A Review on Chromatography-based purification of monoclonal antibody

International Research Journal of Engineering and Technology (IRJET) e-ISSN: 2395-0056

Volume: 09 Issue: 08 | Aug 2022 www.irjet.net p-ISSN: 2395-0072

A Review on Chromatography-based purification of monoclonal antibody

Pranoti Deshpande1

1Student, Dept. of Biochemical Engineering and biotechnology, KIT’s college of engineering (Autonomous), Kolhapur, Maharashtra, India. ***

Abstract - The foundation of downstream processing remains chromatography, especially at the process scale. There are several causes for this, but crucial ones include the superior scalability, robustness, and selectivity that process chromatography provides in comparison to its competitors. For the purpose of removing contaminants and capturing antibodies, chromatography selection is crucial. In this minireview, we attempt to summarize different types of chromatography for monoclonal antibody purification.

Key Words: Downstream process, purification, chromatography, mAbs, biopharmaceuticals.

1. INTRODUCTION

Therearehundredsofmonoclonalantibodies(mAbs)either inuseorbeingdeveloped.DuetothepopularityofmAbsas therapeutics, several businesses have many antibodies. Today, monoclonal antibodies are recognized as a critical treatment approach for a variety of disorders. Companies have steadily increased the total number of mAbs under clinicaldevelopmentthroughouttime.Anappealingstrategy for solving the diseases is to use monoclonal antibodies (mAbs),whichmaybemadetotargetcellsonlywhenthey arespecificallydesiredandcauseawiderangeofreactions when bound. These substances have the ability to either directlykillcellsbydeliveringpoisonoussubstancestothe targetortoorchestratecelldeathinotherways,suchasby activatingimmunesystemcomponents,inhibitingreceptors, or scavenging growth factors. [1] The most common categoryofrecombinantproteintreatmentsisestablishedto be monoclonal antibodies (mAbs). They are often very soluble,exhibithighlevelsofexpressionincellculture,and are comparatively stable after processing. It is generally knownthatmammaliancellculturescanproduceantibodies. Currently, mammalian cells make up around 70% of recombinant therapeutic proteins, with Chinese hamster ovary(CHO)cellsbeingthemostcommonexpressionhost. In addition to purity and process capacity, downstream processdevelopmentplacesastrongemphasisonyieldand productivity.[2]Themostimportantstepinthedevelopment of biopharmaceuticals is the efficient recovery and purificationofmAbsfromthecellculturemedium.Product stabilityisoneofthemostcrucialcharacteristicsthatmust bepreservedthroughouttheprocess.Thehighestpriority shouldbegiventomaintainingproductqualityandboosting purity while overcoming multiple obstacles. To minimize

materiallossandtoreducethepotentialofcontamination, each step of downstream processing should be handled carefully. [3] Removing the majority of the cell culture's insolublecomponentsfromtheproductstreamisthefirst step in recovering secreted antibodies. Whole cells, cell fragments,colloids,andotherkindsofcontaminantsmake upthesecomponents.Harvestsfrombioreactorsareusually clarifiedbycentrifugationand/orfilteringthroughanumber ofdepthfiltersUtilizingcontinuousdisc-stackcentrifugation alongwithdepthfilteringisoneapproachthattheindustry prefers to use for completing this initial separation. Followingadepthfilter,afilterwithanabsoluteporesize rating (usually 0.45 m or 0.2 m) is used to assure the removalofsolidparticles(andbacteriainthecaseofthe0.2 m filter) from the cell culture harvest supernatant. These initial recovery methods are designed to eliminate the majorityofparticlesfromcellbrothinordertoreducethe workloadforthefollowingpurificationsteps.[4][5]Dueto itsgreatresolution,chromatographyisacrucialandoften used separation and purification method in the biopharmaceutical sector. Chromatography separates biomoleculesbymakinguseoftheirphysicalandchemical distinctions. After that, a succession of chromatography procedures,beginningwiththecapturestage,areappliedto theclarifiedcrop.Capturechromatography,atechniquethat involvesbindingandeluting,guaranteesbothadecreasein harvest volume and the safety of the final product by eliminating the majority of contaminants, including potentially harmful proteases. Ion exchange, hydrophobic interaction,andmultimodalchromatographyarefrequently utilizedaspolishingprocessesforthemonoclonalantibody purificationaftercapturingwithProteinA.[6]

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Fig

-1: ProcessflowformAbpurification

2. CHROMATOGRAPHIC PROCESSES

2.1 Protein A chromatography

A native or recombinant proteic ligand derived from Staphyloccocus aureus or Escherichia coli, respectively, is combinedwithanaturalorartificialbasematrixinProteinA affinity chromatography. Five homologous domains of Protein A E, D, A, B, and C can bind to the Fc moiety of immunoglobulin G. (IgG). Because of its strong binding affinity and the high levels of purity that may be attained, Protein A affinity chromatography is a well-known procedure in the pharmaceutical sector. The target antibodies are loaded onto the immobilized Protein A support at neutral pH as part of the purification process, facilitating the interaction of the ligands. Following the separationofthe proteinfromcontaminantslikehostcell proteins(HCP),themobilephase'spHisloweredtofacilitate theproduct'sdesorption.Theresinisthenregeneratedand putthroughaclean-in-placetechnique.[7][8]

Fig -2: ProcessflowforproteinAchromatography

Protein A chromatography is the most expensive stage, making it a suitable place to start. The monoclonal antibody's interaction with immobilized Protein A is the foundation of this affinity chromatography technique. Hydrophobicinteractionsarepredominantlyresponsiblefor thebindingbutionicandhydrogenbondsalsoplayarole. Dependingontheantibodysubclass,thepHrangeof6–9is wheretheantibodybindstoProteinA,andthesaltcontent ofthebindingbuffercanaffectthis.ApHbufferwithalow rangebetween2and4.5ischosentoelutethebinding.Since theantibodyelutionoccursatalowpHlevel,thismethodis alsoutilizedtoinactivateviruses.[9][10][11][12]ProteinA purificationisfocusedandeffective.However,therearestill anumberofproblemsthatneedtoberesolved,suchasthe inadequate removal of contaminants including host cell proteins,DNA,aggregates,etc.Moreover,theimpactofwash buffers on protein. The physicochemical properties of antibodiesarestillnotwellunderstoodafterpurification.A newmonoclonalantibodypurificationmethodthatenhances the physicochemical characteristics of the antibodies by simply adding a basic buffer wash step following the traditionalproteinApurification'scapturestage.[13][14]In comparison to packed columns, Protein A membrane adsorbers and monoliths are more productive because of theirlowbedheightsandhighoperationalflowrates.These gadgets might serve as a model for future increases in protein A productivity even though they are not now practicableforlarge-scaleproduction.[15][16][17]

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Fig -3: AntibodyandProteinAinteraction

2.2 Ion Exchange chromatography

The protein's pI value is used in ion-exchange chromatographytoachieveseparation.Apositivelycharged protein is drawn to a negatively charged solid support in cation-exchange chromatography, whereas a negatively chargedproteinisdrawntoapositivelychargedsupportin anion-exchange chromatography. Elution is often accomplishedbychangingthebuffer'ssaltorpHcontentina stepwise or gradient fashion. Ion-exchange is a quick and affordable approach for purifying antibodies, and it's frequently employed as a polishing step after Protein-A affinity chromatography.[3][18]. For the purification of monoclonalantibodies,cationexchangechromatographyis commonly used in bind-and-elute mode. However, it was discoveredthatbind-and-eluteconditionswereinsufficient foreliminatingappreciableamountsofantibodyaggregate. Thedesiredproductflowsthroughastationaryphaseandis subsequentlyretainedbyamediuminpositivepurification, also known as the retention mode. Protein A affinity and cationexchangechromatographyisemployedinretention modeina broadplatformmAbpurificationprocedure.On theotherhand,theflow-throughmodeisfrequentlyutilized withanionexchangechromatography.Giventhattheflowis continuous,theoperationtechniqueknownasflow-through chromatography (FTC) is regarded as an effective way to separate two components. The desired product is flowed throughamediuminthenegativepurificationprocess,also knownastheflow-throughmode,whilecontaminantsare trappedbyastationaryphase.Targetproteinsareremoved from the chromatography column in FTC without being adsorbed, whereas contaminants are tightly attached. In contrast,tobindandelute,highercolumnloadingsareoften attainableintheflow-throughmode.[11][19][20][21][22]

If applicable, purification using the flow-through mode providesmanybenefitsoverpositivepurificationintermsof the quantity and size of buffers needed, workload, and processingtime.[23]

2.3

Fig -4:Ionexchangechromatography

Multimodal chromatography

Multimodalormixed-modechromatographicmedia(MMC) that demonstrate several binding interactions are more tasteful substitutes for a sequence of processes that each involve a single interaction. MMC is a chromatographic techniquethatutilizesmanysortsofinteractionsbetween thestationaryphaseandthemobilephase,wherevarious solutes are present. [24] In multimodal or mixed mode chromatography, the chromatography ligand interacts selectively with the analyte molecule through a variety of interactions. These contacts can be ionic hydrophobic, hydrogenbonding-based,orevenVanderWaalsinteractions. Inthedevelopmentofmixed-modechromatography,ligands arecrucial.Theadsorbentcanoffersalttolerantqualities, improved separation, and high binding capabilities with which a wide range of medicines can be purified if the hydrophobic and ionic moieties in the mixed mode ligand arebalancedproperly.Electrostaticrepulsionbetweenthe analyteandresinligandcanresultinselectiveelutioninthe caseofhydrophobicchargeinductionchromatographywith the choice of an adequate pKa value. [18] [25]. MMC has selectivitiesandspecificitiesthataredistinctfromthoseof conventional ligands, giving it a versatility that makes it possibletoaddressavarietyofdifficultpurificationissues. [26][27].

3. CONCLUSIONS

Someofthecrucialcomponentsofaplatformdownstream processformAbswerediscussedinthisreview.Inaddition to this, several other technologies are still utilized during mAb purification. The development of chromatographic processes is a crucial step in the manufacturing of

biopharmaceuticals.Itusuallyrequiresalotofresourcesand time,whichaffectshowlongittakestolaunchaproduct.

ACKNOWLEDGEMENT

IwanttothankMr.Nikhil,Mr.Mayur,Mr.Sufiyan,andMrs. Saeema’am,whohelpedmetocompletethismanuscript.

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