a discussion of biotechnology

8/13/2019 A Discussion of Biotechnology

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A Discussion of Biotechnology

The Future of the Pharmaceutical Industry?

A Discussion of Biotechnology ..................................................................................1The Future of the Pharmaceutical Industry?..................................................................1Background and Scoe...................................................................................................!Pharmacogenetics and Pharmacogenomics....................................................................!"ecom#inant Proteins.....................................................................................................$%accines.........................................................................................................................&Theraeutic Anti#odies................................................................................................1!Stem 'ells....................................................................................................................1(The Future of Biotechnology in the )*.......................................................................1+


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Background and Scoe

In the last millennium ,and stretching into this one- a great /enture 0as undertaken

#y colla#orati/e la#oratories in eighteen countries The 2uman 3enome Pro4ect. Theaim of this ro4ect 0as to identify and se5uence all the some !6666 !7666 genesthat make u human D8A. 8ot more than forty years since 9atson and 'rickannounced they had cracked the code of life it is remarka#le that such de/elomenthas occurred in this field.

The 2uman 3enome Pro4ect has imlications in many fields such as D8Aforensics agriculture and anthroology: this essay ho0e/er 0ill concern itselfe;licitly 0ith the harmaceutical industry. 8aturally there are many ethical legaland social issues 0ith any genetic maniulation. This essay 0ill not address any ofthese issues since it is not the urose of this iece of 0riting. Instead it 0ill focuson the strategies used in the harmaceutical industry to use gene technology in the

attemt roduce safer and more efficacious medicines.

Pharmacogenetics and Pharmacogenomics

Pharmacogenetics is the study of ho0 an indi/idualid lead to mi;ed drug resonse #eha/iour in

atients. For some the recommended dose 0as not ade5uate enough to treat thecondition. For others the recommended dose resulted in a manifestation of to;ic

effects. It 0as found that the en>yme i/er 8=acetyltransferase 0as resonsi#le forthe drugyme e;isted in t0oolymorhs. Polymorhism is a henomenon 0here an en>yme is e;ressed in morethan one form each form differing #y erhas a fe0 amino acids. This is not to #econfused 0ith truncated or ill=e;ressed roteins@en>ymes resulting from D8Adamage. Polymorhisms are 0ell esta#lished differences in en>yme structure andfunction i.e. a significant roortion of the oulous e;hi#it the olymorhism not

4ust a fe0 indi/idual cases. In the case of isonia>id the t0o olymorhs of li/er 8=acetyltransferase lead to the different resonses to the drug. ne olymorhmeta#olised the drug too 5uickly for any theraeutic affect to #e noted= indi/idualse;hi#iting this en>yme are du##ed fast acetylators.C The other olymorh did not

meta#olise the drug fast enough thus leading to a #uild u that conse5uently lead tothe re/iously mentioned to;ic effects= indi/iduals e;hi#iting this en>yme are du##edslo0 acetylators.C

Incidences like the isonia>id case and many others lead to a greaterunderstanding of the role genetics lays in drug resonse. The 2uman 3enomePro4ect ser/ed to deeen this le/el of a0areness and no0 the study of

harmacogenetics is 0ell esta#lished. The ne;t 5uestion 0as of course 0hat can #edone a#out it? Pharmacogenomics is the ractical element of using genetics in drug design andholds the romise tailor=made drugs may one day #e a#le to #e rescri#ed to #est suitan indi/idual #ased on the atient


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"8AC ,si"8A- molecules that target areas on D8A to halt transcrition of certaingenes 0here it may #e desira#le to do so.

In summary e;citing ne0 comounds are griing the harmaceutical industry


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and not the desired gene. 'ells that cannot gro0 on the second media ha/e taken ulasmids that ha/e the anti#iotic resistance gene interruted and so do contain thedesired gene.

Fermentation 2ar/esting and Purification

nce the ure culture of cells that contain the recom#inant D8A /ector is o#tained

the material is fro>en in glycerol and stored in a master cell #ank at temeratureslo0er than =176 o'. 9orking cell #anks of smaller samles are then generated fromthe master cell #ank #efore the cultures are remo/ed to ferment industrial /olumes ofthe acti/e harmaceutical ingredient ,API-. 2ere are details of the fermentation

rocess

The amoule is remo/ed from the 0orking cell #ank added to nutrient

#roth and allo0ed to gro0. This is termed the rimary culture hase

Seed culture is transferred to a small scale ,16 166 - fermentor and

allo0ed to gro0. This is termed the seed fermentation hase

The contents of the rimary small scale fermentor are transferred to a large

scale ,1666 166666 - fermentor and the culture is gro0n to a secificdensity. This is termed the roduction scale fermentor. nce thearoriate cell density is achie/ed roduction of the desired rotein isinitiated #y acti/ating the e;ression romoter ,heat and@or chemicals-.

All stages must

o Be erformed under sterile conditions

o Be monitored throughout

o Adhere closely to /alidated rocedures

At the end of fermentation the cells are har/ested ,e.g. centrifugation-. If thedesired rotein is e;tracellular the cells are discarded from the centrifuged solutionand the suernatant medium is rocessed for reco/ery. If the rotein is intracellularthe medium is discarded and the cells are 0ashed and then lysed to release theircontents. The cellular de#ris is then filtered and the filtrate is rocessed for reco/ery.

The rotein of interest is generally resented as a lo0 concentration 0ith a 0iderange of contaminants ranging from small molecules such as anti#iotics and sol/entsto large #iological macromolecules such as other host cell roteins and car#ohydrates.The desired rotein therefore must #e urified and concentrated. Eany techni5uescan #e emloyed to urify the rotein such as ultracentrifugation and electrohoresis.This essay 0ill detail the most 0idely used techni5ue the alication ofchromatograhy to urify the rotein. 'hromatograhical methods in use are

Ion e;change chromatograhy

Si>e e;clusion chromatograhy

2ydroho#ic interaction

Affinity chromatograhy


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Ion Exchange Chromatography (IEX)

I searates the desired rotein #y e;loiting differences in charge. Stronglycharged molecules 0ill #ind more strongly than 0eakly charged molecules thusachie/ing searation.

Size Exclusion Chromatography (SEC)

S' searates the rotein of interest from other imurities #y e;loiting differencein si>e. Smaller molecules are hindered #y the orous #eads and take longer to assthrough the column than larger molecules.


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Affinity Chromatography (AC)

A' is a highly secific techni5ue that utilises rotein secific ligands to effectsearation. Eany rocessed no0 use A' as a single ste urification rocess and ishighly cost effecti/e.

Formulation

nce the API is urified it needs to #e formulated rior to resentation and use.Formulation is a critical stage in the manufacture of #ioharmaceuticals as it ensuresthe efficacy 5uality sta#ility and safety of the roduct rior to reaching the atient.The formulation needs to consider the route of administration for e;amle

Toical the su#stance is re5uired directly to 0here the action is re5uired e.g.

sul#utamol for asthma inhalers

nteral the desired effect is systemic ,non=local-: the su#stance is gi/en /ia

the digesti/e tract ,e.g. oral anti#iotics-.

Parental the desired effect is systemic: the su#stance is gi/en #y routes other

than the digesti/e tract ,e.g. intra/enous anti#iotics-.


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;amles of "ecom#inant Protein Products

The first commercial alication 0as the roduction of human insulin from E. coli#y li illy. ther e;amles are

Protein Year Host Application2uman insulin 1&+! E. coli Dia#etes

2umansomatotroin

1&+7 E. coli 3ro0th hormone

Interferon G!a@ G!# 1&&! E. coli 'ancer

Tissuelasminogeninacti/ator

1&+H E. colieastAnimal cells

Eyocardialinfarction@ulmonaryem#olism

Interleukin ! 1&&$ E. coliAnimal cells

'arcinoma@melanoma

Factor %III 1&&! Eammalian cells 2aemohiliarythrooietin 1&+& Eammalian cells Anaemia ,in AIDS-

Case Stuy ! Somatotropin (Human "ro#th Hormone)

Somatotroin deficiency can arise from se/eral human illnesses such as

Turneryme used to #e isolated from the ituitary glands of cada/ers and 0as /erye;ensi/e. In 1&+7 unusual cases of 'reut>feldt=Jaco# disease ,a degenerati/eneurological disorder- 0ere reorted in indi/iduals 0ho had recei/ed cada/er=sourcedsomatotroin treatment ten to fifteen years re/ious. Based on the assumtion thatinfectious rions 0ere resent in the cada/er=deri/ed 232 it 0as remo/ed from the

market. Then in 1&+7 an E. coli deri/ed recom#inant en>yme 2umatroeK

,somatotroin for in4ection- 0as marketed #y li illy L comany. Somatotroin hasrecently #ecome the first #iosimilarC to #e aro/ed for use.

Protein Sta#ility

Proteins ha/e oor hysical and chemical sta#ility e/en 0hen refrigerated orfro>en. T0o methods are used to imro/e sta#ility

yohilisation ,free>e drying-

P3lyation ,the attachment of a fle;i#le strand or strands of olyethylene

glycol ,P3- to a rotein.-


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$yophilisation

Ad/antages

Einimal damage and thus minimal loss of acti/ity

o0 finial moisture content of roduct therefore longer shelf life

ase of reconstruction

Disad/antages

Slo0

2igh oerating e;enses

2o0e/er the ad/antages out0eigh the disad/antages.

Princiles


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PE"lyation

The si>e 0eight shae and the linkage used to connect the P3 strand to themolecule determine the imact P3lyation has on

Delayed a#sortion

Decreased roteolysis

Increased circulation lifetime

Slo0ed renal clearance

"educed immunogenicity

Imortant notes on P3s

8on=to;ic

Amhohilic

P3lyation roceeds /ia co/alent #onds #et0een a secific acti/e grou on

the rotein and a chemically reacti/e grou on the P3

P3lyation can #e either linear or #ranched

'urrently a/aila#le P3lyated drugs include

P3interferon anti/iral agent

P3figrastim chemotheraeutic

P3adenosine isomerase en>yme relacement

%accines

Immunity

Ac%uire Immunity

;osure of the immune system to antigens leads to roduction of anti#odies ,IgB-#y B lymhocytes M ac5uired immunity. %accination uses modified antigens ,toreduce clinical risk- to stimulate anti#ody roduction. Immunisation may #e eithertheraeutic or rohylactic. ;amles EE" cholera and tetanus /accines.

Passi&e Immunity

Introduction of anti#odies into the system confers assi/e immunity 0hich istransient. Antisera are anti#ody containing serum rearations from animals that react0ith a circulating antigen. This immunisation is often only temorary and can #etheraeutic or rohylactic. ;amles snake /enom antisera sider anti/enins.

EE" 'ontro/ersy

In 1&&+ a aer u#lished #y the ancet ,9akefield et al- sarked fears of an

autism connection 0ith the EE" /accine. This resulted in arents making theotentially dangerous decision to refuse to ha/e their child /accinated. As a result


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incidences of measles ha/e #een on the rise since 1&&+. "ecent reorts ha/e refuted9akefield


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stimulates the immune system against a ossi#le later emergence of the authentic/irus. Initial results sho0 that this form of /accine may #e /ery effecti/e. %P/accines can #e mass=roduced 5uite effecti/ely.

Suunit accines

These are tyically comrised of surface antigens or #acterial olysaccharidecasule fragments that are immunogenic. Eany /accines are con4ugated into roteinsto increase the o/erall immune resonse in those 0ith 0eaker rimary immunesystems ,e.g. children under t0o years old-.

Case Study- Pneumococcal Vaccines

Streptococcus pneumoniae is the largest re/enta#le killer in children under fi/e.The organism causes many tyes of infection such as neumonia sinusitis otitismedia meningitis #acteraemia endocarditis and #rain a#scesses. S. pneumoniae isalso the most common cause of #acterial meningitis in adults and children.

!$=%alent Pneumococcal Polysaccharide %accine ,PP%-

o Eanufactured using casular olysaccharides from !$ /irulent

strainso 2ighly effecti/e in those older than ! years

H=%alent Pneumococcal 'on4ugate %accine ,P'%-

o Eanufactured using casular olysaccharides from H /irulent strains

con4ugated 0ith dihtheria '"E1&H rotein.o Eore effecti/e for immune resonse in toddlers under ! years old.

*ther accines

Recombinant Vaccines

These are created #y utilising #acteria or yeast to roduce large 5uantities of asingle /iral or #acterial rotein. This rotein is then urified and in4ected into the

atient and the atient


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Theraeutic Anti#odies

Theraeutic anti#odies are la#oratory=engineered anti#odies that recognise and #indto a rotein antigen on the surface of a cell. ach theraeutic anti#ody recognises adifferent antigen and in general can #e used alone in com#ination 0ith

chemotheray or as a carrier of su#stances such as to;ins or radiation. After #indingto the targeted site the theraeutic anti#ody can #lock the gro0th of a tumour and@orrecruit the #ody


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+onoclonal Antioies

A mouse is immunised #y an in4ection of antigen to stimulate the roduction ofanti#odies targeted against . The anti#ody forming lymhocyte cells are thenisolated from the mouse


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Problems wit !urine !onoclonal Antibodies

Although murine anti#odies are /ery similar to that of human


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&erceptin%

2ercetin is a humanised mA#. It #inds to 2"! a recetor for eidermal gro0thfactor ,3F- that is found on some tumour cells ,some #reast cancers lymhomas-.To date 2ercetin seems to #e the only mA# effecti/e on solid tumours. Eode ofaction


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ReoPro%

This is manufactured using the antigen #inding fragments and inhi#its the clumingof latelets #y #inding to the recetors on their surface that are normally linkedtogether #y fi#rinogen. It is therefore helful in re/enting reclogging of the coronary

arteries in atients 0ho ha/e undergone angiolasty.

Dia'nostic mAbs

Anti#odies are used in se/eral diagnostic tests to detect small amounts of drugsto;ins or roteins

EA#s to human chorionic gonadotroin ,2'3- are used in regnancy testing

kits.

Diagnosis of certain cancers in tissue #iosies.

9estern Blotting and ISA techni5ues.

Antibody Type Directed against Disease/ condition

8ofetumo#a# Eurine Fa#fragment

'arcinoma=associated antigen

Small lung cellcarcinoma

Arcitumo#a# Eurine Fa#fragment

2uman carcino=em#ryonic antigen

'olorectinal cancer

Imicroma# enetate Eurine Fa#fragment

2uman cardiacmyosin

Eyocardialinfarction imaging

Stem 'ells

Stem cells are e;traordinary #ecause

They can di/ide and make identical coies of themsel/es o/er and o/er again

,self rene0al-

"emain unsecialised 0ith no secific function or #ecomeO

Secialised ,differentiated- 0ith the otential to roduce o/er !66 different

tyes of cells in the #ody.

There are t0o tyes of stem cells

m#ryonic from inner mass if #lastocysts ,a- left o/er from I% fertilisation in

the la#oratory or ,#- from a#orted foetuses.

Adult stem cells Stem cells ha/e #een found all o/er the #ody including the

#lood #one marro0 li/er kidney cornea dental ul um#ilical cord #rainskin muscles sali/ary glands etc.


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m#ryonic %s. adult stem cells

Embryonic Adult

Pluriotent can #ecome almost any cell Eultiotent can #ecome many cells #ut

not allSta#le can undergo many cell di/isions ess sta#le caacity for self rene0al is

limited

asy to o#tain #ut #lastocyst is destroyed Difficult to isolate in adult tissue

Possi#ility of re4ection 2ost re4ection minimised


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Stem 'ell Treatments

2uman diseases such as Parkinson


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Ea4or de/eloments in the )S highlight one of the most imortant issues facingharmaceuticals and #iotechnology data drug ricing. " L D is increasinglye;ensi/e: studies ut the cost of utting a ne0 drug for ma4or chronic diseases ontothe market at u0ards of +66 million ,including the cost of failures-.

a discussion of biotechnology

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