1 chemicals and raw materials€¦ · back during the e-business boom, many people talked about...

8 BioProcess International 2003 INDUSTRY YEARBOOK

Chemicals and Raw Materials

Raw materials, of course, constitute a large andvaried category of products. They have applicationacross the entire development process. Here’s

where you’ll find chemicals and reagents for use in GLPassays for validation and quality testing as well as earlyproduct and process development research. Here tooyou’ll find formulation excipients and viruses for testingpurposes and for use as viral vectors. Detergents,enzymes, amino acids and peptides, antibodies andantigens, growth media, and genes and nucleic acids areall here as well.

Often, companies that produce the raw materialsused in GMP processes must comply with GMPs,themselves. Some use bioprocess techniques andapplications; some use synthetic techniques. Sourcematerials must be documented to support theregulatory scrutiny that end users will receive. And thevendor companies are expected to keep up with theadvancing science and regulatory changes.

SUPPLIER QUALIFICATION

Regulations define raw materials as any ingedients usedin drug manufacture, including those that do notappear in the final product. Cell culture andchromatography media, buffers and sera, and otherchemicals and reagents all come under that definition.They must meet specified criteria for identity, purity,traceability, and suitability for their intended use. ForGMP manufacturing operations, raw materialsacceptance specifications must be developed todocument identity and quality. Those specs are drawnup with input from manufacturing, product andprocess development, and QA/QC groups at acompany. “Raw materials specifications should includethe amounts of material necessary for all relevantsampling and testing information. . . . Testingspecifications should be based on good science, process

needs, and information derived from the manufacturer’sC of A or developed internally” (1). Vendors’certificates of analysis (C of As) are just the beginning.Their critical assay results must be validated by in-housetesting (or analysis done by a contract laboratory) andvendor audits. Vendor audits for suppliers of critical rawmaterials may be nearly as extensive as those forcontract service providers. Good vendor audits “mayadd more safety than that provided by direct QCtesting” (1).

Vendors and their raw materials are often classifiedaccording to their suitability for use in the GMPprocess. Some are rejected outright. Others are allowedonly for “provisional” use, typically in the manufactureof preclinical materials. Full approval for use requiresapproved raw material specifications and ID testmethods, a record of success in development andclinical manufacturing, a completed vendor audit, andthe initiation of C of A testing. Once that testing iscomplete for three lots, and three or more lots havebeen used in the manufacture of clinical batches thathave met product specifications, the raw material hasbeen “qualified” for use. “Certified” suppliers ofqualified materials have good supplier/partnershipagreements in place and a long, successful history with aparticular biotherapeutic/biodiagnostic company.

THE PROCUREMENT PROCESS

Back during the e-business boom, many people talkedabout online procurement of raw materials being “thewave of the future.” That has yet to come to fruition asa major trend in the biopharmaceutical industry. In aregulated industry, raw materials must be rigorouslycontrolled. A slight change of ingredient — or ashipping problem that delays delivery — can affect thewhole manufacturing process and consequently, futureregulatory submissions. Thus, relationships betweenvendor and client are very important. Purchasing andprocurement require a well-documentedagreement/contract rather than just a simpletransaction.

Raw materials arrive at a secure, centralized locationin the company and are stored for inspection, thentagged and quarantined by the quality controldepartment. QC personnel will sample and test theproducts according to the specifications mentionedabove. As described in the sidebar, internationalpharmacopoeias provide valuable references. But theydiffer in their takes on many commonly used materialsand test methods. “Once the necessary testing iscompleted, disposition of the material is determined,stability testing is performed, expiration dates are set,and archive samples are collected and stored” (1).Biotherapeutics manufacturers must be able to

TE C H N O LO G Y OV E RV I E WPH

OTO

DIS

C(W

WW

.PH

OTO

DIS

C.C

OM

)

document the origin of all raw materials used for eachprocess.

Vendor Audits: The basics of vendor audits can befound in “almost any book on the ISO9000 qualitysystem” (2). Long-term relationships between suppliersand customers offer the chance for improvements overtime and provision of extra services as vendorcompanies seek to keep their best customers happy. Avendor audit strategy is therefore useful for workingtoward this kind of competitive advantage. The vendoraudit team often includes purchasing, operations,product/process development, and quality controlpersonnel. It works toward GMP certification ofpotential vendor “partners,” including evaluation offacilities, equipment, shipping/receiving protocols,personnel training and contamination control, and lotcoding, traceability, and recall procedures.

Another relevant acronym comes in at this point:HACCP (hazard analysis and critical control points), “amethodical and systematic application of science andtechnology to evaluate, plan, control, and document thesafe and efficient manufacture of products, from rawmaterial to end use” (3). HACCP came out of the earlyspace program but is now in use throughout the food,cosmetic, and pharmaceutical industries for mappingand identifying (then monitoring and documenting)critical hazard points and activities in manufacturing.Even raw material transport conditions can affect theprocess if, for example, a material is detrimentallyaffected by a lack of temperature control.

As with any relationship, supplier “partnerships”must be continually assessed and improved over time.Trend and comparison reports will keep track ofdeliveries, costs, service, and other important criteria.The vendor audit is not the end of vendor qualification,but the beginning.

RESOURCESIBC Conferences and Professional Development Courses:

1–2 December 2003 Antibody Therapeutics: From ClinicalDevelopment to Business Strategies. San Diego, CA,www.lifesciencesinfo.com.

8–10 December 2003 Biopharmaceutical Manufacturing Processes:Development, Optimization and Validation. Dedham, MA,www.lifescienceinfo.com.

Drug and Market Development (D&MD) Publications:Carter, JM. A Guide to Assay Development. March, 2003,

www.drugandmarket.com/9114.Kanarek, AD. The Bioprocessing Industry: Increasing Capacity,

Production, and Efficiency. December, 2002,www.drugandmarket.com/9095.

Kanarek, AD. A Guide to Good Facility Design Practice: TheDesign of Regulatory-Compliant Facilities for Biologicals andBiopharmaceuticals. March 2003, www.drugandmarket.com/9067

Kanarek, AD. A Guide to Good Manufacturing Practice. January2001, www.drugandmarket.com/9019.

Kanarek, AD. A Guide to Good Validation Practice. October2001, www.drugandmarket.com/9049.

Advancing Your Life Sciences –From Discovery to LaunchTM

Merck KGaA · ProcessingDarmstadt · GermanyPhone: +49(0)6151/72-2009E-Mail: [email protected]

www.merck-lsp.de

Life Science Products – ProcessingYour link to success in the drug development chain

Buffers for

Bioprocessing

Reliable biological buffers are a fundamentalrequirement of the pharmaceutical and bio-technology industries. To guarantee this reli-ability Merck has invested in a new modernproduction facility.Enjoy the security, confidence and versatilityprovided by:

· Guaranteed purity· Absence from interfering contaminants· Tailor made specifications and packaging

Setting the standards…

…securing your futureCircle Reader Service No. 114

Regulatory Guidances:Guidance for Industry: Manufacturing, Processing, or Holding

Active Pharmaceutical Ingredients. Center for Drug Evaluation andResearch, March 1998. www.fda.gov/cber/gdlns/active.pdf

Guidance for Industry: For the Submission of Chemistry,Manufacturing and Controls and Establishment DescriptionInformation for Human Plasma-Derived Biological Products, AnimalPlasma or Serum-Derived Products. Center for Biologics Evaluationand Research, February 1999. www.fda.gov/cber/gdlns/cmcplasma.pdf.

Guidance for Industry: Submitting Type V Drug Master Files to theCenter for Biologics Evaluation and Research. Center for BiologicsEvaluation and Research, August 2001.www.fda.gov/cber/gdlns/dmfv.pdf.

For other regulatory guidances, see also the following web sites:www.fda.gov/cder/guidance/index.htm, www.emea.eu.int/htms/human/bwp/bwpfin.htm, and www.ich.org/ich5q.html. For furtherhelp, go to www.phrma.org, www.pda.org, www.usp.org,www.pheur.org, or http://jpdb.nihs.go.jp.

REFERENCES1 Del Tito Jr., BJ; Tremblay, MA; Shadle, PJ. Qualification of

Raw Materials for Clinical Biopharmaceutical Manufacturing.BioPharm 1996, 9, 10 (November), 45–49.

2 Vesper, JL. Quality and GMP Auditing: Clear and Simple.Interpharm Press: Englewood, CO, 1997.

3 Vega-Mercado, H; et al. HACCP: A Process Validation Toolfor Ensuring Quality of Biotech and Pharmaceutical Products.BioProcess Int’l. 1, 5; 50–57.

Contract Services

AA t a recent conference in Monte Carlo (1), onepresenter said in no uncertain terms thatoutsourced manufacturing in the life sciences

industry is no longer an option, but a necessity.Biopharmaceutical companies are often started byentrepreneurial scientists who have very specificexpertise. They have enough trouble learning how tofinance their new endeavors. It’s just not possible for asmall company surviving by the skin of its venturecapitalist’s teeth to hire in-house all the necessarypersonnel for making an FDA-regulated business work:regulatory and compliance specialists, validation experts,quality assurance people, process developers,formulators and drug delivery scientists, and so on.These days, much of that work is instead “farmed out”to contractors or consultants.

And it isn’t just the manufacturing that’s outsourced.Contract research organizations provide analyticalexpertise to back up research and development effortsand to help work out formulation and stability issues.Clinical management companies understand the ins andouts of clinical trials. And consultants provide myriadservices, from regulatory affairs to training to managingthe outsourcing relationships themselves.

Bioprocess manufacturing capacity has been an issuefor many months now. Although interpretations vary,many people still believe that capacity issues are themain challenge facing the bioprocessing industry.Future applications of protein therapeutics are expectedto put pressure on the capacity for upstream productionand on the scalability of unit operations for downstreamprocessing. Not only are the facilities and equipment inshort supply, but personnel are needed who possess theskills and expertise required to run new bioprocessoperations. Many companies without the necessaryresources must look to competent suppliers andcontract manufacturing organizations for help. Atpresent, signs still indicate that the potential demandmay exceed planned supplies.


LON

ZA

GR

OU

PLT

D. (

WW

W.L

ON

ZA.C

OM

)

THE PHARMACOPOEIAS

The international pharmacopoeias promote publichealth by providing authoritative standards andinformation for diagnostic and pharmaceuticalingredients and development support technologies.The United States Pharmacopeial Convention, Inc.(www.usp.org) states its vision, “to be a leader in . . .creating a unique knowledge base . . . accessible topeople throughout the world.” With the help of morethan 650 elected volunteers, the USP establishesstate-of-the-art standards to ensure the quality ofmedicines and other health products. It alsoparticipates in dietary supplement verification and thedissemination of information. Its members come fromindustry, state associations and colleges of pharmacy,the federal government, consumer organizations, andnational and international professional, scientific, andtrade organizations. Its equivalent in Japan is theJapanese Pharmaceopoeia (http://jpdb.nihs.go.jp),which is government-based.

The European Pharmacopoeia (www. pheur.org) wasfounded in 1964 by Belgium, France, Germany, Italy,Luxembourg, the Netherlands, Switzerland, and theUnited Kingdom. It now involves 26 member statesinvolved in establishing “common standards for thecomposition and preparation of substances used inthe manufacture of medicines.” As with the USP, theEP publishes its standards as monographs. It isrecognized as an authority on quality and safety.Biological medicine standardization efforts are underway in cooperation with the European Union’sregulatory authorities.


US REGULATION

In August 1999, CBER published a guidance forindustry, still in draft form. Its general provisions are inoperation now because of regulations in force thatgovern related activities. It often takes several years for adraft guidance to be finalized, but its provisions arefollowed almost immediately.

The regulation of contract manufacturing changedquite a bit from earilier guidances to accommodate adefinition of manufacturer established in 1996.Companies may now “contract with another entity(ies)to perform some or all of the manufacture of a productas a service to the license applicant” (2). The draftguidance also explains the FDA’s recommendations forcontracts and quality agreements between sponsorcompanies and their contractors. In a contractmanufacturing arrangement, the license holder (sponsorcompany) is solely responsible to the FDA for allcompliance issues and product quality and safetyassurance.

INDUSTRY CONSOLIDATION

Since its resurrection in the mid-1990s, biotechnologycontract manufacturing has seen its share ofconsolidation (3). Between 1996 and 2002, the largestCMOs (those with market capitalization between $15million and $20 million or so) were bought up by finechemicals companies that wanted to get into the lifesciences market. Lonza bought Celltech; DSM boughtGistbrocades/Bio-Intermediair; Diosynth boughtCovance; Cambrex bought BioScience ContractManufacturers and Marathon; Avecia bought ZenecaSpecialties, and so on. The fine-chemical parentcompanies have years of experience in contractmanufacture for other industries. They are well suited tojudge how much capacity is likely to be necessary andwhen, and they have the deep pockets necessary toinvest in new capacity when required. The companiescurrently operate at 2–10,000 L scales, with the eyes oftheir management set on 30,000-L bioreactors in thefuture.

Those mergers — and the continued scale-up theyrepresent — demonstrate a continuing maturing of thebiotechnology market as a whole. Early CMOinvestment was aimed at clinical manufacturing. With anadvancing global biotech drug pipeline, contracting hasbecome an established strategy for many biotechcompanies. CMOs have responded with investments toaddress that market for late stage and licensed productmanufacture.

A different kind of consolidation may be in store forsome smaller players. Several contract researchorganizations (CROs) are vying for a piece of thebiotech pie. As in any other industry, companies in acompetitive market need to offer more or better servicesto stand out from their competitors. In the CROindustry, that competitive pressure could drive a movetoward “molecule to market” services. So the largestCROs may want to acquire manufacturing capacity.

Looking to the future can be tricky. Some industry

experts believe that contract research and manufacturingare such entirely different disciplines that they willalways require different facilities and staff. The CRObusiness may continue to focus primarily on drugdiscovery and clinical investigations.

THE INS AND OUTS

As with any industry, outsourcing follows a pendulumswing pattern. For a while, the majority of companiesdo most of their work in-house. Then some brightmiddle manager trying to work her way up in the worldrealizes that she could save the company a lot of moneyand headaches by hiring a specialist company to do thevalidation. So the company fires all its validation peopleand hires a contractor. And over the course of the nextfive or ten years, the work creeps back in-house. Inbiotechnology (and in classical pharmaceuticals as well)the pendulum is on the outsourcing side right now, butit will inevitably swing back the other way. In the drugindustry, it may already be swinging back in-house.Then, of course, it will swing back to outsourcing again.

Wherever on the pendulum an industry is at anygiven moment, the reasons for insourcing oroutsourcing remain pretty much the same. Companiesuse contractors to get access to specialized talentand/or to save money. They move work back in-housefor more control of their processes (especially if they’vehad a bad experience with a contractor) and/or to savemoney.

Other factors figure into those basic ones. Forexample, the amount of material needed figures into thecost of making it in-house compared with outsourcingit. If you’re doing only two or three production runs ayear, it would be more cost effective to contract thatout than to build a dedicated facility. If you’re runningtwo or three shifts full time, on the other hand, doing ityourself might be more cost effective, especially whencontrol issues are considered.

Another factor is equipment maintenance andupdating. “If you’re not wearing your equipment outbefore it becomes obsolete, you might be better offoutsourcing that work and letting the maintenance andupdating be someone else’s headache,” says NancyChew, president of Regulatory Affairs, North AmericaLLC.

The growth of a company is also a consideration.Often, little biotech start-ups get bought out by bigpharma companies (or bigger biotech companies) thatalready have manufacturing capabilities. For smallcompanies hoping to merge into bigger ones,contracting out current manufacturing needs wouldmake sense.

A GLOBAL MARKET

As the biotech industry has matured, it has spreadaround the world. Although it has always been a globalscience, the commercialization of biotechnology hasspread out from the United States, Europe, and Japanduring the past decade. So far, however, manufacturinghas largely remained concentrated in the United States


and Europe — although Japan, the Far East (especiallyKorea), and Australia each have some manufacturers inoperation as well. So the situation is already starting tochange. Whether large-scale contract manufacturingproduction moves to the developing world remains tobe seen. Will we see a commoditization of biologicsmanufacture moving to India, China, and other placeswhere companies now practice commercial antibioticsmanufacture and organic chemistry?

According to D&MD author Alex Kanarek (4), theoverall value of the outsourcing industry was aroundUS$6 billion in 2003 (up from $4.5 billion in 1999 and$1.5 billion in 1995), with the largest revenues foundin contract research (drug discovery, early developmentresearch, and clinical trials). The global market includesabout 1,300 CROs responsible for about 20% of drugdevelopment activities. Half of them are working inclinical trials, and over half of those remaining focus onregulatory assistance.

Meanwhile, most of the world’s CMOs (50 of about70) are located in the United States. The rest can befound in Europe — with the exception of one in Japanand one in Australia. Most companies developingtransgenic manufacturing methods offer them ascontract services. Validation, glycosylation, adventitiousagents, and downstream processing issues are still thebiggest concerns with both transgenic animals andplants. The biopharmaceutical contract manufacturingindustry is expected to reach US$2 billion in value by2005, up from a billion in 2000 and $780 million in1998. Its customers are everyone from so-called“virtual” biotech enterprises to discovery-focusedentities to large biopharmaceutical companies that runout of manufacturing capacity and don’t have time tobuild. The most outsourced activities are cell-linecharacterization, process scale-up, GMP manufacturing,product safety testing, and clinical trial management.

RESOURCESDrug and Market Development (D&MD) Publications:

Schaub, W. A Guide to Evaluating and Choosing CROs. June2002, www.drugandmarket.com/9097.

Regulatory GuidancesGuidance for Industry: Cooperative Manufacturing Arrangements

for Licensed Biologics. Center for Biologics Evaluation and Research.August 1999, www.fda.gov/cber/gdlns/coopmfr.htm.

Guidance for Industry: Submitting Type V Drug Master Files to theCenter for Biologics Evaluation and Research. Center for BiologicsEvaluation and Research, August 2001.www.fda.gov/cber/gdlns/dmfv.pdf.

For other regulatory guidances, see also the following web sites:www.fda.gov/cder/guidance/index.htm,www.emea.eu.int/htms/human/bwp/bwpfin.htm, andwww.ich.org/ich5q.html.

REFERENCES1 “BioPharMOS: Biotech and Pharma Manufacturing Outsource

Services” event, 26–28 March 2003, Monte Carlo, Monaco; VBInternational (www.vbinternational.mc).

2 Center for Biologics Evaluation and Research. Guidance forIndustry: Cooperative Manufacturing Arrangements for LicensedBiologics. US Government Printing Office, Rockville, MD; August

1999, www.fda.gov/cber/gdlns/coopmfr.htm.3 Steffy, C; Burrill, GS. Partners in Outsourcing: Keys to

Successful Contract Relationships Across Borders. Supplement toBioProcess Int’l. 2003, 1, 9. (This article was adapted from ChapterTwo: “The Evolution of Outsourcing — An Industry ReinventsItself,” pp.12–20.)

4 Kanarek, A. The Bioprocessing Industry: Increasing Capacity,Production, and Efficiency. D&MD Report #9095, January 2003;D&MD Reports, Westborough, MA (www.drugandmarket.com).

DownstreamProcessing

DD ownstream processing steps are fundamental tobioprocessing: capture, separation, andpurification. Living things (whether microbes,

cells, or whole organisms) produce proteins andpeptides very efficiently, but those molecules tend to beexpressed as part of a mixture: fermentation broth, cellculture supernatant, blood plasma, or transgenic milk,for example. The production method determines thedownstream processing steps that will be necessary. Forexample, viral clearance is an important issue to addresswith products derived from transgenic animals or cellculture.

Often, bacterial fermentation is immediately followedby a cell disruption step because bacteria do not releaseexpressed proteins into their surrounding medium.After the cells have been collected, they go through ahomogenizer, mill, or press. A new technology usesultrasonic vibration to break cell walls.

Few consumables are used in downstream processing.The bioprocessing market has been estimated at half abillion US dollars in value (1). As with most industries,big multinational companies tend to discover andswallow up any small companies that come up with newideas. But as with most science-based industries, asteady trickle of academic researchers with newtechniques and refinements continues founding thosesmall organizations.

CENTRIFUGATION

No matter what method of production is used, acentrifugation or filtration step is often necessary earlyon to remove cellular debris and other solids from the

MIL

LIPO

RE

CO

RPO

RAT

ION

(WW

W.M

ILLI

POR

E.C

OM

)

process stream. Continuous flow (bowl and disk-bowl)centrifuges are usually preferable to rotory centrifugesbecause they offer simpler operation. Very large-scaleunits can cost up to a million dollars.

Centrifugal separation technologies continue a slowbut steady improvement, but major breakthroughs areunlikely (1). Construction materials are said to be aboutfully developed, and the drive technology is long-established. Minor design modifications will improveseparation efficiencies, and modular equipment is on theway that could be plumbed directly into a processstream for immediate operation.

FILTRATION

Often, some kind of filtration is involved either early on(just after harvesting) or in between chromatographysteps. Membrane filtration devices are continuouslyimproved by the vendor companies that make them.Extraction and precipitation methods are more commonin the blood products industry but may have applicationelsewhere in bioprocessing (with certain transgenicsystems, for example).

Filters do not trap material simply by size exclusion.Pore size plays a major role, of course, but so doeselectrical charge and other surface properties of thefilter membrane material. Depth filters are used for cellcapture and process fluid clarification, removing largedebris particles. Ultrafiltration can be used for virusremoval or for reduction of bacterial pyrogens in bloodplasma fractionation operations.

The most common filters used in the biotherapeuticsindustry are cartridges containing 5–15 ft2 of stacked orpleated membrane material (1). These cartridges oftencan be cleaned in place by pressurized steam. Disposablecartridges are more common on smaller scales, reusablecartridges on larger scales. Crossflow filtration is foundin fermentation operations that trap intact bacterial cellsfor processing. These large-scale (up to 5,000 Lbatches) filters are also reusable and can be cleaned inplace. New twists on the technology focus on modulardesigns or vibrating stacks of Nylon filter disks.

CHROMATOGRAPHY

The purification (also called polishing) segment of thebioprocessing industry is seeing much development.Chromatographic methods and media are the primaryfocus, especially when dealing with very complex anddelicate large biomolecules. The basic technologiesinvolved separate molecules by size (gel-filtration andsize-exclusion chromatography), electrical charge (ion-exchange), hydrophobicity (hydrophobic interaction orreversed-phase chromatography), or bioaffinity. Muchattention is being given to new and refined types ofchromatography such as perfusion, expanded bedadsorption, simulated moving bed, and hydrophobiccharge induction chromatography. New affinitychromatography media are always being developed —along with improvements to existing separations mediafor the commonly used ion-exchange method.

In biotherapeutic development and manufacturing,



www.merck-lsp.de


Benzonase®

Benzonase® is a recombinant endonucleaseespecially designed for the removal of DNAand RNA in the downstream processing ofproteins – from lab to production scale.

· Increased protein yield by reducing visco-sity caused by nucleic acids in cell lysates

· Efficient purification of cell-derived particles e.g. viruses associated withnucleic acids

· Significant reduction of DNA contamina-tion to meet current regulatory standardsfor biopharmaceuticals

The smart solution…

…for DNA removalCircle Reader Service No. 117


ion-exchange and affinity chromatographies have thewidest application. Nearly 2/3 of all processes includean ion-exchange step (1). Adding salts is the mostcommon way to change the ionic properties of thesamples for capture and elution, depending on whethercation- or anion-exchange is being used. Some filtrationcompanies are making charged membrane filters for usewith these techniques. And a gel-filtrationchromatography step is often included because it is agentle procedure that will not cause proteinaggregation, unfolding, and so on.

Protein activity is based on specific linking betweenthe molecules (protein–protein interaction) or withothers (such as antibodies attaching themselves toforeign agents in the body). Affinity chromatographycan be used “whenever a suitable ligand is available forthe protein(s) of interest” (1). High-resolutionseparations come from the selective nature of theseinteractions, so large volumes of process fluid can bepurified. Some manufacturers of affinity media createcustom media based on their clients’ particular proteins,and they’re making new stock resins and dyes availableall the time. However, affinity chromatography is themost expensive type, not only because of the ligandsthemselves, but also difficulties encountered in columnregeneration and cleaning.

A driving force here is reducing the number ofchromatography steps required to yield a product ofacceptable purity. Each step requires more labor andmaterials and involves some product loss — as well asrequiring specific validation for GMP compliance.Companies that develop successful abbreviated processesfor purifying their products will see significant costsavings. They should also be able to validate theirprocesses more easily, thus facilitating the regulatoryapproval process.

Submicron and ultrafiltration steps are often foundbetween chromatography steps in downstreamprocesses. Flow-through, tangential flow, and cross-flowfilters are used to concentrate process fluids, desaltfollowing ion-exchange steps, and exchange buffersbetween different chromatographic methods.Continuous diafiltration washes out buffer salts throughthe addition of water at the same rate that filtrate isgenerated. Discontinuous diafiltration involves repeateddilution or concentration of samples removing moreand more unwanted salts, solvents, or smaller molecules.Ultracentrifugation (operating speeds up to 40,000 rpmproviding a centrifugal force 100,000 times the strengthof Earth’s gravity) can be used in a gradient separationmethod based on molecular sizes. Viral vaccinemanufacturers use it to purify inactivated viruses fromtheir sources.

OTHER METHODS

Other separation techniques are less commonly found inthe industry. These include electrophoresis, extractionand fractionation, and magnetic beads. Electrophoresisand magnetic beads are commonly used in researchapplications. Extraction and fractionation are ubiquitousin the blood products industry.

Magnetic beads carrying affinity ligands can capture aprotein of interest. Then they themselves are capturedby magnetism. This method has been adapted fromresearch and diagnostic applications as a gentleseparation method for cells, proteins, and fragments ofmessenger RNA. More than 35 companies in theUnited States and Europe are involved in developingand promoting this technology.

Electrophoresis is well known to molecularbiologists, but its use in downstream processing hasbeen limited by difficulties with the media and voltagesrequired. A few companies, however, are looking atways to make it work on large scales using continuouselution or disposable gel cartridges. Capillaryelectrophoresis is “overtaking the traditional form, butprincipally as an analytical tool” (1).

Solvents and detergents are used in fractionationprocesses, and they are finding application for viralinactivation as well. Several other techniques have beenused for this very important aspect of downstreamprocessing: pH changes, heat, filtration andultrafiltration, affinity chromatography, and ultravioletlight.

FURTHER PROCESSING

Big biomolecules like proteins and nucleotides aresensitive to their environment. Their bioactivity dependsas much on their physical structure as on their chemicalcomposition. So they must be protected fromdegradation in their final product form. Parenteralformulations (those intended for injection) are mostcommon. They usually begin with a buffer such asphosphate, citrate, or a TRIS-based salt. Gelatin orhuman serum albumin are often added along with thetherapeutic protein for shelf-life stability. A fewcompanies are working on sustained-releaseformulations for less frequent injection, embedding thedrug in biodegradable polymers. Most formulations,however, are proprietary recipes kept secret by thebiotherapeutics companies that develop them. Few areeven patented.

Lyophilization: Proteins are most stable kept at lowtemperatures. So many vaccines and proteintherapeutics are prepared as lyophilized (freeze-dried)powders. Single- or multidose vials of product areshipped to clinics, where the drugs are reconstitutedbefore injection into patients. Sugars, sugar alcohols,and polymers are used as stabilizers.

Because they are the link between the manufacturingprocess and the patient recipients of the products, fillingand lyophilization equipment must rigidly adhere toGMP requirements. The lyophilization process is veryexacting and must be closely monitored and controlled.


Five companies dominate the industry worldwide bymeeting all those requirements.

Other Delivery Systems: Most advanced among thealternatives to parenteral formulations are inhalationconcepts. Traditional metered-dose and dry-powderinhalers as well as liquid-jet and ultrasonic nebulizers donot deliver large enough doses intact to the deep lungsto be feasible for use with large biomolecules. Somecompanies are working on refinements that will addressthose problems. A July 2002 D&MD report,Overcoming Formulations Challenges: Novel Techniquesfor Optimum Results by Pamela Bassett, addresses manyof these technologies.

RESOURCESIBC Conferences and Professional Development Conferences:


8–10 December 2003 Cell Culture and Upstream Processing:Improve the Yield and Efficiency of Your Cell Culture ProductionProcesses. San Diego, CA, www.lifesciencesinfo.com.

Drug and Market Development (D&MD) Publications:Kanarek, AD. A Guide to Good Manufacturing Practice. January

2001, www.drugandmarket.com/9019.Kanarek, AD. A Guide to Good Validation Practice. October

2001, www.drugandmarket.com/9049.

Regulatory Guidances:Guidance for Industry: Q5A Viral Safety Evaluation of

Biotechnology Products Derived From Cell Lines of Human or AnimalOrigin. International Conference on Harmonisation, September1998, www.fda.gov/cder/guidance/Q5A-fnl.PDF.

Guidance for Industry: Content and Format of Chemistry,Manufacturing and Controls Information and EstablishmentDescription Information for a Vaccine or Related Product. Center forBiologics Evaluation and Research, January 1999,www.fda.gov/cber/gdlns/cmcvacc.pdf.

Guidance for Industry: For the Submission of Chemistry,Manufacturing and Controls and Establishment DescriptionInformation for Human Plasma-Derived Biological Products. AnimalPlasma or Serum-Derived Products. Center for Biologics Evaluationand Research, February 1999. www.fda.gov/cber/gdlns/cmcplasma.pdf.

Guidance for Industry: Q6B Specifications: Test Procedures andAcceptance Criteria for Biotechnological/Biological Products.International Conference on Harmonisation, August 1999,www.fda.gov/cder/guidance/Q6Bfnl.PDF.

Guidance for Industry: Analytical Procedures and MethodsValidation Chemistry, Manufacturing, and Controls Documentation.Center for Biologics Evaluation and Research, August 2000,www.fda.gov/cber/gdlns/methval.htm.

Guidance for Industry: Good Manufacturing Practice Guide forActive Pharmaceutical Ingredients. International Conference onHarmonisation, August 2001, www.fda.gov/cder/guidance/4286fnl.htm.

Guidance for Industry: Container Closure Systems for PackagingHuman Drugs and Biologics Questions and Answers. Center for DrugEvaluation and Research, May 2002. www.fda.gov/cber/gdlns/cntanrq&a.pdf.

Guidance for Industry: INDs for Phase 2 and Phase 3 StudiesChemistry, Manufacturing, and Controls Information. Center forDrug Evaluation and Research, May 2003, www.fda.gov/cder/guidance/3619fnl.pdf.

Guidance for Industry: Sterile Drug Products Produced by AsepticProcessing — Current Good Manufacturing Practice. Center for Drug

Evaluation and Research, August 2003, www.fda.gov/cder/guidance/1874dft.htm.

For other regulatory guidances, see also these web sites:www.fda.gov/cder/guidance/index.htmwww.ich.org/ich5q.htmlwww.emea.eu.int/htms/human/bwp/bwp.htm

REFERENCE1 Kanarek, A. The Bioprocessing Industry: Increasing Capacity,

Production, and Efficiency. D&MD Report #9095, January 2003;D&MD Reports, Westborough, MA (www.drugandmarket.com).

2 Schwartz, L. Diafiltration for Desalting or Buffer Exchange.BioProcess Int’l. 2003, 1, 5 (May), 43–49.

Expression/Production

AA decade ago, discussion of biotechnologyproduction issues was limited to fermentation andcell culture alone — primarily Escherichia coli and

Chinese hamster ovary cells. Now the options extendfrom bacteria and yeast fermentation through animaland insect cell culture to transgenic plants (primarilytobacco and corn, but also rice and barley), mammals(mainly cattle, sheep, and goats), and even hens’ eggs.Vaccines are grown in potatoes, tomatoes, and bananas.And Genencor International Inc. is looking at adaptingits filamentous fungi enzyme production methods tothe manufacture of antibodies and antibody fragments.

For the vast majority of manufacturing processes,fermentation or cell culture are still the norm. Theyhave a longer regulatory track record than any of thenewer ideas, and the science of culturingmicroorganisms and animal cells is well established. Celllines are genetically engineered and optimized,characterized and banked, then cultivated andharvested. Thus, companies throughout our yearbookprovide items and services you may need in developingproduction processes — particularly in the laboratoryproducts and equipment and the chemicals and rawmaterials sections. Under this production/expressioncategory, however, only companies focusing primarilyon the production side of development will be found;that is, those selling fermentors and bioreactors,cryopreservation systems, culture media, monitors forgrowth and other conditions, and equipment for cellharvesting, disruption, separations, and sorting.

BAY

ERA

G(W

WW

.BAY

ER.C

OM

)


Posttranslational Modifications: Talk about expressionsystems, and one word will inevitably come up:glycosylation. It is the biggest question for planttransgenics, and it is a major reason that animal cells arecultured to produce the most complex recombinantproteins. Post-translational modifications such asglycosylation can only be made by eukaryotic cells (so-called “higher organisms”); simple prokaryotes such asbacteria and yeasts do not have the cellular equipmentto perform such functions. And many complex proteinsare inactive without certain sugars hanging off of themin specific places.

To further complicate the issue, nearly every specieshas its own take on these modifications, so a proteinbased on the same DNA sequence can have differentproperties if made by different animals: sheep, cattle,pigs, humans, and so on. Plants and animals make verydifferent posttranslational changes, and fungi such asyeast fall somewhere in between. The immune system ofa patient exposed to a protein recognized as “foreign”may react by destroying it, making the patient sick orworse. Biotherapeutics made by nonhumanized celllines may require special downstream processing stepsto correct their glycosylation. Many companies areworking on different approaches to the problem,“humanizing” cell lines, for example.

A QUESTION OF SCALE

Some transgenic products are quite advanced indevelopment, even as far as phase III clinical trials and arecently submitted common technical document inEurope. But all currently approved biotherapeutics arederived either from fermentation or cell cultureprocesses or directly from bodily fluids or tissues (such

as blood plasma fractionation products). The “CellLines” sidebar lists the most commonly used cell linesfor producing recombinant proteins. Traditionally, onlyantibiotics were made at very large scales (using 10,000 L bioreactors and so on); but now companiesare expecting similar levels of production for somerecombinant proteins in the development pipeline. Suchdrugs won’t treat only rare disorders but commonproblems as well, so the market demand for them willbe higher than it ever was for growth hormone, forexample. Imagine the response to a biologic treatmentfor pollen allergies or obesity!

Most animal cells are attachment dependent,meaning that they will not grow suspended in a fluidmedium like bacteria and yeast. At large scales, they willrequire specially engineered bioreactors that are quitedifferent from the large fermentors used by antibioticsmanufacturers. You’ll find some of them described inthis section of our yearbook. On small scales, VEROand CHO cells are often grown in flat-sided flasks, eachabout 1 L in size. You can add more and more racksholding those flasks, but that would be prohibitivelydifficult at the kind of scales now being considered forrecombinant proteins. Some answers have includedrolling bottles that make use of the entire surface of theflask rather than just one side, stacking culture surfaceswithin vessels, using microcarrier beads for suspensionculture of cells attached to them, and growing cells inor on hollow tubes (hollow fibers) in packed bundlesthat act as a sort of circulatory system for the flow ofnutrients and waste products.

The world market for fermentors and bioreactors isprobably worth about US$250 million. The largestcompanies involved are well known, and many of the

COMMONLY USED CELL LINES

Specialized microbe and animalcell strains for recombinant proteinand vaccine production aredeveloped by vendor companies toaddress yield problems caused byproteinase degradation or toincrease the production life andability of the cells. These cell linescan be purchased and thenengineered to make a protein ofinterest — but some vendorsprovide that service. The US marketfor cell lines is worth an estimated$50 million per year (1). For animalcell lines, epithelial cells are oftenchosen because of their resiliencyin culture. Hybridomas (cell linesderived from cancerouslymphocytes) are often used toproduce antibodies. Here are a fewcommonly used cell lines in thebiotherapeutics industry:

Escherichia coli (bacteria)

Bacillus subtilus (bacteria)

Saccharomyces cerevisiae (brewer’syeast)

Schizosaccaromyces pombe(another brewer’s yeast)

Pichia pastoris (yeast)

SF-9 (Spodoptera frugiperdabutterfly larva cells)

VERO (kidney cells of Africangreen monkeys)

HeLa (human cervix cells)

PER-C6 (diploid cells from thehuman eye)

CHO (Chinese hamster ovary cells)

NSO (murine myeloma cells)

BHK (baby hamster kidney cells)

CEF (chicken embryo fibroblasts)

MRC-5 (human diploid lungfibroblasts)

KB (human epithelial carcinoma)

UMSCC (human squamouscarcinoma)

For more information, see the website of the American Type CultureCollection (www.atcc.org). Begunover 70 years ago, the organizationkeeps and sells thousands ofbacterial, fungal, animal, and othercell lines and hybridomas. TheEuropean Collection of CellCultures (www.ecacc.org) keeps asimilar collection, and many largecountries maintain their own.

smaller players do customized work, such as building“smaller but highly instrumented custom reactors formammalian cell culture” (1). Scalability is, of course, aprime factor in the choice of vendor — along withcleaning, process validation, and quality issues vital toregulated manufacturing processes. Even the computerprograms that control fermentation and cell cultureprocesses must comply with regulations such as 21 CFRpart 11, “Electronic Records, Electronic Signatures,” inthe United States. Hollow-fiber, small-scale solutions,and disposable devices represent a small but fast-growingsegment of the market for production equipment.

Advances in growth media have been the biggest newson the production front for a few years — ever since theadvent of transmissible spongiform encephalopathies(TSEs), infectious diseases that appear to cross speciesbarriers, caused concern about the sources of manyculture medium ingredients. Vendor companies haveresponded to industry demands by developing serum-free, animal-product free, and/or protein free chemicallydefined growth media for use in bioprocessing. They alsoprovide source information for more traditional products,buying raw materials that only come from those countrieswhere TSEs have not been reported. Biopharmaceuticalcompanies are making process changes to accommodatethe changing science of bioprocessing, and such changeswill have regulatory implications when it comes time tofile for marketing approval.

The US market for growth media is estimated to beover $250 million for 2002, with much research anddevelopment going on to address the above concerns.Add the European and Asian markets, and BPI advisorAlex Kanarek has suggested that $500 million “may bean underestimate” of the size of the global market (1).Powder and liquid media, most in nonproprietaryformulations, may be prepared by the biotherapeuticcompany itself (if large enough) or premixed purchasedat smaller volumes from specialist vendors. Processdevelopment scientists expect great consistency in lotsof media; inconsistencies can affect product yields andeven composition. And regulatory authorities expectthose suppliers — whether selling media or theingredients thereof — to have adequate quality controlsand documentation proving them. For example, a drugmaster file with the US FDA helps ensure productquality.

Despite the preference for serum-free media, fetalbovine serum (still the most commonly used mediasupplement) is a near-$150 million internationalbusiness (1). Preferred sources for the ingredient areNew Zealand, Canada, and the United States, whichhave yet to report any incidences of bovine spongiformencephalopathy. Europe, South America, and SouthAfrica cannot supply FBS. You can expect to pay$200–$1,200 per liter depending on how muchscreening has been done — or negotiate a volumediscount in return for promised large demand.

Companies are changing their processes to make useof serum-free media and such, but that does not happen



www.merck-lsp.de


New robustand Rapid Resins

Fractoprep® DEAE is the first member of this new tentaclemedia family for efficientcapture. It allows you to:· use higher flow rates at

low pressures· pack larger columns· run more cycles· purify proteins and other biomolecules· remove DNA, endotoxins and viruses.

To increase…

...chromatographic productivity: Today!

Circle Reader Service No. 121


overnight. And some managers may choose the“calculated risk of using an efficient FBS-basedmedium” over delays in product development (1).

LOOKING AHEAD

Transgenic milk appears to be no more difficult topurify than the products of fermentation and cellculture (2). Costs remain low, even figuring indownstream processing: $1–100 per gram of finalproduct. Companies using hens’ eggs for transgenicexpression of recombinant proteins claim simplerpurification (expression is directed to the egg whites)and lower costs: $0.10–0.25/gram. And transgenicplants can express enzymes, antibodies, and vaccines ina variety of tissues at costs roughly equivalent to the lowend for transgenic milk. Tobacco and alfalfa leaves,potatoes, and the seed grains of corn, canola, safflower,rice, and barley have shown varying degrees of promise.Yield and regulatory acceptance appear to be majorconcerns here.

Major trends in fermentation and cell culture pointtoward raising expression levels, increasing cell densities,simplifying culture media, and controlling cultureprocesses and conditions. All those improvements areaimed at reducing the cost of producing the largeamounts of target molecules needed for commercialoperations and achieving the optimum productivity ofGMP facilities. Current estimates for the production bycell culture of a gram of a relatively simpleprotein/MAb run between $200 and $300. For morecomplicated molecules expressed in, for example, CHOcells, it may reach $1,000 or more per gram (1).

RESOURCESIBC Conferences and Professional Development Conferences:

1–2 December 2003 Antibody Therapeutics: From ClinicalDevelopment to Business Strategies. San Diego, CA,www.lifesciencesinfo.com

8–10 December 2003 Cell Culture and Upstream Processing:Improve the Yield and Efficiency of Your Cell Culture ProductionProcesses. San Diego, CA, www.lifesciencesinfo.com

15–16 December 2003 Protein Therapeutic Drug Development: ARoad Map from Discovery to Product Launch. Washington, DC,www.lifesciencesinfo.com

26–29 April 2004 TIDES 2004: Oligonucleotide and PeptideTechnology Conferences. Las Vegas, NV,www.lifesciencesinfo.com/tides.

Drug and Market Development (D&MD) Publications:Das, RC and Morrow, J. Antibody Therapeutics: Production,

Clinical Trials, and Strategic Issues. November 2001,www.drugandmarket.com/9039

Weck, E. The Transgenic Plant Market: Profits from New Productsand Novel Drugs. August 2002, www.drugandmarket.com/9070

Regulatory Guidances:Guideline for Industry: Quality of Biotechnological Products:

Analysis of the Expression Construct in Cells Used for Production ofr-DNA Derived Protein Products, ICH Q5B. International Conferenceon Harmonisation, February 1996, www.fda.gov/cder/guidance/ichq5b.pdf.

Points to Consider in the Manufacture and Testing of MonoclonalAntibody Products for Human Use. Center for Biologics Evaulationand Research, February 1997, www.fda.gov/cber/gdlns/ptc_mab.pdf.

Guidance to Industry: Derivation and Characterisation of CellSubstrates Used for Production of Biotechnological/Biological ProductsQ5D. International Conference on Harmonisation, July 1997,www.ich.org/pdfICH/q5d.pdf.

Guidance for Industry: S6 Preclinical Safety Evaluation ofBiotechnology-Derived Pharmaceuticals. International Conference onHarmonisation, July 1997, www.fda.gov/cder/guidance/1859fnl.pdf.

Guidance for Industry: Q6B Specifications: Test Procedures andAcceptance Criteria for Biotechnological/Biological Products.International Conference on Harmonisation, August 1999,www.fda.gov/cder/guidance/Q6Bfnl.PDF.

Guidance for Industry: Drugs, Biologics, and Medical DevicesDerived from Bioengineered Plants for Use in Humans and Animals.Center for Biologics Evaluation and Research, September 2002,www.fda.gov/cber/gdlns/bioplant.htm.

For other regulatory guidances, see also these web sites:www.fda.gov/cder/guidance/index.htmwww.emea.eu.int/htms/human/bwp/bwpfin.htmwww.ich.org/ich5q.html.


Production, and Efficiency. D&MD Report #9095, January 2003;D&MD Reports, Westborough, MA (www.drugandmarket.com).

2 Goldman, M. Processing Challenges for Transgenic Milk.BioProcess Int’l. 2003, 1, 10 (October); 60–63.

InformationTechnology

BB ioinformatics. . . genomics . . . proteomics . . .systems biology: These are all buzzwords thathave been passed around the industry for years,

and they represent a meeting of two sciences (lifescience and computer science) that is expected to yieldamazing results. In its 2003 report on thebiotechnology industry, Burrill & Company states,“The Bio+IT equation has yet to reach anything closeto its full potential.” However, people involved inbiopharmaceutical process development are morefamiliar with laboratory information managementsystems, manufacturing control, bioprocess monitoring,and documentation software. Only now are thebuzzwords working their way into process developmentlaboratories for quality assurance and control, processoptimization, and validation use. “The use of

NO

VA

RTIS

AG

(WW

W.N

OV

ART

IS.C

OM

)


microarray technology has moved from the fringe to themainstream, and in 2002 several new array platformswere introduced” (1).

THE DOCUMENTATION DILEMMA

Regulators, scientists, industry, and the medical/clinicalprofession are all seeking a way to standardize and shareinformation. It’s not easy when each makes its own useof data and faces its own priorities and history. “It’s aconfusing subject,” writes BPI associate editor ChristinaPrier Steffy in a September article (2), “and as oftenhappens, attempts to clarify it have only increased theconfusion.” For example, manufacturers are stillstruggling with the unclear requirements of the 21 CFRPart 11 regulation. In August 1997, the US Food andDrug Administration (FDA) adopted the final version ofthat regulation for electronic records and electronicsignatures. Despite its being in force for years,confusion about Part 11’s requirements was still sowidespread that in February 2003, the FDA withdrewits guidances on the subject. In late summer, a newguidance redirecting the regulation appeared.

“A major sticking point in Part 11 compliance issoftware validation,” Steffy writes. “Validatingprocesses, assays, and equipment is well understood bypeople in regulated industries, although not always easyto practice. But despite its 20-year history, softwarevalidation still confuses people. The only regulatoryspecifics on the subject are in Part 11 and in variouscodes regarding medical devices. Even the new Part 11guidance itself refers to a final guidance about softwarevalidation for medical devices for basic softwarevalidation principles. The problem is that although theconcepts may be similar, the terminology is not, andpeople expert in biopharmaceutical manufacturing maynot be fluent in device or systems terminology. The newguidance only partly clarifies the continuing confusionover exactly which systems and records need to bevalidated.”

One thing’s for sure: You can’t just take yourvendor’s word that a program or suite of programs is“Part 11 compliant” — no more than you can leaveGMP compliance up to your contract manufacturer.

ENGINEERING AND PROCESS CONTROL

Bioreactor performance can be controlled “off-line,”using laboratory bioassays that can take too much time.In biotherapeutic and biodiagnostic development, timeis money. On-line monitoring and control is preferablebecause it occurs in “real-time.” Dependent processvariables (such as cell growth rates, productconcentration, apoptosis, and so on) are regulated bycalculated adjustments made to independent variables(such as feed and mixing rates). Dissolved gases, celldensities, product concentration, and carbon levels maybe monitored using sensors and automation to aid inprocess optimization. Computer control rooms cansend signals to make adjustments based on complexalgorithms, all of which must be validated to work ofcourse. Process modeling can help engineers set up

good systems to begin with (4). Many softwarepackages are available to help.



1–2 December 2003 Digital Imaging with Automated MicroscopySystems: A Guide to Their Use in Drug Discovery. Washington DC,www.lifesciencesinfo.com.

1–2 December 2003 Statistics: Basic Principles & Applications inDrug Discovery. San Francisco, CA, www.lifesciencesinfo.com.

4–5 December 2003 Proteomic Analysis in Discovery: NewProteomics Tools for Solving Problems in Drug Discovery. Cambridge,MA, www.lifesciencesinfo.com.

8–9 December 2003 Molecular Diagnostics: Novel ProteomicsAnalysis Technology Tools for Diagnostics Applications. Cambridge, MA,www.lifesciencesinfo.com.

8–10 December 2003 Design and Synthesis of Target-BasedCompound Libraries: Using Novel Synthetic Technologies, Target DrivenChemistry, and Chemical Biology to Improve the Quality ofPharmaceutical Compound Libraries. La Jolla, CA,www.lifesciencesinfo.com.

11–12 December 2003 Validating Commercial Off-the-ShelfSoftware for 21 CFR Part 11 Compliance. San Diego, CA,www.lifesciencesinfo.com.

Drug and Market Development (D&MD) Publications:Bioinformatics: The Next Generation of Market Opportunities.

March 2001, www.drugandmarket.com/DMD9025.Carter, JM. A Guide to Assay Development. March 2003,

www.drugandmarket.com/9114.Focus Report: The Bioinformatics Age: Evolution of a New

Direction for Drug Discovery and Development. May 2001,www.drugandmarket.com/9050.

Rubenstein, K. Biochips: Progress and Prospects. January 2001,www.drugandmarket.com/DMD9016.

Sheehan, SM. Technology Commercialization: Technology Transferfor Business. January 2001, www.drugandmarket.com/9024.

Skinner, NG. High Density Multiplexed Assays: Obstacles &Opportunities for Bioscience Markets. August 2003,www.drugandmarket.com/9124.

Other Publications:Galvanauskas, V; Simutis, R; Lubbert, A. Model-Based Design of

Biochemical Processes: Simulation Studies and Experimental Tests.Biotechol. Letters 1997, 19; 1,043–1,047.

Galvanauskas, V; et al. Model-Based Design of a BiochemicalCultivation Process. Bioprocess Eng. 1998 (MAR), 18; 227–234.

Sonnleitner, B. Bioprocess Automation and Bioprocess Design. J.Biotechnol. 1997, 50; 175–179.

Gregory, ME; et al. A Visual Programming Environment forBioprocess Control. J. Biotechnol. 1994, 33, 3; 233–241.

Bastin, G; Dochain, D. On-Line Estimation and Adaptive Controlof Bioreactors. Elsevier Press: Amsterdam, 1990.

Levine, WS. The Control Handbook. CRC Press: Boca Raton, FL,1995.

Oggunaike, BA; Ray, WH. Process Dynamics, Modeling, andControl. Oxford University Press: New York, NY, 1994.

Van Impe, JF; Vanrolleghem, PA; Iserentant, D. AdvancedInstrumentation, Data Interpretation, and Control of BiotechnologicalProcesses. Kluwer Academic Publishers: Dordrecht, the Netherlands,1998.

Regulatory Guidances:Guidance for Industry: Part 11, Electronic Records; Electronic

Signatures - Scope and Application. Center for Drug Evaluation and


Research, September 2003, www.fda.gov/cber/gdlns/prt11elect.htm.For other regulatory guidances, see also these web sites:

www.fda.gov/cder/guidance/index.htmhttp://esubmission.eudra.orgwww.ich.org/ich5m2.html

REFERENCES1 Burrill, GS. Biotech 2003 (17th Annual Report on the Industry):

Life Sciences — Revaluation and Restructuring. Burrill & CompanyLLC: San Francisco, CA, 2003.

2 Steffy, CP. Focus on Compliance: Computer Validation Basics.BioProcess Int’l. 2003, 1, 9 (September), 24–28.

3 Kanarek, A. The Bioprocessing Industry: Increasing Capacity,Production, and Efficiency. D&MD Report #9095, January 2003;D&MD Reports, Westborough, MA (www.drugandmarket.com).

Laboratory Productsand Equipment

FF ormulation. Raw material testing. Lot release.Product characterization. Process optimization.Scale-up. Validation. Quality control. None of

these things can happen at a biotherapeutic orbiodiagnostic company without the efforts of itsanalytical laboratories.

Biotechnology is an industry based on life science. Assuch, it is heavily dependent on scientific advances andinformation, on scientific principles and techniques, andultimately on highly specialized equipment andinstrumentation. “It has been estimated that more than60% of the manufacturing cost of a biological is due toquality control procedures rather than to actualproduction” (1). The analytical laboratories thatperform early research and discovery, preclinical testing,formulations, process development, validation, andquality work must be well-equipped and operatingunder GLP and/or GMP conditions. Their equipmentmust be qualified and their processes validated to work.

Design qualification (DQ) is often done by vendorcompanies. It confirms that an instrument or piece ofequipment has been manufactured in line with itsoriginal design. Installation qualification (IQ) is doneon site when new equipment or instrumentation arrives.

It should prove that the item was properly installed.Operational qualification (OQ) and performancequalification (PQ) are intended to show that a machinedoes the job it’s supposed to do in the manner it’sexpected to do it — and that its performance ismeasurable, predictable, and repeatable. Validation isthen applied to the processes and protocols themselves,making use of documented standard operatingprocedures (SOPs). Some of the most sophisticatedanalytical methods are also the most sensitive to thedetails of experimental conditions. That can make theirresults hard to reproduce and thus difficult orimpossible to validate. Process validation willdemonstrate scalability and reproducibility to regulatoryauthorities when it comes time for submitting acommon technical document.

REGULATORY OVERSIGHT

Laboratory equipment can be complex (such as theelectron microscopes, spectrometers, calorimeters, andspectroscopes used in bioanalytical methods) or simple(such as refrigerator/freezers, incubators, andcentrifuges) in design and operation. The USOccupational Safety and Health Administration(OSHA) requires the presence of certain types ofequipment, such as the ubiquitous eyebaths and fireprotection. GLPs and GMPs require others, such asisolators and pressure gauges to monitor airflow.Ergonomics come into play for repetitive tasks likepipetting (many of which are being automated thesedays whenever and wherever possible). Andenvironmental laws apply when a laboratory makes useof potentially dangerous chemicals or organisms.

Any related products — such as pumps, largecentrifuges, piping, and various process vessels — thatare actually used in the manufacturing stream (havingcontact with the product rather than just samples takenof it) must undergo rigorous GMP testing and cleaningprocedures. The earlier in development a particular taskis performed, the less stringently the regulations apply.Therefore, very little discovery work is GLP compliant,but preclinical testing must be documented well enoughto support regulatory application for moving forwardinto human clinical studies. But following GLPs is oftencommon sense and good science — and will ultimatelycontribute to better quality data and reproducibleresults.

Many preparative separations methods were adaptedfrom analytical techniques: chromatography andultracentifugation, for example. The laboratory productsindustry serves a much larger market than just the R&Dand QA/QC laboratories of biotherapeuticsmanufacturers. They also cater to the rest of the lifesciences world, from professors and their graduateassistants to government research institutions to theR&D laboratories of companies making foods,pharmaceuticals, cosmetics, and all the supportingindustries that make the products and equipment theyuse! Analytical advances are made by those userscombining and refining methods as much as by the

TTP

LABTE

CH

LTD. (

WW

W.T

TPLA

BTE

CH

.CO

M)


vendor companies inventing something new. Sometimesa user becomes a vendor by developing and patenting anew invention.

For quality control laboratories in our industry,certain criteria must be applied to any analytical methodchoice: specificity, sensitivity, robustness andreproducibility, regulatory acceptability, and cost in timeand effort (1). Chemical analysis methods include aminoacid analysis and peptide mapping as well as analyticalchromatographies like HPLC and capillarychromatography. Many such methods are destructive tothe samples being tested. Methods of physical analysisinclude centrifugation, electrophoresis, microscopy, andmass spectrometry. They test physical properties ofsamples: molecular size and weight, electric charge, celland organism appearance, and so on.

Biological methods include simple microbial testingusing filters or culture plates, bioassays using laboratoryanimals, and immunoassays using enzymes andantibodies. About 50 companies supply laboratoryanimals to the research world, ranging from largemultinationals to single rabbit farms. In thebiotherapeutics industry, animal testing is used primarilyfor preclinical safety and potency testing of drugs. Drugdevelopers prefer more controlled in vitro testingwhenever possible. Immunoassays can often provide asolution using microplates (with 96, 384, or 1,536individual wells for many simultaneous experiments),automated plate handling robots, spectrophotometers,and computer control and analysis.

Table 1 lists some common analytical methods andtheir application in the industry. High-performanceliquid chromatography is probably the most commonlyused analytical method in GMP laboratories. Anothervery common technique is electrophoresis. Over 70% ofEuropean life science researchers use it, spending overUS$40 million on gel media alone (1).



1–2 December 2003 Cell-Based Assay Technologies for DrugDiscovery. Cambridge, MA, www.lifesciencesinfo.com.

1–2 December 2003 Statistics: Basic Principles & Applications inDrug Discovery. San Francisco, CA, www.lifesciencesinfo.com.

3–4 December 2003 Overcoming Statistical Challenges withMicroarrays: Processing and Analysis. San Francisco, CA,www.lifesciencesinfo.com.

8–10 December 2003 Design and Synthesis of Target-BasedCompound Libraries: Using Novel Synthetic Technologies, Target DrivenChemistry, and Chemical Biology to Improve the Quality ofPharmaceutical Compound Libraries. La Jolla, CA,www.lifesciencesinfo.com.

8–10 December 2003 Foundation in Pharmaceutical Analysis,IBC’s 2nd International Foundation Course. Cambridge, UK,www.ibc-lifesci.com/pafoundation.

11–12 December 2003 Fluorescence: Basic Principles andApplications in Drug Discovery. San Francisco, CA,www.lifesciencesinfo.com.

15–16 December 2003 Protein Therapeutic Drug Development: A

TTaabbllee 11:: Analytical methods commonly used in biotherapeutic andbiodiagnostic development

Technology Applications

Amino Acid Analysis Protein characterizationQuantifying protein in final product

Biocalorimetry Biomolecular interaction studiesAntibody QC and characterizationDrug design and screeningEnzyme kineticsProtein formulation stability studiesLipid transition studiesNucleic acid studies (uncoiling)

Circular Dichroism Protein structure studiesStability studies

Electron microscopy Virus identification and titer estimatesContaminant identificationCell line characterization

Electrophoresis QA/QCGenome sequence analysisProtein identity, purity, and structure studies

Fluorescence In-Situ Genetic stability testingHybridization (FISH)

High-Performance QA/QCLiquid Protein characterization and identificationChromatography Active ingredient quantitation(HPLC) Impurity identification and quantitation

Immunoassays Protein identificationCell typingHybridoma screening for MAb productionDiagnostics and drug monitoringLocation of antibodies in cells and tissues

Isoelectric Focusing Protein identification/purityFormulation development, stability studiesMethod validation and process validationLot-release testing

Limulus Amebocyte Pyrogen/entotoxin analysisLysate (LAL) Assay

LC/MS/MS In vivo drug concentration testing forpreclinical and clinical studies

Near-Infrared (NIR) Fermentation monitoring and control

Spectroscopy Raw materials inspectionProduct testing (dosage forms, etc.)

Nuclear Magnetic Standards calibration (for other methods)Resonance Purity assaysSpectroscopy NMR/HPLC for analyzing complex mixtures

Excipient compatibility (formulations)Drug interaction studies

Peptide Mapping Batch and lot-release testingGenetic stability testingQA/QC

Polymerase Chain Pathogen detection/identificationReaction (PCR) DNA sequencing

Gene expression analysis and cloning

Surface Plasmon Biomolecular interaction and affinity studiesResonance (SPR)

Total Organic Carbon Cleaning validation(TOC) Analysis Water analysis

Ultracentrifugation Molecular binding studiesProtein heterogeneity/conformation testingQA/QC, process development, formulation

Ultraviolet/Visible Protein concentration studies (QA/QC)Spectroscopy HPLC detection

Lot-release testing

Road Map from Discovery to Product Launch. Washington, DC,www.lifesciencesinfo.com.

19–20 January 2003 Immunogenicity of Recombinant BiologicalTherapeutics: A Practical Approach. Dedham, MA,www.lifesciencesinfo.com.

26–29 April 2004 TIDES 2004: Oligonucleotide and PeptideTechnology Conferences. Las Vegas, NV,www.lifesciencesinfo.com/tides.

Drug and Market Development (D&MD) Publications:Carter, JM. A Guide to Assay Development. March 2003,

www.drugandmarket.com/9114.Kanarek, A. A Guide to Good Laboratory Practices for Start-up and

Growing Laboratories in Industry and Academia. October 1999,www.drugandmarket.com/9004

Nabhan, A; et al. Cell-Based Assays: Commercial Opportunities,Legal Trends, & Technology Analyses. March 2003,www.drugandmarket.com/9116.

Nabhan, A; et al. Protein Arrays: Commercial Opportunities, LegalTrends, and Technology Analyses. June 2003,www.drugandmarket.com/9117.

Rubenstein, K; Coty, C. High-Throughput Screening: Redefiningthe Mission, 2nd Ed. May 2001, www.drugandmarket.com/9032.

Skinner, NG. High-Density Multiplexed Assays: Obstacles andOpportunities for Bioscience Markets. August 2003,www.drugandmarket.com/9124.

Regulatory Guidances:Guideline for Industry: Quality of Biotechnological Products:

Analysis of the Expression Construct in Cells Used for Production ofr-DNA Derived Protein Products, ICH Q5B. International Conferenceon Harmonisation, February 1996, www.fda.gov/cder/guidance/ichq5b.pdf.

Guidance for Industry: Analytical Procedures and MethodsValidation Chemistry, Manufacturing, and Controls Documentation.Center for Biologics Evaluation and Research, August 2000,www.fda.gov/cber/gdlns/methval.htm.

Guidance for Industry: Monoclonal Antibodies Used As Reagents inDrug Manufacturing. Center for Drug Evaluation and Research,Center for Drug Evalutation and Research, March 2001,www.fda.gov/cder/guidance/3630fnl.htm.

For other regulatory guidances, see also these web sites:www.fda.gov/cder/guidance/index.htmwww.emea.eu.int/htms/human/bwp/bwpfin.htmwww.ich.org/ich5s.html.


Production, and Efficiency. D&MD Report #9095, January 2003;D&MD Reports, Westborough, MA (www.drugandmarket.com). ��

BioProcess I N T E R N A T I O N A L

TM

Career Center Online

Find Candidates. Find Employment.

Biotechnology Pharmaceuticals Medical Devices Life Sciences

Serving the biosciences:

www.bioprocessintl.com

Member of the BioJobNetwork

Recruiters - Advertise on the fastest growing recruitment service for the biosciences! Reach thousands of registered candidates with a Standard Job Listing or a Premium Job Listing. The service is fast, easy and affordable. Job Seekers - Register to receive new job alerts and to apply online to hundreds of new job listings.

Circle Reader Service No. 125

1 chemicals and raw materials€¦ · back during the e-business boom, many people talked about...

Documents