bioprocesses, however,could become a major factor in commodity grainmarkets if bioprocesses find a place in large-scalefuel or chemical production.

Less important raw materials are some byprod-ucts of agricultural and food processing, such as“corn steep liquor” and “distillers solubles. ” Pe-troleum hydrocarbons are little used because oftheir high cost. The potential for relatively purecellulose (e.g., delignified wood) remains unreal-ized. * For various carbohydrate wastes—agricul-tural, food, industrial, or municipal—in spite offrequent claims of their availability and low cost,no economical bioprocess applications have yetbeen found.

Biocatalyst

The substances that actually cause chemicalchange in bioprocesses are the enzymes producedby a living cell. For simple enzymatic conversions,isolated enzymes can be used as biocatalyst.When biological transformation of the substrateinvolves several sequential and interrelatedchemical reactions, each catalyzed by a separateenzyme, however, whole cells (most commonly,but not exclusively, micro-organisms-bacteria,yeast, or fungi) are used as biocatalyst. Bioproc -esses used for the synthesis of complex molecularstructures (e.g., antibiotics or proteins such as in-sulin), for example, require entire systems of en-zymes, Such systems do not yet function in con-cert outside a living cell. Indeed, when the desiredproduct is the cell itself (e.g., baker’s yeast orsingle-cell protein), all the enzymes comprising thecell’s growth machinery are components of thecatalytic system.

An inspection of the immense spectrum of orga-nisms whose biochemical capabilities have beenreasonably well defined reveals that bioprocessesemploy only a small, select group of biocatalyst.If one eliminates those organisms considered“natural populations” in food fermentation orbiological waste treatment, the range of biocata-lyst employed in bioprocesses is even morelimited. Some animal cells and tissues areemployed for vaccine production and relatedactivities, but the catalytic capabilities of plantcells, except for some algae, have not yet beenemployed commercially. It is possible that biotech-nology will provide a means whereby importantcatalytic activities from poorly understood

● See Chapter 9: Commodit}r Chemicals and Energv Production,

52 . commercial Biotechnology: An International Analysis

organisms can be transferred to cells whose large-scale growth is well understood.

Wider availability of thermotolerant biocatalystis important for all industries using bioprocesses.Recent research on the development of thermo-tolerant biocatalytic agents has advanced thepotential efficiency of bioprocesses. The advan-tages of thermotolerance include:

●

●

●

●

reduced susceptibility to contamination;easier removal of metabolic heat;more complete and rapid conversions whenvolatile inhibitors are present (but oxygenvolubility is reduced); andeasier recovery of volatile products (e.g.,ethanol).

Biocatalyst that can withstand high pressuremay also be useful industrially. For instance,higher pressures will increase the volubility ofoxygen.

Finally, research investigating the relationshipbetween the structure and the function of en-zymes is proceeding. Ultimately, the aim is to beable to design, with the help of computers, anenzyme to perform any specific catalytic activityunder given conditions. Although this procedurewill not be done routinely for many years, it willsoon be possible, using rDNA technology, tomodify the structure of an enzyme to improveits function in a given condition, such as at a par-ticular pH or temperature. Thus, biotechnologycould greatly affect the efficiency of bioprocesses.

Bioprocess monitoring andassociated instrumentation

Despite the need for close control of processvariables during a bioprocess operation, the tech-niques available for making measurements on-lineare extremely limited. Existing equipment canreadily monitor only temperature, pH, dissolvedoxygen concentration, and evolution of gases. Al-though many other sensors have been developedto measure other variables (e.g., glucose levels),all are sensitive to steam sterilization. Thus, theirusefulness in monitoring most bioprocesses is lim-ited. Many critical variables are able to be moni-tored only by withdrawing samples from the reac-tion vessel and analyzing them off-line, and, even

then, it is difficult to determine key characteristicsaccurately. When measuring cell mass (an indi-cator of growth), for example, most process oper-ators simply note such crude indicators as packedcell volume, turbidity, or, at best, dry weight.

It is possible to measure the compositions andflow rates of gaseous streams entering and leavingthe reactor and to use the values obtained fromsuch measurements to help estimate key processconditions indirectly. Such procedures have beengreatly facilitated by the use of computers. Thereal potential of computer control, however, willnot be realized until a greater range of reliableon-line sensors becomes available. *

A number of European, Japanese, and Amer-ican groups have developed improved sensors forbioprocess control, but, so far, most devicesrequire removal of samples for off-line analysisbecause the sensors cannot withstand steriliza-tion. Continuous sampling combined with varioustypes of rapid instrumental analyzers offers areasonable compromise, but, with this approach,there is a time lag between the actual sample timeand the time at which the assay informationbecomes available.

Sophisticated instrumentation will have increas-ing use in bioprocess monitoring. High perform-ance liquid chromatography, for example, is usedto identify particular compounds in a mix of com-pounds and is one of the fastest growing instru-mentation fields. Flow cytometry has potential usein measuring process variables such as cell size(an indicator for adjusting nutrient flows) and cellviability. Other instrumentation will surely beused as bioprocess monitoring becomes morewidely investigated.

Computer-coupled bioprocesses can greatly im-prove monitoring and controlling the growth con-ditions during a bioprocess run. Computers canbe used to analyze the data from sensors andother monitoring instrumentation and respondto these data by adjusting process variables, suchas nutrient flow. Additionally, computer inter-faces can be used:

● to schedule efficiently the use of equipment;. to alarm operators when necessary;

*For a discussion of biosensors, see Chapter 10: Bioeleetronics.


Photo credif: Porfon/LH /nternationa/

100-liter pilot plant bioreactor with computer controls

● to log, store, and analyze data; and● to inventory raw material depletion

product synthesis.

These functions optimize the methodology

because of increased interest in bioprocesses byand electronics experts, as evidenced by the recent

joint venture between Genentech and Hewlett-Packard. *

andorganization of bioprocessing within a plant. Com - The automation of bioprocessing will be of crit -panies are only now starting to use computer- ical importance in the future as companies com -controlled bioprocesses because of cost, lack of pete for shares in biotechnology product markets.good sensors, and interfacing problems. Yetadvances in this field are sure to occur soon ‘See Chapter 4: F’irrns Commercializing Biotechnolo@.


As automation reduces the labor intensity oflaboratory tasks, the pace of competition willquicken, and countries with sophisticated soft-ware to direct the automation will possess an ad-vantage in the commercialization of biotech-nology.

Separation and purificationof products

Separation and purification techniques used inbioprocesses are the aspect of bioprocess engi-neering most in need of attention, especially forthe production of novel products such as proteins.Research is needed to find highly selective recov-ery techniques that leave as little residual productas possible in the medium and thus lessen the la-bor intensity associated with downstream proc-essing. An example of the effort expended indownstream processing is provided by the newplant Eli Lilly built to produce human insulin(Humulin”). The plant employs 220 people, 90percent of whom are involved in recoveryprocesses.

Some of the possibilities for improving recoverytechniques now under consideration include thefollowing:

● Ultrafiltration. Membranes and other filtra-tion systems, such as porous metals, offermany advantages, and considerable ex-perience in other areas of chemical tech-nology is already available. Some U.S. com-panies, such as Millipore, Amicon, andNucleopore are making advances in this area.

● Continuous chromatography and highperformance liquid chromatography. If theseapproaches, already available on the labora-tory scale, could be scaled-up, it would bepossible, in principle, to collect a crude prod-uct from the medium and then, by selectiveelution, recover product, reusable nutrients,and inhibitory substances separately, oneAmerican manufacturer (Waters, a Milliporesubsidiary) claims to have developed a pilot-scale chromatographic unit.

● Electrophoresis. Electrophoretic methods,especially continuous flow, can separate pro-teins, peptides, and nucleic acids on the basis

●

of their electrical charge. The advantage ofthis separation method over some others isthat it can run continuously and can effec-tively separate molecules in large samplevolumes. The potential of continuous-flowelectrophoresis for producing commercialquantities of high purity substances such aspharmaceuticals was demonstrated on arecent space shuttle mission. The electro-phoresis experiment, cosponsored by Mc-Donnell Douglas and the National Aeronau-tics and Space Administration, demonstratedthat under weightless conditions an electro-phoresis system, identical to one tested onEarth, separated about 700 times more ma-terial in a given period of time and alsoachieved four times the purity while process-ing 250 times more material.Monoclinal antibodies. Immobilized MAbsare being used as purification agents for pro-tein products (see “Monoclinal AntibodyTechnology” section above). This techniquebest suits large molecular weight and high-value-added products such as proteins,

Genetic modifications of micromganisms usedin bioprocessing could also aid in recovery proc-esses. Two changes in particular would greatlyimprove the yield and ease recovery of proteins.First, microorganisms could be developed thathave minimal intracellular proteindegrading en-zymes. The presence of these enzymes will de-crease the yield of protein product. Second, a pro-tein is much more easily purified if it is secretedfrom the cell into the surrounding medium. Thegenetic incorporation of protein secretion mech-anisms will lower production costs dramatically.

Although purification and separation protocolshave been developed for existing bioprocesses,new bioprocesses will present new challenges.For example, rDNA technology has led to a newset of bioprocesses that synthesize proteinproducts, and substantial work is needed to im-prove recovery strategies for large-scale proteinpurifications. In addition, one of the factors thatrestricts the use of bioprocesses for producingcommodity chemicals is the expense of recoveringthese low-value-added chemicals from diluteaqueous solutions.


Culture of higher eukaryotic cells

The organisms used most extensively in large-scale bioprocesses are prokaryotes (e.g., bacteria)or simple eukaryotes (e.g., yeast). These are hardyorganisms which grow to high cell densities andconsequently give high product yields.

Certain products can be obtained in some situa-tions only from the large-scale cultivation ofhigher eukaryotic cells. As noted in table 2, forinstance, many proteins that are potentially use-ful (e.g., in medicine) have not been isolated inlarge enough quantities to study adequately. Ifeukaryotic cell culture made these proteins avail-able in larger quantities, their amino acid se-quence could be determined, their genes cloned,and even more of the proteins could be produced.Furthermore, some proteins probably need “post-translational modifications” (changes in proteinstructure after the protein is made from mRNA)that only higher eukaryotes can perform. Thesemodified proteins may onty be made in eukaryoticcells, Also, in many cases, the production of sec-ondary metabolizes in plant cells is a function ofseveral enzymatic functions, most of which arenot known. Therefore, the growth of plant cellsin culture might be the easiest way to produceuseful plant compounds. Finally, many individualsthink that the growth of hybridomas would beeasier and more economic in culture if the culturetechnology were better developed (see “Mono-clinal Antibody Technology” section above). Asbiotechnology becomes more integrated into theindustrial structure, the development of moreefficient and economic bioprocess technologiesfor higher eukaryotic cells will increase inimportance.

Photo credit: Sci&Jce Photo Library and Porion/LH International

Laboratory tissue culture production

The technologies developed for the growth ofmicroorganisms have limited applicability to thegrowth of higher eukaryotic cells because of dif -ferences between microbial and mammalian cells(see table 3). Mammalian cells are larger, morefragile, and more complex than microbial cells. *

● Most cell culture research has been done with mammalian cells,so the work reported here focuses on those cells. Problems withplant cell culture are similar to those of mammalian cell culture.

Table 2.—Situations Potentially Requiring Large-Scale Eukaryotic Cell Culture

Cell culture system Reason for large-scale eukaryotic cell culture

Cells producing useful proteins . . . . . . . . . . . Not large enough quantity to determineamino acid sequence; therefore cannot userDNA technology

Cells producing modified proteins . . . . . . . . Modification systems present only in highercells

Plant cells producing useful secondarymetabolizes. . . . . . . . . . . . . . . . . . . . . . . . . . . Enzymatic pathways for metabolic production

not well understood; therefore cannot userDNA technology

Hybridomas . . . . . . . . . . . . . . . . . . . . . . . . . . . . Mouse acites system has limited capacityand applicability


.


Photo cmdlt: Porton/LH International

Bioreactor specially designed for the growth of plantor animal cells

Furthermore, mammalian cells have very complexnutritional requirements, which have not beencompletely defined. They require serum fromblood for growth, and the essential compositionof serum is not well characterized. In contrastto microbial cells, mammalian cells are not nor-mally exposed to the environment, but are con-stantly surrounded by a circulatory system thatsupplies nutrients and removes wastes. Whenthese cells are grown in culture, the medium isinitially clean and nutritionally balanced; as thecells take up the nutrients and excrete wasteproducts, however, the medium becomes muchless like the cells’ normal environment. This prob-lem, along with the problem of fragility, requiresmodified reactor design (39).

Some mammalian cells grow in suspension likemicrobial cells, but most higher cells must attachto a solid surface. A major problem with large-scale cell growth of mammalian cells has been theavailability of large, accessible surfaces for cellgrowth. The attachment of cells to microcarriers,or very small beads, has begun to solve many ofthe problems associated with large-scale mam-malian cell culture. The beads provide a largeamount of surface area and can be placed in acolumn where either a continuous-flow or fluid-ized-bed bioreactor can be used for cell growthand product formation (see box A). Either of thesebioreactors is gentler than a stirred tank reactor.Additionally, because of the continuous natureof these bioreactors, fresh nutrients are addedand wastes are removed continuously.

The instrumentation requirements for mam-malian cell growth are different than those formicrobial growth. The lower rates of metabolismand lower density of mammalian cells requiremore sensitive sensor systems than for microbialcell growth. Additionally, because the nutritionalrequirements are so much more complex, dif-ferent strategies are needed to monitor andcontrol cell growth. These problems are justbeginning to be addressed (4o).

Priorities for future research

Priorities for future generic research in bio-process engineering that would be applicable


Table 3.—Comparison of Microbial and Mammalian Cells

Characteristic Microbial cells Mammalian cells

Size (diameter) . . . . . . . . . . . . 1-10 microns 10-100 micronsMetabolic regulation . . . . . . . Internal Internal and hormonalNutritional spectrum . . . . . . . Wide range of substrates Very fastidious natureDoubling time . . . . . . . . . . . . . Typically 0.5-2.0 hours Typically 12-60 hoursEnvironment . . . . . . . . . . . . . . Wide range of tolerance Narrow range of toleranceOther characteristics. . . . . . . Limited life span of normal

cellsLack of protective cell wall

SOURCE: Office of Technology Assessment, adapted from R. J. Fleischaker, Jr., “An Experimental Study in the Use of instru-mentation To Analvze Metabolism and Product Fermentation in Cell Culture,” thesis, Massachusetts Institute ofTechnology, Cambridge, Mass., June 19S2.

to all industries using biotechnology includeresearch in the following areas:

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continued work on the practical use of anddesign of bioreactors for immobilized cell andenzyme systems;development of a wider range of sterilizablesensors for process monitoring and control;improved product recovery techniques,especially for proteins;general reactor design and practicalapproaches to better oxygen transfer;inhibition of intracellular protein-degradingenzymes;improving the genetic stability oforganisms;

Chapter 3 references

Recombinant DNA technologyreferences

rDNA

“ Hvferemw ot gerwral ]nterest are marked wrth asterw.ks

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protein secretion mechanisms;improved methods for heat dissipation dur-ing bioprocessing; andbiochemical and physiological mechanismsfor temperature and pressure tolerance.

The large-scale culture of eukaryotic cells isbeginning to receive some research attention.Because of the complex nutritional requirementsof eukaryotic cells, the cost of the medium is high.If industry is going to adopt eukaryotic cell culturetechnology, the development of economic artifi-cial media is important. Also important is thedevelopment of new bioreactor design and instru-mentation for the control of cell growth.

1. Guarente, L., Roberts, T., Ptashne, M., et al., ‘(ATechnique for Expressing Eukaryotic Genes inBacteria, ” Science 209:1428-30, 1980.

2. Ris, H., and Kubai, D. F., “Chromosome Struc-ture, ” Ann. Rev. Genetics 4:263-94, 1970.

3. Shortle, D., DiMaio, D., and Nathans, D., “DirectedMutagenesis,” Ann. Rev. Biochem. 15:265-94,1981.

Monoc]onal antibody technologyreferences *

*4. Baskin, Y., “In Search of the Magic Bullet,”Technology Review, October 1982, pp. 19-23.

*5. Business Week, “Biotechnology’s New Trust inAntibodies,” May 18, 1981, pp. 147-156.

6. Cavagnaro, J., and Osband, M., “A StatusReview—Human-Derived Monoclinal Antibod-ies, ” Genetic Engineering News, May/June 1983,Pp. 6-7.

7. Chemical and Engineering News, “Method MayBoost Monoclinal Antibody Output,” Jan. 11,1982, p. 22.

*8. Chisholm, R., “On the Trail of the Magic Bullet,”High Technology, January 1983, pp. 57-63.

9. Croce, C. M., Linnenbach, A., and Hall, W., et al.,“Production of Human Hybridomas Secreting An-tibodies to Measles Virus,” Nature 288:488, 1980.

10. Diamond, B. A., Yehon, D. E., and Scharff, M. D.,“Monoc]onal Antibodies: A New Technique forProducing Serological Reagents, ” New Eng. J.Med. 304:1344, 1981.

11. Fox, P. L., Bernstein, E. H., and Berganian, R. P.,

25-561 0 - 84 - 5


“Enhancing the Frequency of Antigen-SpecificHybridomas)” Eur. J. Immune]. 11:431, 1981.

12. Galfre, G., Milstein, C., and Wright B., “Rat x RatHybrid Myelomas and a Monoclinal Anti-Fd Por-tion of Mouse IgG,” Nature 277:131, 1979.

*13. Gatz, R. L., Young, B. A., Facklam, T. J., andScantland, D. A., “Monoclinal Antibodies: Emerg-ing Product Concepts for Agriculture and Food, ”Bioflechno]ogy, June 1983, pp. 337-341.

14. Gefter, M. L., Margulies, D. H., and Scharff, M.D., “A Simple Method for Polyethylene Glycol-Promoted Hybridization of Mouse MyelomaCells,” Somat. Cc/] Genet. 3:231, 1977.

15. Herzenberg, L. A., and Herzenberg, L. A.,“Analysis and Separation Using the FluorescenceActivated Cell Sorter (FACS),” Handbook of Ex-perimental Immunology, vol. 2, D. M. Weis (cd.)(London: Blackwell Scientific Publications, 1978).

16. Hoffman, R. A., and Hansen, W. P., “Immuno-fluorescent Analysis of Blood Cells by FlowCy-tometry,’’lnt. J. Immunopharmac. 3:249) 1981.

17. Karpas, A,, Fischer, P., and Swirsky, D., “HumanPlasmacytoma With an Unusual Karyotype Grow-ing in Vitro and Producing Light-Chain Immu-noglobulin, ” Lancet 1:931, 1982.

18. Kindel, S., “The Birth of Bioindustry,” Forbes, July18, 1983, pp. 130-132.

19. Klausner, A., “Genentech Makes MonoclinalPrecursors From E. coZi,” Bioflechnofogy, July1983, pp. 396-397.

20. Kohler, G., and Milstein, C., “Continuous Culturesof Fused Cells Secreting Antibody of PredefineSpecificity,” Nature 256:495, 1975.

21. Kohler, G., and Milstein, C., “Derivation of SpecificAntibody-Producing Tissue Culture and TumorLines by Cell Fusion,” Eur. J. Immunol. 6:511,1976.

*22. Langone, J., “Monoclonals: The Super Anti-bodies,” Discover, June 1983, pp. 68-72.

23. Lewin, R., “An Experiment That Had To Succeed,”Science 212:767, 1981.

24. Melchers, F., Potter, M., and Warner, N. L. (eds.),“Lymphocyte Hybridomas,” Curr. Top. Microbioi.Immunol., vol. 81 (New York: Springer-Verlag,1978).

*25. Milstein, C., “Monoclinal Antibodies,” ScientificAmerican 243:66-74, 1980.

26. Montagnon, B. J., et al., “Large-Scale Culture ofVero Cells in Microcarrier Cuhure for Virus Vac-cine Production: Preliminary Results for KilledPolio Virus Vaccine, ” Inter. Deve]. Biol. 47:55,1980.

27. Olsson, L., and Kaplan, H., “Human-HumanHybridomas producing Monoclinal Antibodies of

31

*32.

35,

36.

37.

38.

39.

40.

41.Microbiology,” Scientific American, September1981, pp. 181-196.

“ *Most of these references are of general interest for those wishing to pur.sue the subject of bioprocess technology further,


42. Gaden, E. L., “Bioprocess Technology: An Analysisand Assessment,” contract paper prepared for theoffice of Technolo~V Assessment, U.S. Congress,July 1982.

43. Gaden, E. L., University of Virginia, personal com-munication, 1982.

44. Hochhauser, S. J., ‘(Bringing Biotechnology tohlarket,” High Technology, February 1983, pp.55-60.

45. Johnson, 1. S,, ‘(Human Insulin From RecombinantDNA Technology,” Science 219:632-637, 1983.

46. Kindel, S., “Enzymes-The Bioindustrial Revolu-tion)” TechnoZo~, November/December 1981, pp.62-74.

47. Klibanov, A. M., “Immobilized Enzymes and Cellsas Practical Catalysts,” Science 219:722-727, 1983.

48. National Academy of Engineering, “Genetic En-gineering and the Engineer” (Washington, D. C.:National Academy Press, 1982).

49. Wang, D. I. C., Cooney, C. L., Demain, A. L., etal., Fermentation and Enzuyme Technolo~ (NewYork: John Wiley & Sons, 1979).

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Commercial Biotechnology: An International Analysis (Part 5 of 35)