commercial manufacturing scale formulation and analytical characterization of therapeutic...

Research Overview

Commercial Manufacturing Scale Formulation andAnalytical Characterization of Therapeutic Recombinant

AntibodiesReed J. Harris,1 Steven J. Shire,2 and Charles Winter3

1Analytical Chemistry Department, South San Francisco, California2Pharmaceutical Research and Development, South San Francisco, California

3Recovery Sciences, Genentech, Inc., South San Francisco, California

Strategy, Management and Health Policy

Enabling

Technology,

Genomics,

Proteomics

Preclinical

Research

Preclinical Development

Toxicology, Formulation

Drug Delivery,

Pharmacokinetics

Clinical Development

Phases I-III

Regulatory, Quality,

Manufacturing

Postmarketing

Phase IV

ABSTRACT Stable therapeutic antibody dosage forms present production technology challenges,particularly when high-concentration formulations are needed to meet the elevated dose requirements thatare generally required for successful antibody therapy. Solid dosage forms, such as lyophilized powders,are generally more stable than liquid formulations. High-concentration drug products can be achieved byreconstitution of the lyophilisate in a smaller volume than its initial (pre-lyophilization) volume, butrequires a significant vial overfill. High-concentration liquid formulations are becoming feasible as newtechniques and technologies become available. Analytical methods to detect subtle molecular variationshave been developed to demonstrate manufacturing consistency. Some molecular heterogeneity iscontributed by conserved sites, such as Asn297 glycosylation and the loss of heavy chain C-terminal Lysresidues. Characteristics that affect potency, stability, or immunogenicity must be elucidated for eachtherapeutic antibody. Drug Dev. Res. 61:137–154, 2004. �c 2004 Wiley-Liss, Inc.

Key words: monoclonal antibody; formulation; ultrafiltration; analytical characterization

INTRODUCTION

By the end of 2002, twelve therapeutic anti-bodies, antibody fragments, or conjugated antibodieshad been licensed by the US Food and DrugAdministration (FDA) to treat a wide range of humandiseases (Table 1). Antibodies represent one of thefastest-growing segments of biotechnology, with newclinical and manufacturing lessons learned during thedevelopment of each new therapeutic candidate. IgG1-type antibodies have two heavy chains and two lightchains, with carbohydrates at a conserved Asn in eachheavy chain (Fig. 1). All recombinant MAbs areheterogeneous due to common features such asglycosylation and C-terminal lysine processing.In addition, each MAb has unique sources of

heterogeneity due to protein degradative processes,production conditions, and heterologous expressionartifacts.

Monoclonal antibodies (MAbs) are producedusing mammalian cell lines; the MAb must be purified,concentrated, and exchanged into an appropriateformulation, and then be demonstrably stable overthe proposed shelf life. Ten of the 12 currently

DDR

nCorrespondence to: Reed J. Harris, Genentech, Inc. (#62),1 DNA Way, South San Francisco, CA 94080.E-mail: [email protected]

Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/ddr.10344

DRUG DEVELOPMENT RESEARCH 61:137–154 (2004)

�c 2004 Wiley-Liss, Inc.

FDA-approved monoclonal antibodies are adminis-tered by the intravenous (IV) route. High-concentra-tion liquid MAb formulations for subcutaneousadministration are desirable for chronic disease thera-pies and outpatient treatment to improve patientconvenience and compliance, but the development ofsuch formulations is constrained by protein-proteininteractions (e.g., aggregation, viscosity) and processingequipment limitations.

This report reviews key challenges in the devel-opment and production of stable, convenient MAbdosage forms. Useful physicochemical methods thatdemonstrate product quality and consistency, alongwith methods for the identification of desired orundesired forms, are also discussed.

FORMULATION CHALLENGES

The challenges in formulation of therapeuticantibodies are quite similar to those faced with otherprotein therapeutics, i.e., the large number of func-tional groups susceptible to chemical degradation

routes coupled with the requirement of maintainingnative protein conformation [Rouan, 1996; Chang andHershenson, 2002]. In addition to assuring sufficientpharmaceutical stability, the formulation also mustallow for successful administration. Almost all ap-proved monoclonal antibodies are administered by theIV route (Table 1). Several ongoing monoclonalantibody development programs are targeting diseasesthat may require outpatient or home administration,and hence require the development of alternatedelivery routes. Administration routes such as oral,transdermal, and pulmonary have been difficult toachieve because of the instability and size of mono-clonal antibodies. For example, one monoclonal anti-body given by the pulmonary route had low systemicdistribution and was clinically ineffective [Fahy et al.,1999]. This leaves mainly injection routes of adminis-tration such as subcutaneous (SC) injection that can beused in a physician’s office/clinic or by the patient athome. The high doses (on the order of mg/kg) oftenrequired for antibody therapy require the development

TABLE 1. Monoclonal Antibody Therapeutics Approved by the FDA

Product Approval date Type of MAb Dosage form and routeof administration

Target Indication

Orthoclone OKT3(muromonab-CD3)

1986 IgG2 Murine 1mg/ML liquid/IV CD3 antigen onT lymphocytes

Acute transplantrejection

ReoPro (abciximab) 1984 Fab Chimeric 2mg/ML liquid/IV GP llb/llla on platelets Platelet aggregationinhibitor forpercutaneous cornaryintervention

Rituxan (rituximab) 1997 IgG1 Chimeric 10mg/ML liquid/IV CD20 antigen onB lymphocytes

Non-Hodgkin’slymphoma (relapsed orrefractory low-grade)

Zenapax (daclizumab) 1997 IgG1 Humanized 5mg/ML liquid/IV Interleukin 2 receptor onactivated T lymphocytes

Acute rejection ofkidney transplants

Herceptin(trastuzumab)

1998 IgG1 Humanized Lyophilizate/IV p185HER2 Metastatic breast cancertumors that overexpressp185HER2

Remicade (inflixibmab) 1998 IgG1 Chimeric Lyophilizate/IV Tumor necrosis factor Rheumatoid arthritis,Crohn’s Disease

Simulect (basiliximab) 1998 IgG1 Chimeric Lyophilizate/IV Interleukin 2 receptor onactivated T lymphocytes

Acute rejection ofkidney transplants

Synagis (palivizumab) 1998 IgG1 Humanized Lyophilizate/IM F protein of respiratorysyncytial virus (RSV)

Respiratory tract diseasecaused by RSV infectionin children

Mylotarg (gemtuzumab,ozogamicin)

2000 IgG4 Humanized(calicheamicinconjugate)

Lyophilizate/IV CD33 antigen onleukemia cells

CD33 positive myeloidleukemia

Campath(alemtuzumab)

2001 IgG1 Humanized 10mg/mL liquid/IV CD52 antigen on Band T lymphotocytes

B-cell chroniclymphocytic leukemia

Humira (adelimumab) 2002 IgG1 Human 50mg/mL liquid/SC Tumor necrosis factor Rheumatoid arthritisZevalin (ibritumomab,tiuxetan)

2002 IgG1 Murine(Yttrium-90chelate)

1.6 mg/mL liquid/IV CD20 antigen onB lymphocytes

Non-Hodgkin’slymphoma (relapsed orrefractory low-grade)

138 HARRIS ET AL.

of SC formulation at concentrations exceeding 100mg/mL because of the small volume (o1.5mL) that can begiven by the SC route. Development of antibodyformulations at high concentrations poses stability,manufacturing, and delivery challenges related to thepropensity of antibodies to aggregate at the higherconcentrations. These challenges will be discussed aswell as appropriate analytical techniques for assess-ment of high concentration monoclonal antibodyformulations.

CHOICE OF DOSAGE FORMS

The indication, patient population, route andfrequency of administration, and stability of the proteindrug often dictate the choice of dosage form. Liquidformulations for protein drugs have been the preferreddosage form since theoretically they are cheaper tomanufacture and are more convenient for the end-user.However, it can be difficult to obtain the 1.5–2-yearshelf life needed to manage inventories because of thevariety of physical and chemical degradations thatoccur in proteins stored in solution [Manning et al.,1989; Chang and Hershenson, 2002]. An understand-ing of the physical and chemical properties of proteins

using preformulation studies helps the developer inmapping out the solution conditions such as pH andionic strength to control the major degradation routesthat may affect activity and safety of the molecule. Thedifficulty in developing a marketable liquid formulationis that several degradation routes may need to becontrolled [Kroon et al., 1992], and thus it may requirea choice of formulation conditions that are sub-optimalfor some of the individual reactions. In addition, liquidformulations require careful studies of compatibilitywith container/closure surfaces as well as agitationsince solutions of proteins are susceptible to surface-and shear-generated denaturation [Thomas et al., 1979;Watterson et al., 1974].

Solid dosage forms produced using technologysuch as lyophilization and spray drying to remove waterresults in slower kinetics for many of the hydrolyticreactions in proteins. Crystallization has proven to beeffective in the development of insulin formulations,and shows great promise for the development of stablemonoclonal antibody dosage forms [Harris LJ et al.,1995; Shenoy et al., 2001]. Although development of asolid dosage form can lead to increased stability andlonger shelf life, this result comes at the expense of

Fig. 1. Space-filling model of trastuzumab. Amino acid residues are colored as follows: heavy chainconstant and framework regions, silver; heavy chain CDRs, yellow; light chain constant and frameworkregions, gold; light chain CDRs, green. Carbohydrate residues are shown in pink.

ANTIBODY FORMULATION AND CHARACTERIZATION 139

convenience for the end-user, which ultimately canlead to issues with respect to patient compliance.Recent advances for easier use of lyophilized drugssuch as dual-chambered syringes or cartridges andconvenient reconstitution devices have helped phar-maceutical companies mitigate potential patient com-pliance issues. In the end, if it is not possible to developa stable liquid formulation there is little choice but toproceed with solid dosage form development. Althoughthe common protein degradation rates in solution aregenerally slower in the solid state, process-dependentstresses on the protein during the drying process canresult in additional degradation pathways.

Spray drying has been used to produce free-flowing powders that could be used for aerosoladministration [Costantino et al., 1998; Andya et al.,1999] or possibly for bulk storage [Masters, 1985;Rouan, 1996; Maa et al., 1998]. During spray drying,the protein is subjected to nebulization followed byair-drying; thus excipients are chosen to minimizedamage due to exposure of air-water interfaces andhigh temperature [Tzannis and Prestrelski 1999].Particle morphology and protein stability as a functionof spray drying parameters such as input flow rates andtemperature have been described [Maa et al., 1997].

All approved monoclonal antibody solid dosageformulations have been developed using lyophilization(Table 1). During lyophilization, the protein is sub-jected to stresses during freezing and then subsequentdrying by sublimation. Excipients that can serve ascryo- and lyo-protectants are added to minimize thedegradations that can occur as a result of thelyophilization process [Carpenter et al., 1993; Pre-strelski et al., 1993]. Sugars have been commonly usedas excipients as it has been found that non-reducingdisaccharides such as sucrose and trehalose areeffective in minimizing degradation due to thelyophilization process [Ressing et al., 1992; Breenet al., 2001; Cleland et al., 2001]. Reducing sugars suchas maltose can result in alteration of a monoclonalantibody by the Maillard reaction whereby ketoamineadducts of the reducing sugar are formed at side chainamino groups of lysine [Kroon, 1994]. Poor selection ofbuffer component and pH can result in generation ofreducing sugars from non-reducing sugars and impactthe stability of the freeze-dried formulation [Townsendand DeLuca, 1988]. Moisture also needs to be carefullycontrolled since high moisture levels in freeze-driedpreparations can result in loss of stability due to alowering of the glass transition temperature,Tg0 [Duddu and Dal Monte, 1997; Breen et al.,2001].

An example of how effectively lyophilization cancontrol protein degradation is shown for a recombinant

humanized monoclonal antibody (rhuMAb) stored for3months in solution at 21–81C. This monoclonalantibody when stored in solution undergoes isomeriza-tion of an aspartic acid residue in one of thecomplementary determining regions (CDR). Thedegradation products, which result in decrease ofbinding affinity, can be monitored by hydrophobicinteraction chromatography (HIC) of a pepsin digest[Cacia et al., 1996]. After freeze-drying, the rate of theisomerization is greatly reduced as shown by the HICanalysis of lyophilized and liquid formulations ofrhuMAb (Fig. 2). The main degradation route insolution, Asp isomerization, is controlled by lyophiliza-tion, whereas aggregation is a major degradation routein the lyophilized formulation for this monoclonalantibody (Fig. 3) [Andya et al., 2003]. Size exclusionchromatography of the unformulated (excipient-free)rhuMAb detects aggregates that formed as a result ofthe lyophilization process, along with those thatdeveloped subsequently. Appropriate addition of ex-cipients, including lyoprotectants, can prevent theformation of aggregates during the lyophilizationprocess or during storage of the final product. A keyparameter for effective control of aggregation is themolar ratio of lyoprotectant to the monoclonal anti-body. At 501C storage, as the lyoprotectant:MAb molarratio approached a value of 500:1, the pseudo firstorder rate constants begin to level off at a minimumvalue, indicating the optimal formulation is obtained atmolar ratios 4500:1 (Fig. 4). Similar analysis foranother antibody has shown that a molar ratio of

Fig. 2. Hydrophobic interaction chromatography after pepsin diges-tion of a MAb. Solid lines are from a pH 5.2 acetate-NaCl liquidformulation and dashed lines are from a lyophilized formulation after3 months of storage at 2–81, 251, or 401C. Peaks 1–5 represent F(ab0)2forms with Asp32 residues bearing these modifications: 1, Asp/Asp; 2,Asp/isoAsp (isoAsp in one Fab); 3, Asp/Asu (Asu: succinimide); 4,isoAsp/isoAsp or isoAsp/Asu; 5, Asu/Asu.

140 HARRIS ET AL.

lyoprotectant to the monoclonal antibody at 360:1 canprovide long-term storage at room temperature orabove, and thus the appropriate minimum molar ratiomay depend on the individual monoclonal antibody[Cleland et al., 2001].

IMPACT OF DOSAGE FORM ON MANUFACTURINGOF HIGH CONCENTRATION FORMULATION FOR

S.C. DELIVERY

Development of monoclonal antibody formula-tions for SC delivery imposes additional challenges for

liquid formulation development. Typical cell cultureand recovery processes yield purified product poolconcentrations on the order of 5–10mg/mL, requiringa process operation that not only exchanges buffer andsolvent components from the last column chromato-graphy step, but also concentrates the protein to finalconcentrations exceeding 100mg/mL. The main tech-nology that has been used in a scale-up mode isultrafiltration (UF), discussed in this report in refer-ence to a subset of tangential flow filtration technologythat uses a UF membrane barrier [Genovesi, 1983;Shiloach et al., 1998; van Reis and Zydney, 2001].Continuous circulation through piping, valves, andpumps may generate sufficient shear, or may causemicrocavitation, such that protein unfolding results[Watterson et al., 1974; Thomas et al., 1979]. Thechallenges and development strategy to design UFsystems to generate high-concentration liquid formula-tions are further discussed below in Production of theFormulated Bulk Drug Substance.

Although UF is the industry standard forconcentration of macromolecules at commercial pro-duction scales, there are alternatives. In particular, anydrying technique can be used with a subsequentreconstitution with lower volume in the final vialconfiguration. Using the lyophilizer as a concentratorwhereby a protein loading volume VL at loadingconcentration CL is lyophilized and then reconstitutedwith a diluent volume VR, where VRoVL leads to afinal drug product concentration CF for SCadministration:

CF¼ CLVL=ðVRþVSÞ ð1Þ

VS is the volume contributed by the remaining solids,and can be estimated from the sum of the partialspecific volumes of all the excipients and the protein,although the best way is to reconstitute the finalvials with a series of VR values to determine theappropriate volume to attain the desired target CF.In addition, sufficient volume needs to be used in thereconstitution process to ensure that the requiredvolume for administration can be withdrawn. Thus,experiments may need to be designed to determine therequired overage in the fill by varying VL and/or CL.The effects of loading concentration on reconstitutiontimes and morphology of the lyophilized solid asdetermined by scanning electron microscopy showsthat these parameters can have a major impact on thereconstitution properties of the final product [Breenet al., 1998].

In the study discussed in Figure 5, formulationswere prepared that maintained the same weight ratio ofprotein to excipients. Each vial was loaded with thesame total protein mass and formulation excipients so

Fig. 4. Aggregate formation after 3 months MAb storage at 501C asfunction of lyoprotectant (sucrose) composition. Percent aggregate wasdetermined as described in Figure 3.

Fig. 3. Aggregation evaluated by size exclusion chromatography of alyophilized excipient-free MAb stored 1 year at 401C. Chromatogra-phy conditions: TSK-gel G3000SWXL column (TosoHass, Montgo-meryville, PA), flow rate: 0.5mL/min., mobile phase: 20mM sodiumphosphate, 150mM NaCl, pH 7. Chromatographic peak area percentvalues are shown.


that the lower CL required greater VL values. Theformulations were investigated for solution clarity anddissolution of solids 10min after reconstitution to125mg/mL. Reconstitution occurs more readily as theprotein loading concentration is decreased. Investiga-tion of the morphology of the lyophilized solid byscanning electron microscopy (SEM) shows a verydense compact structure when a 110-mg/mL bulk wasfilled, whereas the lyophilized cake from a 40-mg/mLbulk fill appears to consist of a loosely packed layer offlakes. Thus, the ease of wetting of the cake appears tobe related to the differences in this morphology. Theintermediate concentration SEMs (not shown) show agradual transition from a dense solid to the moreloosely packed structure, and further supports thiscorrelation.

The process described above leads to designing aformulation that contains all the required excipients forensuring stability and appropriate tonicity. Isotonicity isnot necessarily required for SC administration [Gatlinand Gatlin, 1999]; however, it may be desirable in orderto have a formulation that does not generate pain at thesite of administration. The main problem with thisapproach is that all excipients in the formulation arealso concentrated as the protein is concentrated afterlyophilization and reconstitution. Thus, as shown inTable 2, excipient:MAb molar ratios of 500:1 will result

in hypertonic preparations if the final protein concen-tration is targeted for 4100mg/mL. If the desire is toachieve an isotonic formulation, then a choice of lowermolar ratio of excipient:MAb may result in a potentiallyless stable formulation. In an unpublished example, amonoclonal antibody formulation with an excipient toprotein molar ratio of 250:1 was significantly less stableat 301C storage than one at a 500:1 molar ratio.However, at 21–81C storage, the stabilities at 250:1and 500:1 molar ratio were comparable. Althoughstability is an important feature of a formulation,other features such as ease of administration orminimization of potential pain on injection may dictatethe choice of excipients that lead to an overall

Fig. 5. Lyophilization of a monoclonal antibody as a function of bulkloading concentration. Top left: Each 10-mL glass vial was loaded withthe same MAb mass (208mg) and excipients, with vial loadingconcentrations from left to right of 40, 60, 80, 100, and 110mg/mL

MAb. Bottom left: Scanning electron microscopy of lyophilized solidfor the 40 and 110mg/mL MAb loading concentration samples. Right:Drug product appearance 10min after reconstitution of the same vialsto 125mg/mL MAb with 1.3mL of sterile water for injection.

TABLE 2. Concentration of Excipient* as a Function of FinalReconstituted Protein Concentration (CF) and Molar Ratio ofExcipient to Proteinn

Excipient: protein molar ratio

MAb (mg/mL) 250:1 500:1

50 83 167100 167 333150 250 500200 333 666

nExcipient concentration (mM).

142 HARRIS ET AL.

optimization of the formulation. In the case ofthis particular antibody where the target productprofile and frequency of administration allowed for21–81C storage, a 250:1 molar ratio of sugar to proteinwas chosen leading to an optimization of the formula-tion that fit the intended usage of this monoclonalantibody.

COST OF GOODS AND DRUG DELIVERYCHALLENGES

Though high-concentration formulations have thecost-saving advantages of decreasing bulk storage spaceor number of product fills, they have undesirableoverall cost of goods because of unrecoverable fixedvolume losses from ultrafiltration units, filling equip-ment, and product containers. The vial size ismandated by the amount of bulk material needed inthe final dosage form; if the lyophilized material isreconstituted in a lower volume, this results in a vialthat is relatively large compared to the reconstituteddrug product, requiring an overfill of material at highconcentration. Unrecoverable product losses in thefinal dosage form could be minimized with the use ofsmaller vials, narrower based vials, or prefilled syringeconfigurations.

Protein formulations at high concentrations mayalso have physical properties that impair delivery of theprotein drug. Protein association/aggregation is ex-pected to be concentration-dependent and can resultin the generation of covalently linked higher orderaggregates [Townsend and DeLuca, 1991]. Aside from

these stability concerns, physical properties such ashigh viscosity may result in preparations that aredifficult to administer by injection. Unless the viscositycan be reduced by appropriate formulation excipients,the high concentration required for SC delivery maynot be attainable. As an example, the time required toload 1mL of a monoclonal antibody into a syringeequipped with a 27 g needle correlates with theviscosity of the formulation as a function of antibodyconcentration (Fig. 6). Addition of NaCl greatlyreduced viscosity (Fig. 7), and when combined withthe use of a 25 g needle, yielded reasonable times todraw the drug product into the syringe. Syringes for SC

Fig. 6. Viscosity (open circles) and syringe (27g needle) loading time (solid triangles) as a function of MAb concentration. Inset: Linearregression fit of viscosity vs. 27 g syringe loading time.

Fig. 7. Viscosity (open circles) and 25g needle syringe loading time(open triangles) of a 125-mg/mL monoclonal antibody as a function ofNaCl concentration.


injection are often equipped with 25 g, 26 g or 27 gneedles.

ANALYTICAL CHALLENGES AT HIGH PROTEINCONCENTRATIONS

Physical properties that are highly concentration-dependent such as viscosity may need to bedetermined at the concentration of the formulation.Properties such as reversible protein self-association at100–200mg/mL are difficult to assess by commonlyused techniques such as size exclusion chromatography(SEC) because of dilution effects. The use of on-linelight scattering detectors coupled with SEC can beused to quantify molecular masses in solution [Andyaet al., 2003], but cannot circumvent the problem ofaggregate dissociation due to dilution during chroma-tography. As an example, three closely related huma-nized monoclonal antibodies (with the same constantregions) that have the same SEC retention timeexpected for a monoclonal antibody with a molecularmass ofB150 kDa were compared. These antibodies inthe same excipients formulation were freeze-driedusing the same lyophilization cycle at bulk concentra-tions of 25, 50, and 100mg/mL. The reconstitutiontimes for MAb2 and MAb3 were similar, and both aresignificantly shorter than for MAb1. Comparison of theviscosities after reconstitution to 85mg/mL revealedthat MAb1 has B3-fold higher viscosity than MAb2and MAb3 (data not shown). This higher viscosity maybe a prime reason why the reconstitution takes longerfor MAb1 since efficient mixing of the diluent with thelyophilized solid may not be as efficient in the higherviscosity solution.

The viscosity of a macromolecule in solution isdependent on the concentration, shape, and molecularweight of the molecule. The viscosity of a proteinsolution, Z can be related to the solvent viscosity, Z0,and concentration in g/mL of the protein, cp, by apower series [Cantor and Schimmel, 1980]

Z ¼ Zoð1þ k1cpþk2c2pþk3c

3pþ . . . . . .Þ ð2Þ

where k1 is associated with the contribution fromindividual solute molecules, and k2, k3, and higherorder coefficients are related to effects from interac-tions of additional protein molecules. This equation canbe rewritten in terms of the specific viscosity,

Zs ¼ðZ� ZoÞ=Zo

½�� Zo=Zo�=cp¼ Zs=cp¼ k1þk2cpþk3cp2þ . . . . . .

ð3Þ

In the limit of infinite dilution where cpapproaches 0, this equation reduces to the intrinsic

viscosity, which is equal to k1. The intrinsic viscosity isdependent on the shape of the protein and thusconformational information of macromolecules can beobtained through determination of intrinsic viscosity.However, at the higher concentrations, the higherorder terms of the power series in equation (2) willdominate and the virial coefficients, which are relatedto molecular interactions, will contribute to theviscosity.

To determine if molecular self-association couldaccount for the observed differences in viscositybetween the three antibodies discussed above, it wasimperative that protein molecular weight in solution bemeasured at the high concentration of the formula-tions. Analytical ultracentrifugation using a preparativecentrifuge and a microfractionator was used asdescribed [Minton, 1989; Darawshe et al., 1993;Darawshe and Minton, 1994]. In this method, for-mulated protein at high concentration is centrifuged toequilibrium in a swinging bucket rotor and the contentsare collected in 10-mL fractions using a Brandellmicrofractionator [Attri and Minton, 1986]. Thecollected fractions are placed into a 96-well plate forUV absorption spectroscopy analysis. Analysis of thegradients to obtain weight average molecular weights iscomplicated because of the high protein concentra-tions, which results in a thermodynamically non-idealsolution.

The main contributors to non-ideality areexcluded volume and primary charge effect [Roarkand Yphantis, 1971; Minton, 1983; Zimmermanand Minton, 1993]. The data can be analyzedin a semi-empirical fashion by assuming thatMAb2 and MAb3 are not associating to any greatextent in solution. Adjustment of the formulations to150mM NaCl will decrease the non-ideality due tocharge, and thus the correction to obtain 150 kDamolecular mass will be mainly due to excluded volumecorrections. The correction for the charge effect canthen be obtained at 0mM NaCl by subtracting thealready determined correction for excluded volume.Applying these corrections to MAb1 results in thecorrected molecular weight as a function of concentra-tion (Fig. 8). These corrections are estimates andprobably do not account for the complete correction,but the analysis clearly demonstrates that MAb1undergoes self-association as a function of concentra-tion. These interactions are greatly reduced afteradjustment of the formulation to 150mM NaCl. Thus,the addition of NaCl would be expected toreduce viscosity, and, in fact, this was the case asshown previously (Fig. 7) where viscosity decreasesas a function of NaCl for formulated MAb1 at125mg/mL.

144 HARRIS ET AL.

PRODUCTION OF THE FORMULATED BULK DRUGSUBSTANCE

Large-scale production of recombinant antibo-dies at commercial scales is well established in thebiotechnology/biopharmaceuticals industry. Full lengthrecombinant antibodies have been produced in bior-eactors ranging from 10 liter to 10,000 liter volumes[van Reis and Zydney, 2001]. A typical productrecovery process consists of a separation of cells andcell debris from the supernatant that contains therecombinant protein (referred to as the harvest step), apurification train comprised of chromatography andultrafiltration steps, and a finally a formulation step(Fig. 9). The formulated bulk is then frozen for storageand/or filled for use. It is the aspects of the formulationstep that will be discussed below with respect to high-concentration recombinant antibodies. Cell cultureproduction, harvest, and purification steps are notusually affected by the need for high-concentrationformulations.

The formulation step exchanges the purified bulkdrug substance into the final excipient composition andMAb concentration. There is typically no purificationachieved at this step except small molecule removal;the governing aspects are yield, buffer exchange, andstep robustness, defined as the reproducibility of thestep within the expected variability of the upstreamconditions. Several methods have been investigated forthe formulation of biological macromolecules, includ-ing large-scale SEC and UF [Athalye et al., 1992]. SEChas limitations with respect to eluant concentration, asconsiderable dilution is incurred at this step, making itimpractical for high-concentration recombinant anti-body formulations. A comprehensive analysis by vanReis et al. [1997a] comparing formulation by SEC andUF concluded that, unless the protein is prone to

aggregation or denaturation brought on by extensive(450) pump passes through the UF system, UF has aclear advantage over SEC with respect to smallmolecule removal, material and labor cost, buffervolumes, plant space, operating time, and throughput[Kurnik et al., 1995].

Ultrafiltration has been widely adopted as themethod of choice for protein formulation in thebiotechnology industry [van Reis and Zydney, 2001].However, there are many challenges in applying UFto high concentration protein solutions, includingproduct solubility, restrictive bulk and mass transportof protein solutions, and product losses duringrecovery. Figure 10 shows a UF system schematic.The protein-containing feed is pumped through theUF system and back to the recycle vessel. The UFmembrane retains the protein as filtrate (or permeate)is driven through the membrane by pressure. Thepressure is called the transmembrane pressure (TMP)and is typically controlled using a retentate pressurecontrol valve. The relationship between TMP andfiltrate flux (filtrate rate per membrane area) isfundamental to TFF separations [Porter and Nelson,1982].

The general strategy for UF process developmentfor high concentration recombinant antibodies isconsistent with other recombinant proteins. Membranepore size and chemistry selection have been describedby Huisman et al. [2000] and van Reis and Zydney[1999], and selections are usually based on themolecular weight of the protein as well as thepermeability regeneration characteristics of the mem-brane. The incoming pool is first concentrated byultrafiltration to an intermediate volume, and thenbuffer-exchanged by diafiltration into the desiredformulation. The number of volumetric equivalents(diavolumes) necessary to remove buffer components

Fig. 8. Corrected weight average molecular weight determined bysedimentation equilibrium analytical ultracentrifugation for MAb1with (solid triangles) or without added NaCl (solid squares).

Fig. 9. Recombinant antibody production process flow.


from the pool can be calculated, and is confirmed byappropriate methods such as NMR [Kao et al., 2001].The determination of the concentration at which toperform diafiltration, and the number of diavolumes,are determined by process time (economic factors) andsmall molecule concentrations, respectively. Figure 11shows the relationship of process time and diafiltrationconcentration for a particular formulation. The localminimum is due to the fact that at low concentrations,the process time increases because of the magnitude ofeach diavolume, whereas the governing factor isdecreasing flux at high concentrations.

Product solubility of high-concentration rhuMAbformulation via ultrafiltration is additionally challengingin that the concentration at the membrane wall, Cwall,can be significantly (e.g., 2–5-fold) higher then the bulkconcentration, as explained through the stagnant filmmodel theory [Michaels, 1968]. Control strategies havebeen developed to maintain a constant Cwall during UFoperations to minimize dynamic local protein concen-trations within the system [van Reis et al., 1997b].Whether utilizing these advanced control strategies oremploying more traditional control methods, such asmaintaining constant transmembrane pressure, con-firmations of product solubility at the higher Cwall

concentrations should be performed at lab-scale earlyin the development process. Highly viscous solutionsoffer a particular challenge for UF bulk fluid transportand mass transport [Aimar and Field, 1992]. Pumpcapacities and pressure limitations may become asignificant factor as the bulk protein concentrationincreases.

Production of formulated antibodies by ultrafil-tration to high final bulk concentrations requirescareful consideration of system design. System sizes,mixing, sampling, and product recovery all play an

important role in achieving a robust process andreproducibly high overall yields. In addition, processingin a single UF system or transferring to multiple UFsystems needs to be decided, as discussed below.System size is arguably the most important factor insystem design because of the excessive concentrationfactor usually required to get to high final bulkconcentrations. The minimum system volume willdefine the maximum bulk concentration for a givenprotein bulk size. Figure 12 presents the relationshipbetween final bulk concentration and minimum systemholdup volume for given protein bulk masses. Theminimum system volume is defined as piping andmembrane holdup volume plus the minimum tankvolume at which the dip tube is submerged. A generalrule for the minimum system holdup for a givenmaximum membrane surface area available to a UFsystem is approximately 100mL/ft2. This rule is basedon a 3-inch pipe size for a 1,000-ft2 system, 1.5 inchpipe size for a 250-ft2 system, and 0.5 inch pipe for a20-ft2 system. With the example of a 1,000-ft2 system,the minimum bulk volume would be B100L; if theprocess yields a 10,000-g protein bulk, then themaximum concentration would be 100 g/L.

If higher final bulk concentrations are requiredthan those indicated in Figure 12, two accommodationscan be considered: employing multiple UF systems fora single pool or combining multiple pools for one largerUF system. The use of multiple UF systems allowsconcentration of material on the larger primary system.Product recovery is then performed and this material is

Fig. 10. TFF system schematic. Key operating parameters: Flux (J):Filtrate rate (L/hr)/Area (m2); Transmembrane pressure (TMP): (PFe þPR/2) – Pfi. Feed-side pressure drop (DP): PFe – PR.

Fig. 11. Processing time vs. monoclonal antibody concentrationduring diafiltration. Time required to process an antibody productionlot through a 10-fold concentration, with 8 diavolumes and 200 g ofMAb per square meter of UF membrane.

146 HARRIS ET AL.

transferred to a smaller UF system, with a smalleroverall holdup volume. The disadvantages to thisapproach are cost, time, product yield, and additionalrisk of contamination. The added capital costs for twosystems, and the cost and time of running essentiallytwo unit operations instead of one should be factoredinto the planning. The yield losses incurred duringproduct recovery now exist on two systems. Finally,given the challenges to ensure a sterile system, the riskof bioburden contamination during UF operations isnow doubled with the additional system. Combiningtwo upstream pools to process together on one systemhas several advantages. The capital costs and processtimes are minimized. Yield losses from productrecovery are reduced by half, as is the risk of bioburdencontamination. To use this approach, the pool from thepreceding step must be stable enough to be held until asubsequent run is processed. In addition, a strategy fortesting and combining pools must also be identified.

Other challenges also appear during UF systemdesign for high-concentration formulations, includingmixing bulk volumes effectively throughout a 10- to 50-fold concentration step. The final system designconsideration is product recovery, where most UFyield losses occur because the product pool can neverbe completely removed from the system. After the UFprocess is complete, the product is recovered from thetank, piping, and membrane using a combination ofpumping, gravity draining, and/or gas blow-down. Withthese methods, it is inevitable that some product poolwill remain in the system, clinging to surfaces, trappedin dead-end ports, or held up in local low points. High-viscosity protein solutions will add to the recovery

challenge. In addition, a high product concentrationpool will add to the incentive of recovering every lastdrop. Sloping all pipes toward a single recovery port,using zero-static valves, and/or eliminating dead-endports will greatly aid efficient product recovery. A post-recovery buffer flush, where buffer is recirculatedthrough the system and then is transferred to the pooltank, could be considered, but the minimum volume ofthis buffer flush would again be equal to the minimumsystem hold-up, resulting in a potentially unacceptabledilution of the final bulk.

High-concentration protein dosage forms(4100mg/mL) can be achieved through reconstitutionof lyophilized drug product at a reduced volume priorto administration. However, reducing overall cost ofgoods, eliminating reconstitution issues, and develop-ing opportunities for pre-filled cartridge or syringedevices for more convenient administration can beachieved by introducing a high concentration formula-tion step during bulk production instead of relying onlow-volume reconstitution.

ANALYTICAL ISSUES

IgG1-type antibodies have two heavy chains andtwo light chains that are linked by interchain disulfidebonds [Edelman et al., 1969]. Approximately 95% ofthe primary sequences are conserved in all humanIgG1-type immunoglobulins, with common sources ofheterogeneity due to glycosylation at heavy chainAsn297, partial carboxypeptidase processing of C-terminal Lys residues, or conversion of heavy chainN-terminal glutamine residues to pyroglutamate. Theremaining 5% of the primary sequences confer antigenspecificity, introducing molecular variations that re-quire reoptimization of available analytical methods.Additional molecular heterogeneity can be introducedby degradative modifications such as methionineoxidation, asparagine deamidation, aspartate isomer-ization, and/or by unexpected posttranslationalmodifications or unpaired cysteines. Successful devel-opment of a therapeutic antibody requires defining thedesired mixture of forms based on analytical informa-tion gained from clinical, non-clinical (animal), manu-facturing consistency, and stability samples. Thesponsor of a regulatory licensing application needs toprovide sufficient analytical details to demonstratethoroughness and competence to the regulatoryagencies, while identifying sources of heterogeneitythat could affect safety or efficacy.

CARBOHYDRATE HETEROGENEITY

The oligosaccharides found at the conservedAsn297 site in the Fc region of IgG1-type recombinantantibodies produced in Chinese hamster ovary or

Fig. 12. Maximum theoretical final bulk protein concentration as afunction of TFF system volume and total bulk size.


myeloma cell lines are typically fucosylated biantennarycomplex-type oligosaccharides, with measurable lowlevels of truncated or intermediate forms such as Man5[Lifely et al., 1995]. The Asn297 oligosaccharides arean integral part of the Fc structure, allowing glycosyla-tion variations to affect the overall Fc structure, whichin turn may influence complement C1q and Fcreceptor interactions [Wright and Morrison, 1997;Jefferis and Lund, 2002; Krapp et al., 2003]. Terminalgalactose levels correlate with complement-mediatedcell killing in vitro [Tsuchiya et al., 1989], while corefucosylation inversely correlates with antibody-depen-dent cellular cytoxicity (ADCC) [Shields et al., 2002;Shinkawa et al., 2003]. Recent studies have indicatedthat ADCC is a significant contributor to the in vivoefficacy of cancer therapeutic antibodies such astrastuzumab and rituximab [Clynes et al., 2000;Cartron et al., 2002].

For antibodies that derive their clinical benefitfrom simply blocking or binding to an antigen target(rather than by cell killing), a key efficacy considerationis total drug exposure, measured using pharmacoki-netic parameters. The neonatal receptor FcRn protectsantibodies from catabolism, thus higher affinity FcRnbinding correlates with slower clearance [Junghans andAnderson, 1996]. The Asn297 oligosaccharides do notappear to influence the IgG1-FcRn interaction; evenaglycosyl forms produced by site-directed mutagenesisat Asn297 do not differ from glycosylated antibodieswith respect to serum clearance [Tao and Morrison,1989]. Irrespective of potential biological conse-quences, regulatory agencies may require a demonstra-tion of oligosaccharide consistency acrossmanufacturing campaigns or following processchanges, in part because glycosylation may reflect cellculture conditions, thereby serving as a bellwether forchanges in other (undetected) sources of heterogeneity.

Detailed glycoanalytical methods often utilize acombination of enzymatic release using PNGaseF,which releases all typical IgG1 oligosaccharide typesfrom the denatured antibody, followed by HPAEC-PAD, capillary electrophoresis, or NMR [Townsendet al., 1988; Ma and Nashabeh, 1999; Yu Ip et al.,1994]. Mass spectrometry techniques can be used forconfirmation of Asn297 glycans if one assumes thatconventional structures are present, but linkages andanomeric configuration assignments require additionalanalyses. Mass spectrometric analyses of intact anti-bodies, glycopeptides, and released oligosaccharideshave also been described [Roberts et al., 1995; Bourellet al., 1994; Papac et al., 1998].

Carbohydrate compositional analysis is of limitedvalue, given the challenges of obtaining accuratevalues, particularly for the sugar residue at the

reducing terminus, which tends to resist acid hydrolysis[Hardy et al., 1988]. Mucin-type O-linked carbohy-drates are occasionally found in the hinge region[Smyth and Utsumi, 1967], but the absence of N-acetylgalactosamine in hydrolysates precludes thepresence of such oligosaccharides. Sialic acid is rarelyfound above trace levels on recombinant antibodies.Glycation, a modification of lysine residues by glucose[Furth, 1988], has been noted for serum-derivedantibodies with increased levels of glycation fordiabetic patients [Lapolla et al., 2000], and we haveobserved low levels of glycated recombinant antibodyforms by electrospray ionization mass spectrometry(ESI-MS) and by boronate chromatography [Shen etal., 1999]. Glycation is only a product quality issue if itaffects potency, such as if lysines essential for antigenbinding are modified.

SIZE DETERMINATIONS

Analysis of antibodies by SDS-PAGE or capillaryelectrophoresis sieving methods are complicated by thegeneration of artifacts during sample preparation, asthe interchain disulfide bonds are broken duringsample heating in buffers that contain SDS, resultingin the appearance of free light chain, free heavy chain,heavy chain dimer, and other light/heavy chaincombinations [Hunt and Nashabeh, 1999]. Analysis ofsamples after reduction eliminates the artifacts, andallows resolution of the glycosylated and non-glycosy-lated heavy chains by capillary electrophoresis.

Size-exclusion chromatography (SEC) is capableof resolving most aggregated forms, as well asfragments generated by hinge-region cleavage of oneheavy chain, producing Fab-like and Fab-Fc forms[Alexander and Hughes, 1995]. Non-covalent aggre-gates that may be challenging to detect by SEC can bedetected by analytical ultracentrifugation, and havebeen implicated in the relatively high rates ofimmunogenicity observed for early infliximab produc-tion lots [Siegel, 2003]. A remarkable aspect of IgG1-type antibody structure is the maintenance of theconventional MAb structure with two light chains plustwo heavy chains; even samples whose cysteineresidues have been reduced and alkylated will yield asingle peak by size-exclusion chromatography usingnon-denaturing conditions (unpublished observation).

Electrospray mass spectrometry of intact forms isuseful for confirming overall covalent structure, andmay also be used to confirm the major oligosaccharideforms [Roberts et al., 1995]. Analysis after reductionallows independent examination of the light and heavychains, and may allow discrimination of glutamine vs.pyroglutamate forms (�17Da mass difference), theloss of heavy chain C-terminal Lys residues (�128Da),

148 HARRIS ET AL.

or the presence of glycated forms (þ162Da). Similarly,digestion with papain followed by reduction andelectrospray mass spectrometry can yield detailedinformation about the heavy chain component of theFab fragment, without the heterogeneity introduced bythe Asn297 oligosaccharides. The electrospray processgenerates charged antibody forms with relatively highmass to charge values (m/z 2,000–3,000), requiring theuse of quadrupole detectors to obtain accurate values;ion-trap mass detectors are generally inadequate.MALDI-TOF mass spectrometry usually does notprovide enough mass accuracy to identify minor formsof molecules as large as antibodies.

CHARGE HETEROGENEITY

Charge heterogeneity can be introduced by avariety of modifications. Sites in the complementarity-determining regions that are altered tend to have thegreatest effect on cation exchange chromatographicproperties due to their enhanced solvent accessibility.Assignment of sources of charge heterogeneity ischallenging for antibodies because they are comprisedof four polypeptide chains; the underlying structuraldifference might be present on only one light or heavychain. Cation exchange chromatography after papaindigestion can help with such assignments, as the Faband Fc are usually well resolved from each other, oftenallowing minor Fab or Fc forms containing thevariation to be isolated [Moorhouse et al., 1997].

The expected heavy chain IgG1 C-terminalsequence is -Pro-Gly-Lys, but the Lys residues arepartially removed by the action of basic carboxypepti-dases [Harris RJ, 1995], resulting in a mixture ofantibody forms bearing zero, one, or two C-terminalLys residues that can easily be resolved by cationexchange chromatography [Weitzhandler et al., 2001].This C-terminal processing does not affect antigenbinding, and these Lys residues are likely to be removedpromptly by plasma or extracellular carboxypeptidases,limiting any in vivo effect such as distribution orclearance. Plasma-derived antibodies lack the C-term-inal Lys encoded by the IgG1 heavy chain gene [Ellisonet al., 1982]. For many charge-based analyses, it may beprudent to pre-treat samples with CpB to remove theLys residues entirely, which can also allow for otherbasic forms to be revealed and/or monitored.

Additional basic forms may be present due toconversion of N-terminal glutamate residues to pyr-oglutamate, dehydration of aspartate residues to thesuccinimide intermediate [Geiger and Clarke, 1987], oralternate cleavage of a signal peptide that results in thepresence of additional basic residues. In trastuzumab,conversion of aspartate to isoaspartate (isoAsp) shiftedelution to an apparently more basic position; such a

change does not actually modify the total charge, butaspartate isomerization moves the side-chain’s chargeorientation and introduces a methylene group into thepolypeptide backbone, so in this example it appearsthat these secondary structural effects are responsiblefor the resolution of the isoAsp-bearing form [Harriset al., 2001].

Acidic forms have been assigned for severalantibodies due to asparagine deamidation, conversionof the heavy chain N-terminal glutamine to pyrogluta-mate, or sialylation of the Asn297 oligosaccharides.Incubation of samples with sialidase can remove thesialyl forms, reducing the complexity of the remainingacidic forms. Deamidation of an asparagine to aspartatein a light chain complementarity-determining region 1(CDR-L1) had only a minor potency effect fortrastuzumab [Harris et al., 2001]; in general, theconversion to isoaspartate is more deleterious becauseof the charge orientation shift.

Deamidation in the Fc region is commonlyobserved, typically at two underlined sites in the –SNGQPENNYK– region; the first Asn is deamidated toisoaspartate, and the second Asn is deamidated toaspartate. This Fc deamidation is also observed withplasma-derived antibodies (V. Katta, personal commu-nication). Glycation could affect charge distribution,with the glucose modification neutralizing an aminogroup on the N-terminus or Lys side chain, butmaterial we prepared that was glycated with an averageof one glucose moiety per mol did not resolve fromnon-glycated material by cation exchange or isoelectricfocusing gel electrophoresis [Shen et al., 1999].Modification by lactose was revealed by chargedifferences only when a large number of sites werelactosylated [Andya et al., 1999].

Cation exchange chromatography is a preferredcharge-based analytical method because it is alsopreparative, allowing minor forms to be collected forpotency assessments and structural assignments. Ca-pillary- and gel-based isoelectrophoretic focusing (IEF)techniques are available [Hunt et al., 1996], and can beused without the lengthy optimization often requiredfor the chromatographic methods [Bai et al., 2000], andthus are advantageous when screening new molecules.The imaged capillary IEF method is particularly facile[Fang et al., 1998]. However, IEF-based techniques donot resolve the Asp/isoAsp isomers.

COVALENT MODIFICATIONS

Several molecular variations that introducecharge heterogeneity have been described above. Inaddition, antibodies are subject to incomplete disulfideformation [Zhang and Czupryn, 2002]. Unpairedcysteines are detected using Ellman’s reagent [Riddles


et al., 1979] or other thiol-reactive compounds. Assign-ment of unpaired cysteine sites may be possible usingthiol-specific reagents. Oxidation of methionine resi-dues to the sulfoxide can occur in solution, particularlyif peroxide-contaminated polysorbates are used asexcipients [Herman et al., 1996]. OKT3 ampoules areformulated with inert gas in the headspace to stabilize amethionine in CDR-H1 whose oxidation led to adramatic drop in potency [Rao and Kroon, 1993]. InIgG1 constant regions, the methionines that are themost susceptible to oxidation are found in the Fcregion at Met252 and Met428; oxidation of theseresidues can be monitored by hydrophobic interactionchromatography after papain digestion [Shen et al.,1996].

Reversed phase HPLC is seldom useful for intactantibodies; the light chain can produce a single sharppeak, but the heavy chain is often a broad peak withoutresolved forms. Increasing the temperature improvesRP-HPLC resolution [Battersby et al., 2001], but maylead to cleavage of an acid-labile Asp/Pro bond in theheavy chain. RP-HPLC of peptide digests (peptidemapping) is useful for obtaining peptide fractions toconfirm amino acid sequences and modification sites.Two different types of genetic heterogeneity (mutationand recombination) have been identified by peptidemapping [Harris et al., 1993; Wan et al., 1999],justifying the use of this technique for demonstratingcell line stability. Hydrophobic interaction chromato-graphy of intact, pepsin-digested, or papain-digestedantibodies can be a useful alternative to RP-HPLC. Forexample, Fab forms of a recombinant IgG1 antibodywith Asp, isoAsp, or the succinimide at a site in lightchain CDR1 were resolved by HIC after papaincleavage [Cacia et al., 1996].

Classical protein chemistry techniques havelimited applications for recombinant antibodies. Aminoacid analysis lacks sufficient resolution to provide

useful information, except perhaps for determinationsof absorptivity coefficients [Anders et al., 2003; Macchiet al., 2001]. Edman degradation is hampered by animmunoglobulin’s large molecular size and multimericnature, which combine to generate ‘‘background’’ thatobscures the yield of PTH-amino acids from minorsequences. Circular dichroism generates an averagevalue for the pool of material, and cannot identifysamples that contain minor, closely-related forms.

REGULATORY ASPECTS

Key considerations for the development oftherapeutic antibodies include clinical safety, efficacy,and manufacturing consistency. A minimal immuneresponse against the therapeutic protein is desirable toavoid adverse efficacy and/or pharmacokinetic effects.OKT3, a murine monoclonal antibody, leads to rapidanti-product response that limits its utility [Chatenoud,1993]; much of this response is directed against themurine Fc region [Stein, 2002]. Chimeric, humanized,and ‘‘human’’ antibodies elicit anti-product antibodiesin only a subset of patients (o1–10%) [Stein, 2002].Lyophilization conditions appear to have enhanced theimmunogenicity of one recombinant antibody [Tascheret al., 2001]. Reduced efficacy due to inhibition by theanti-product response was noted for infliximab [Baertet al., 2003], but such adverse effects are generally rareand end upon therapy discontinuation.

The US FDA, the European Medicines Evalua-tion Agency (EMEA), and the combined regulatoryauthority/biopharmaceutical industry group referred toas the International Conference on Harmonization(ICH) have published license application guidancedocuments that describe current expectations andrequirements for stability programs, therapeutic pro-teins in general, and monoclonal antibodies specifically(Table 3). Licensing applications are reviewed on anindividual basis, balancing the perceived therapeutic

TABLE 3. Therapeutic Antibody Regulatory Guidance Documents

Agency Enacted Website Title

FDA/CBER 8/1996 www.fda.gov/cber/guidelines.htm For the submission of chemistry, manufacturing, and controls informationfor a therapeutic recombinant DNA-derived product or a monoclonalantibody for in vivo use.

FDA/CBER 2/28/1997 www.fda.gov/cber/guidelines.htm Points to consider in the manufacture and testing of monoclonal antibodyproducts for human use.

ICH 1999 www.ich.org/ich5q.html Topic Q6B. Specifications: test procedures and acceptance criteria forbiotechnological/biological products.

ICH 1996 www.ich.org/ich5q.html Topic Q5B. Quality of bilotechnological products: analysis of the expressconstruct in cell lines used for production of r-DNA-derived proteinproducts.

ICH 1995 www.ich.org/ich5q.html Topic Q5C. Quality of biotechnological products/biological products.EMEA 1995 pharmacos.eudra.org/F2/eudralex/

vol-3/home.htm3AB4A. Production and quality control of monoclonal antibodies.

150 HARRIS ET AL.

benefit against known or unknown safety risks, andinclude an assessment of the sponsor’s ability toproduce commercial lots representing the range ofanalytical characteristics observed in the clinical lotsused to establish safety and efficacy.

FUTURE DIRECTIONS

The high doses generally used for monoclonalantibody therapeutics will continue to present manu-facturing, formulation, and drug delivery challenges.Although UF/DF technology is the primary methodused to manufacture high-concentration protein for-mulations, alternate technology such as spray drying orbulk lyophilization may be useful to produce formula-tions for s.c. delivery. The high-concentration issue mayalso be successfully handled by developing suspensionsof crystallized monoclonal antibody for delivery. Suchan approach has been successfully developed for theadministration of insulin.

The high concentrations of antibody required forlow volume s.c. delivery will impact bulk storage of theantibody, especially during the freeze-thaw operationsoften used for storage and preparation of bulkintermediates. During such operations, the antibodymay be concentrated severalfold due to ice formation,which may result in stability problems, especially whenthe MAb is in contact with stainless steel surfaces.These stability problems may indicate a need foradditional technology such as field flow fractionation tocharacterize the production of insoluble as well assoluble aggregates.

The challenge of identifying single-site modifica-tions in monoclonal antibodies may be aided byapplying proteomic analysis concepts, such as compar-ing the observed mass data set from a peptide digestwith the theoretical data set. However, assignment oftrue molecular variability in such analyses will requireheightened rigor to avoid the introduction duringsample preparation of artifacts such as oxidizedmethionine residues or deamidated asparagine resi-dues. Structural features that elicit immunogenic (anti-product) responses for recombinant proteins maycontinue to be elucidated, which may allow these tobe controlled and/or eliminated.

The ultimate goal will be to produce stablemonoclonal, antibody formulations that are easilyadministered as outpatient therapeutics. This willinvolve developing formulations that are compatiblewith prefilled syringes or cartridges that can be usedwith injection devices such as autoinjectors or needle-less injectors. This direction will open up manyindications that can be treated by monoclonal antibodytherapy without necessarily increasing the number ofvisits to the hospital or clinic.

ACKNOWLEDGMENTS

The authors thank Dr. Robert van Reis in theDepartment of Recovery Sciences for input on UF/DFtechnologies, and Dr. Chung Hsu in the Department ofPharmaceutical R&D for discussions on bulk dryingprocesses. Analytical ultracentrifugation and size ex-clusion chromatography data on monoclonal antibodieswas kindly provided by Dr. Jun Liu and James Andya,respectively. Insights regarding mass spectrometricanalyses were contributed by Victor Ling andDr. Viswanatham Katta.

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