REVIEW. Aggregates in Monoclonal Antibody Manufacturing Processes. Introduction

Transcription

1 REVIEW Aggregates in Monoclonal Antibody Manufacturing Processes María Vázquez-Rey, 1 Dietmar A. Lang 2 1 Manufacturing Science and Technology, Lonza Biologics Porriňo SL, A Relva s/n, 36410, Porriño, Pontevedra, Spain 2 Technology Development, Lonza Biologics plc., 228 Bath Road, Slough, SL1 4DX, UK Qiagen Manchester Ltd., Skelton House, Lloyd Street North, Manchester, M15 6SH, UK; telephone: ; fax: ; dietmar.lang@qiagen.com. Received 5 December 2010; revision received 6 March 2011; accepted 30 March 2011 Published online 7 April 2011 in Wiley Online Library (wileyonlinelibrary.com). DOI /bit ABSTRACT: Monoclonal antibodies have proved to be a highly successful class of therapeutic products. Large-scale manufacturing of pharmaceutical antibodies is a complex activity that requires considerable effort in both process and analytical development. If a therapeutic protein cannot be stabilized adequately, it will lose partially or totally its therapeutic properties or even cause immunogenic reactions thus potentially further endangering the patients health. The phenomenon of protein aggregation is a common issue that compromises the quality, safety, and efficacy of antibodies and can happen at different steps of the manufacturing process, including fermentation, purification, final formulation, and storage. Aggregate levels in drug substance and final drug product are a key factor when assessing quality attributes of the molecule, since aggregation might impact biological activity of the biopharmaceutical. In this review it is analyzed how aggregates are formed during monoclonal antibody industrial production, why they have to be removed and the manufacturing process steps that are designed to either minimize or remove aggregates in the final product. Biotechnol. Bioeng. 2011;108: ß 2011 Wiley Periodicals, Inc. KEYWORDS: aggregates; monoclonal antibody; manufacturing processing Introduction Currently, therapy with monoclonal antibodies (mab) is the largest growth area in the pharmaceutical industry. The FDA has approved 26 mab for clinical use against cancer, Crohn s disease, rheumatoid arthritis, and antiviral prophylaxes among other diseases (Birch and Racher, 2006; Scolnik, Correspondence to: Dietmar A. Lang Qiagen Manchester Ltd., Skelton House, Lloyd Street North, Manchester, M15 6SH, UK; telephone: ; fax: ; Wang et al., 2009) widely used in antitumor therapy because of their specificity. mabs are directed to their tumoral targets (antigen) without affecting healthy tissues or with minimal effects on them. A standard mab manufacturing process using mammalian cell culture is depicted in Figure 1. Cells from a cell bank are thawed and cultured in small flasks. Over a period of approximately 2 weeks, cells are grown in increasingly larger volumes to provide a seed culture (inoculum) for the fermentation tanks. Once cells are inoculated into the production bioreactor (up to 20,000 L scale), they are grown under controlled conditions to an optimal density for maximum productivity. At the end of the production phase, the harvest step separates the cells from the cell culture supernatant to recover the product in the liquid phase. This is typically done by either centrifugation and/or microfiltration (Sommerfeld and Strube, 2005). The next step is in most cases a capture step using Protein A chromatography and, since mab elutes at low ph values, a virus inactivation step follows at low ph. Additional process steps include typically ion exchange chromatography operations for polishing and/or impurities removal (Faherner et al., 2001), in particular host cell proteins (HCP), DNA, endotoxin, leached protein A, and aggregates. In addition, membrane filtration plays an important role for product concentration and buffer exchange. To guarantee product safety, potential viral contaminants are removed by filtration using nanofilters that has traditionally been accepted as a robust method for virus clearance (Ray and Tarrach, 2009). Once product impurities have been removed and conditioning buffer has been added, product is filled into bags, bottles, or stainless steel tanks for subsequent storage until filled into vials. Protein aggregation (oligomerization) behavior was first studied in the 1960 s and was described by the simple Lumry Eyring model (Lumry and Eyring, 1954). Protein aggregation is a common phenomenon during protein drug 1494 Biotechnology and Bioengineering, Vol. 108, No. 7, July, 2011 ß 2011 Wiley Periodicals, Inc.

2 Figure 1. Outline of steps involved in a general mab manufacturing process comprising (1) inoculum expansion, (2) product fermentation, (3) product primary recovery, (4) product purification, (5) product formulation. development but the mechanisms of aggregation are poorly understood (Wang, 2005). Aggregates can initially exist as small dimers or fragments and progress toward larger structures, such as sub-visible or visible particles, if such a transition becomes thermodynamically favorable (Córdoba- Rodríguez, 2008). Also, protein molecules may be unfolded or partially unfolded, that is, the higher order structure of the protein may be disrupted and, thereby, the protein s hydrophobic regions are exposed, promoting intermolecular interactions leading to aggregation or subsequently to precipitation phenomena (Kiese et al., 2008). Protein aggregates can be classified in several ways, including soluble/insoluble, covalent/non-covalent, reversible/non-reversible, and native/denatured according to Cromwell et al. (2006). There is, however, no uniform terminology for aggregate sizes and types so that Philo (2006) introduced the classification: (1) rapidly reversible non-covalent small oligomers (dimer, trimer, tetramer, etc.); (2) irreversible non-covalent oligomers; (3) covalent oligomers (e.g., disulfide-linked); (4) large aggregates (>10 mer), which could be reversible if non-covalent; (5) very large aggregates (diameter 50 nm to 3000 nm), which could be reversible if non-covalent; and (6) visible particulates ( snow ), which are probably irreversible. Aggregation can arise from non-covalent interactions or from covalently linked species (Manning et al., 2010). Vázquez-Rey and Lang: Aggregates in mab Manufacturing Processes 1495 Biotechnology and Bioengineering

3 Soluble aggregates have been defined as those that are not visible as discrete particles and that may not be removed by a 0.22 mm filter (Cromwell et al., 2006). On the other hand, insoluble aggregates are those that are often visible, and can be removed either by 0.22 mm filtration or by centrifugation at mild conditions (Knutson et al., 1979). Generally, they can often be related to the precipitation of HCP impurities rather than the product itself (Yigzaw et al., 2006). Covalent aggregation is associated to chemical binding between two or more monomers or with the chemical linking of partially unfolded molecules with each other. Disulfide bonds between previously unpaired free thiols are a common mechanism for covalent aggregation (Andya et al., 2003). Oxidation of tyrosine residues may also result in covalent aggregation through the formation of bi-tyrosine (Creed, 1984). For some proteins, a covalent interaction between monomers is required to form a stable protein structure. A simplified model of non-covalent aggregation is given in Mahler et al. (2005). Non-covalent aggregates are formed when proteins associate and bind based on structural regions of charge or polarity (Patel et al., 2011). Reversible protein aggregation typically results from relatively weak non-covalent protein interactions such as hydrophobic/ hydrophilic interactions with short distances between them. The reversibility is sometimes indicative of the presence of equilibrium between the monomer and higher molecular weight protein variants. This equilibrium may shift as a result of a change in solution conditions such as a decrease in protein concentration or a change in ph. A weak, reversible self-association of this type has been observed in a monoclonal antibody to vascular endothelial growth factor (VEGF; Moore et al., 1999). On some occasions, reversible protein self-association produces an increase in the viscosity of the protein solution (Liu et al., 2005). The kinetics of protein aggregation has been extensively investigated. Lumry and Eyring (1954) polymerization model has been used as a starting point to study protein aggregation. In the type of situation described by a Lumry Eyring model (Sánchez-Ruiz, 2010), the thermodynamically stable protein with respect to unfolded and partially unfolded states may undergo irreversible alteration processes that lead to some kind of a final state not being able to fold back to the native one. One of the first models to describe protein aggregation was developed based on the understanding that changes in protein conformation can be responsible for creating an altered state of the protein that is then susceptible to aggregation (Lumry and Eyring, 1954). Non-native aggregation can generally be considered as any process which creates protein aggregates with secondary structures at the monomer scale that are significantly different from the dominant structures in the native state (Roberts, 2007). The general model that explain the kinetics of non-native aggregation considers that the protein (a monomer fully folded) unfolds to form an unstable, intermediate state that is in equilibrium with the native protein structure. The unstable state serves as an intermediate for leading irreversibly to the aggregated state (Andrews and Roberts, 2007; Powers and Powers, 2006). This model has formed the basis of most subsequent advances in understanding protein aggregation. Recently Roberts (2007) has shown in more detail the general understanding of aggregation as a multi-stage process and how most available kinetic models of aggregation can be grouped hierarchically in terms of which stage(s) they include. Recent reviews about aggregation kinetics (Morris et al., 2009; Roberts, 2007; Sánchez-Ruiz, 2010) revealed that protein aggregation is usually a higher order reaction due to the fact that multiple conformational altered protein molecules interact to create aggregated species. Despite this general fact, situations in which the aggregation kinetics can be of first order were also described (Kendrick et al., 1998a,b) representing the case if the initial formation of unfolding intermediates is rate limiting. Protein heterogeneity also can be a contributing factor for protein aggregation, as the probability of multiple protein forms interacting with their environment is increased. In the case of Epratuzumab, disulfide bond scrambling favored covalent aggregate formation (Remmele et al., 2006). Importance of Aggregate Removal Differences in biological activity of the aggregates compared to the activity of the monomeric protein can significantly impair the potency of a protein-based drug. In such cases, product efficacy may be compromised (Cromwell et al., 2006). In general, a risk-based assessment of aggregates may warrant specific studies which are helping to elucidate which types of aggregates have a higher biological relevance. A thorough analysis of the different conditions including manufacture, storage, shipping, freeze and thaw cycles, oxygen exposure, light, and physical stress to which the drug is exposed from manufacturing until being administered, can provide a rationale for special degradation studies such as seeding or spiking of a specific aggregate or impurity into a protein solution to observe the potential for further aggregation (Córdoba-Rodríguez, 2008). The extent to which aggregate levels impact the biological activity of the protein is determined by multiple factors such as molecular weight, structure, and solubility of aggregates (Rosenberg, 2006). Individual susceptibility plays a key role, whether determined by genetics or disease or a combination of both (Singh, 2010). Protein aggregates pose risk in terms of generation of immune responses to the therapeutic protein product (Sharma, 2007). Of principal concern are those immune responses associated with adverse clinical effects such as neutralizing antibody that inhibits the efficacy of the product (Franco et al., 1999) or, worse, crossreactivity neutralizes an endogenous protein counterpart, causing severe hypersensitivity responses such as anaphylaxis (Rosenberg, 2006). Protein aggregates come in a variety of forms such as fibrils (ordered aggregates), particulates 1496 Biotechnology and Bioengineering, Vol. 108, No. 7, July, 2011

4 (irregular or spherical), skin, gels, or a combination of these. The final form of aggregates seems to depend on the aggregation pathway (Wang et al., 2010). The presence of particulates in drug substances that are administered intravenously has been shown to decrease microcirculation as a consequence of the mechanical blockage of capillaries (Lehr et al., 2002). Preclinical studies with properly designed models may be useful to rank the factors as to their potential for impacting immunogenicity (Singh, 2010). A first animal model transgenic mice already showed that protein aggregates are able to break the immune tolerance for a protein interferon beta. The potency of the aggregates to break tolerance not only depends on aggregate percentage but also largely on their physical properties such as degree of denaturation, molecular orientation, and size (Van Beers et al., 2010). Further investigations into what types of aggregates are immunogenic, which protein types are most prone to immune problems, what dosing is required and how well do results from animal models predict effects in humans are subjects for future research. There is no consensus on the maximum allowable aggregate levels in protein-based pharmaceutical products because some proteins may be largely stable and safe despite certain levels of aggregates, while for other proteins very small changes in aggregate levels may significantly affect protein stability, and even safety (Córdoba-Rodríguez, 2008; Cromwell et al., 2006). The only group of aggregates that has maximum allowable limits based on United States Pharmacopeia, USP <788> (also European Pharmacopoeia [Ph. Eur.] and Ph. Eur ), is the group of sub-visible particles. Solutions for injection must be clear and practically free from particles. Limits for soluble protein aggregates have to be set on a case-by-case basis as there are no regulatory limits for aggregates in biotherapeutic preparations (Mahler et al., 2009). The acceptance criteria for aggregates levels should reflect values that maintain the safety and efficacy of the product (ICH. Q6B Specifications: Test Procedures and Acceptance Criteria for Biotechnological/Biological Products, 1999). In the absence of information on clinical relevance and process control, many specifications are instituted with narrower than necessary acceptance ranges based on manufacturing experience (Kozlowski and Swann, 2006). The European Pharmacopoeia (2006a) specifies in the monograph parenteral preparations, the requirements for sterile solutions administered by injection like protein solutions. Solutions for injection must be clear and practically free from particles. They have to comply with the test for sterility, the test for particulate contamination: sub-visible particles, test for uniformity of content, and the test for bacterial endotoxins or pyrogens. As protein degradation often results in aggregation and precipitation, particles in the solution present besides other points a critical aspect. The test for visible particles according to the European Pharmacopoeia (2006b) describes a simple procedure for a visual inspection of parenteral solutions. The aim is to assess the quality of the solution in respect to particulate contamination consisting of mobile undissolved particles other than gas bubbles. The visual inspection is performed with the help of a viewing station consisting of a matt black and a non-glare white panel in vertical position next to each other and a suitable white-light source. Non-labeled containers that are clean and dry on the outside are inspected for particles by gently swirling and observing 5 s in front of the white panel and in front of the black panel. As it is a nondestructive method a 100% control can be performed. Thetestforsub-visibleparticlesaccordingtotheEuropean Pharmacopoeia (2006c), which has to be performed for solutions for injection, is conducted using the light obscuration particle count test. The method allows a determination of a size distribution using the principle of light blockage. A suitable instrument calibrated with spherical particles of known size between 10 and 25 mm is used to examine a statistically relevant number of test specimens after sample preparation. Solutions for injection with a nominal volume of equal or less than 100 ml comply with the test if the average number of particles in the tested units does not exceed 6,000 per container equal to or grater than 10 mm and 600 per container equal to or greater than 25 mm. Solutions with a nominal volume of more than 100 ml comply with the test if the average number of particles in the tested samples does not exceed 25/mL equal to or greater than 10 mm and 3/mL equal to or greater than 25 mm. The microscopic particle count test is available as a second backup method in the European Pharmacopoeia (2006c). Aggregates Formation During Monoclonal Antibody Manufacturing Processes There are many environmental factors that can lead to aggregation (Remmele et al., 2006). Environmental conditions of production process such as temperature, protein concentration, ph, oxygen, shear forces, and the ionic strength may affect the amount of aggregate observed. The presence of certain ligands, including specific ions, may enhance aggregation. Stresses to the protein such as freezing, exposure to air, or interactions with metal surfaces may result in undesired post-translational molecule modification even molecule unfolding, which then leads to the formation of aggregates. Finally, mechanical stresses may cause protein aggregation. Each of these environmental factors is typically encountered during bioprocessing (Patel et al., 2011; Wang, 2005; Wang et al., 2010). Aggregation During Cell Culture Protein aggregation during cell culture is a widely reported phenomenon and, in fact, aggregate levels up to 30% have been reported for some mabs in mammalian cell culture (Kramarczyk et al., 2008). During cell culture, protein aggregates might be formed (i) within the cell following protein expression and (ii) once the protein is secreted into Vázquez-Rey and Lang: Aggregates in mab Manufacturing Processes 1497 Biotechnology and Bioengineering

5 the cell culture medium (Cromwell et al., 2006). During expression, accumulation of high amounts of protein may lead to intracellular aggregation owing to either the interactions of unfolded protein molecules or to inefficient recognition of the nascent peptide chain by molecular chaperones responsible for proper folding (Zhang et al., 2004). It is possible to influence the amount of aggregates produced during the cell culture process by carefully selecting the optimal cell line and optimizing cell culture conditions such as media components that will impact media osmolality and conductivity, feed strategy, temperature, and ph (Gabrielson et al., 2007). Mice cells cultured in a hollow fiber bioreactor, exhibited a lower amount of aggregates in the protein secreted when media ph and osmolarity were increased (Franco et al., 1999). Cell culture temperature influence in aggregate formation during cell culture was investigated by Cromwell et al. (2006). They have reported that the greater the length of time the protein was held in the cell culture medium at elevated temperature, the greater the amount of aggregates observed (Cromwell et al., 2006). Secretion of the protein into the cell culture medium exposes the protein to environmental stress such as unfavorable ph conditions that may destabilize the protein. Franco et al. (1999) studied a mab with an isoelectric point (pi) of ph that was secreted into a cell culture medium at ph At the medium ph, the mab theoretically carried an overall negative charge, but there are still some positive charges on the molecule. The electrostatic forces between positive and negative charges on other mab molecules favor the formation of aggregates in solution. To limit this phenomenon Franco et al. (1999) increased the ph to such a degree that the net charge of the antibody should be sufficient to effect repulsion between the molecules in solution. Furthermore, to enhance the solubility, they increased the ionic strength of the medium by adding a solution of sodium chloride (98 g/l) to achieve an osmolarity value of 350 mosm/kg H2O (Franco et al., 1999). Aggregation During Purification When a therapeutic protein has been produced, it has to be purified in order to reduce or eliminate any viruses, aggregates, DNA (nucleotides in general), HCP, and other process-related impurities. Protein purification processes comprise multiple steps where different techniques are employed, based on different separation principles such as affinity, charge, size, hydrophobicity, or other properties of the target protein compared to impurities (Sommerfeld and Strube, 2005). As mentioned above, the result may be that the protein experiences a wide range of ph, ionic strength, protein concentrations, and contact materials during the process. In addition to that, it has to be considered that at large scale the protein is exposed to mechanical stresses such as agitation in tanks and pumping (Thomas and Geer, 2011). Once the protein has been purified, it is typically filled into bottles, bags, or cryo-vessels for further freezing and storage. Each condition experienced by the protein may affect the degree of aggregation observed. In this section, the effect of the above mentioned conditions on protein aggregation will be discussed. Exposure to Low ph Condition Protein A chromatography is the capture step of choice for purifying mab after harvest (Faherner et al., 2001). It was shown that a highly conserved histidyl residue in the center of the Protein A binding site of IgGs faces a complementary histidyl residue on Protein A (Chen, 1992). These residues have a positive charge at low ph, thus repelling each other and dissociating the Protein A IgG hydrophilic interaction. As a result, operating conditions for Protein A columns typically require the use of low ph conditions (between ph 3 and 4). At this low ph proteins might undergo structural changes that could contribute to product aggregation (Chen, 1992; Krishnamurthy and Manning, 2002). In the preparation of CamPath-1H 1 (Alemtuzumab), which elutes from the Protein A column with 0.1 M Sodium Citrate, ph 3.2, the eluate contained approximately 25% aggregated mab (Phillips et al., 2001). Strategies to minimize aggregate formation during Protein A chromatography have been considered and include the use of urea a chaotropic agent at moderate concentrations (<2M) as an effective stabilizer or to carry out the separation under low temperature conditions (Chen, 1992). Low ph conditions are favorable purification conditions for Protein A chromatography as well as for the viral inactivation step. The viral inactivation step is intended to modify the surface chemistry of viruses to render them inactive or denatured. Low ph treatment has been shown to successfully inactivate retroviruses for a variety of biotechnology products but also originates aggregates in the protein pool due to the low ph exposure (Doms et al., 1985; Sofer, 2002). The following manufacturing step requires the neutralization of the solution which supports stabilizing the product. The use of strong bases (such as sodium hydroxide) is avoided despite the advantage of low volume addition due to the risk of product denaturation in the localized region where the solution is added. Due to that the use of higher concentrations of weaker bases (e.g., Tris base solution) is preferred (Shukla et al., 2007). Some mab solutions might exhibit a turbid appearance following neutralization. (Shukla et al., 2007). Exposure to ph Conditions Near Protein Isoelectric Point Proteins have ionizable groups such as carboxyl groups and amino groups. Since the charge of these groups depends on ph, a protein molecule depending on its pi may have different charges at different ph values during manufacturing (Xia, 2007). When a protein structure equally includes both positively and negatively charged groups (i.e., at ph values close to the pi), anisotropic charge distribution on the protein surface 1498 Biotechnology and Bioengineering, Vol. 108, No. 7, July, 2011

6 can create dipoles. In such cases, protein protein interactions are frequent, making assembly processes such as aggregation energetically favorable (Striolo et al., 2002). Agitation Agitation is the mechanism used to achieve homogeneity of protein pools during mab purification manufacturing processes. Mixing is especially critical in those purification stages where buffers or solutions are added to product solutions to adjust ph and/or conductivity for the next separation steps. During agitation of protein solutions a gas/ liquid interface is created at which aggregation predominantly occurs. There have now been many studies specifically investigating how proteins behave at gas liquid interfaces (Thomas and Geer, 2011). Mahler et al. (2005) claimed that agitation in the presence of a (hydrophobic) gas liquid interface caused aggregation of immunoglobulin-g1 (IgG1). Antibodies such as IgG1, might also be susceptible to damage at air liquid interfaces. Harrison et al. (1998) showed that a recombinant scfv antibody fragment suffered a first order loss of activity in a partially filled, agitated vessel. During agitation insoluble aggregates are released into bulk solution (Carpenter et al., 1999; Fesinmeyer et al., 2009). Fesinmeyer et al. (2009) reported that agitation of 70 mg/ml mab solutions for a period of 65 h resulted in increased solution turbidity. If stored statically, the solutions became clear, with a layer of protein aggregate at the bottom, consistent with the formation of insoluble aggregate particles (Fesinmeyer et al., 2009). Protein aggregation is commonly a second- or higherorder process. It is expected to increase with higher protein concentration, however, Treuheit et al. (2002) has shown that aggregation decreased with higher protein concentration if induced as a result of agitation (Treuheit et al., 2002). This unexpected result may be explained by the rate-limiting effect on aggregation of the air/water interface and the critical nature of the air/water interface to protein ratio that is greatest with decreased protein concentration (Treuheit et al., 2002). Apart from agitation, stirring and shaking are other methods used to achieve homogeneity during mab production. These methods are used at large scale to homogenize bottle contents, especially during final filling operations. Mechanical stress associated to stirring and shaking results in the formation of different species, sizes, and amounts of non-covalent aggregates (Kiese et al., 2008). Stirring has been reported to yield many insoluble, visible, and sub-visible particles and high turbidity, whilst shaking is reported to induce higher amounts of soluble aggregates (Kiese et al., 2008). Buffer Characteristics During manufacturing, mabs are suspended in various buffer solutions. Salts and buffers have complex effects on protein stability. Depending on the type and concentration of the salt, the charged groups of the protein and the type of ionic interactions between them, salts may have a stabilizing, a destabilizing, or no effect (Kendrich et al., 2002). In this section, the influence of buffers composition on aggregates formation is explained. Kameoka et al. (2007) have reported that the aggregation effect of buffer species is associated to the specific molecular interaction between buffer and IgG. It was indicated that the specific interaction between buffer molecules (like citrate or phosphate ions) and the Fc domain of IgG is the leading mechanism in the aggregation of IgG (Kameoka et al., 2007). Surfactants, such as polysorbates, are frequently added to pharmaceutical protein solutions to mitigate the risk of agitation-induced aggregate formation as they interfere with hydrophobic interactions at the gas/liquid interface (Fesinmeyer et al., 2009). Although surfactants are typically used to minimize aggregate formation, for some proteins like recombinant human growth hormone (rhgh), surfactants bind more strongly to the native state and they decrease the free energy of denaturation which could contribute to product degradation (Bam et al., 1998). Antimicrobial preservatives, such as benzyl alcohol and phenol, are often used in protein liquid formulations to ensure sterility in multi-use products where by necessity the container-closure must be breached multiple times in the process of taking doses from the vial. Although the use of this preservatives is not very extended because most of the mab are not multi-dose product, the preservatives (if presents) often induce aggregation of proteins in aqueous solution. For example, preservatives (e.g., phenol, m-cresol, and benzyl alcohol) have been shown to induce aggregation of granulocyte colony stimulating factor (Chi et al., 2003) or recombinant interleukin-1 receptor (Remmele et al., 1998). Since the contribution to solution ionic strength increases with the valence of an ion, the use of polyvalent salts could present significant challenges for the development of aggregate-free protein therapeutic products (Fesinmeyer et al., 2009). When the electrostatic coupling strength is increased by increasing valence or lowering the dielectric permits, this is accompanied by a strong accumulation of counter ions close to the protein. Increasing salt concentration will induce protein aggregation and depending on the ion valence less trivalent or far more monovalent salt is required to suppress repulsion (Lund and Jonsson, 2003). Ions resulting from salt dissolution can have a deleterious effect on protein stability and promote aggregate formation. A clear correlation between increased ionic strength and increased aggregation was observed by Fesinmeyer et al. (2009) and Gokarn et al. (2008), however, and as mentioned at the beginning of this section, high concentrations of salt as observed in other proteins can stabilize proteins through the preferential exclusion mechanism. The effect correlates with the Hofmeister series for anions: citrate 3 /citrate 2 > PO 3 4 HPO 2 4 SO 2 4 > OAc, F > Cl > Br > I > ClO 4 (Kendrick et al., 2002). Vázquez-Rey and Lang: Aggregates in mab Manufacturing Processes 1499 Biotechnology and Bioengineering

7 Equipment Contact Materials A general review of the mechanisms of protein adsorption on a solid surface is to be found in Nakanishi et al. (2001). Stainless steel, a ubiquitous surface in bioprocessing, has been shown to cause aggregation of mabs. Two different IgG4 mabs were found to aggregate according to first-order kinetics when exposed to stainless steel under high shear conditions (Biddlecombe et al., 2007). In a different study, exposure of an IgG to stainless steel particulates caused the generation of much larger particles even when formulated with a non-ionic surfactant (Tyagi et al., 2009). Bee et al. (2009a) found that stainless steel induce aggregation of a mab with a second-order dependence on the steel surface area and a zero-order dependence on the mab concentration (Bee et al., 2009a). These studies demonstrate that stainless steel microparticles can cause the aggregation of a mab. Also, the solution conditions are a critical parameter for aggregates formation when mabs are in contact with stainless steel. Fe ions leached from steel have been reported to cause oxidation of proteins resulting in aggregation (Lam et al., 1997; Wang et al., 2007). Stainless steel is a much extended construction material in large-scale equipment used to manufacture mabs, however, along the processes other materials such as Teflon, glass, or titanium can be found. These construction materials also can lead to aggregates formation. A broad study of the adsorption of 18 proteins on a titanium oxide can be found in Imamura et al. (2008). Sluzky et al. (1992) studied the aggregation of insulin in hydrophobic solid surfaces such as Teflon. The monomer was denatured at the surface followed by formation of microaggregates. Colombie et al. (2001) suggested that polytetrafluoroethylene (PTFE) was much more effective at causing aggregation of lysozyme than glass, which confirms the view that hydrophobicity is the governing factor in these phenomena. Ultrafiltration Ultrafiltration (UF) has been widely adopted as the method of choice for protein formulation in the biotechnology industry (Van Reis and Zydney, 2001). This purification step is typically performed to exchange the buffer, reduce conductivity, and to increase the protein concentration in solution (Kiefhaber et al., 1991; Shire et al., 2004). However, there are many challenges in applying UF to high concentration protein solutions including product solubility, restrictive bulk and mass transport of protein solutions, and product losses during recovery. During UF, proteins are exposed to physical stress due to pumping, with a typical process requiring at least 50 passes through the pump (Harris et al., 2004). This stress may result in an increase in protein aggregation (Van Reis et al., 1997). Multiple passes through valves and the concomitant microcavitation and air bubble entrainment, rather than shear, have also been cited as a cause of aggregation during filtration (Narendranathan and Dunnill, 1982). The extensive contact to membrane surfaces during UF is another factor that contributes to aggregates formation. The concentration of protein at the membrane surface may be much higher than that of the bulk solution. This phenomenon may lead to membrane fouling and to boundary layer polarization near the membrane which are considered to promote aggregation (Bodalo et al., 2004; Kiefhaber et al., 1991). Pumping During downstream processing of mab, the use of pumps is quite extensive, especially in those steps that require flow control. Typically lobular pumps are used to load product onto chromatography units, during viral filtration steps, UF, and during final filling (Gomme et al., 2006a; Thomas and Geer, 2011). It is quite extended the knowledge that these pumping processes expose mab to mechanical shear forces that might result into aggregates formation because most damage to proteins during processing will increase with higher fluid flow rates, for example, in passage through a pump, however, it has not always been clear that any effects are truly attributable to shear in the fluid mechanical sense, which is caused by velocity gradients in moving liquids (Thomas and Geer, 2011). Cavitation is an additional pumping stress that aggressively creates and destroys microbubbles inside pumps and valves (Bee et al., 2009b; Van Reis and Zydney, 2007). During rotary lobe pumping of human albumin, a pump with larger self-lubricated clearances caused more aggregation than a pump with smaller clearances (Gomme et al., 2006b). This was attributed to gap cavitation occurring and causing aggregation (Gomme et al., 2006b; Neumaier, 2000). Furthermore, there are studies that support that shear stress is the factor that less influences aggregate formation during pumping. Aggregation observed during the production of protein therapeutics is, therefore, not likely caused by shear per se, but rather by the gas/liquid interface that is created, exposure to solid surfaces, contamination by particulates, or pump cavitation that is often associated with shear (Bee et al., 2009b). The type of pump to be used during manufacturing is another factor influencing protein aggregation behavior. Meireles et al. (1991) studied the effects of different pump heads and observed an increase in turbidity of an albumin preparation with pumping time at room temperature by using a screw pump (Meireles et al., 1991). Likewise, it was observed that the use of a peristaltic pump enhances aggregate formation (Chandavarkar, 1990). In a recent study, strong evidence was found of aggregation of viruslike particles adsorbed to an adjuvant during recirculation studies involving a peristaltic pump. In these studies, increased back pressure in scaled down equipment demonstrated a proportional increase in aggregation, as measured by light scattering. While this result was initially attributed to pump shear increasing as a result of higher back pressures, additional studies showed that if a different tubing material was used, the levels of aggregation were 1500 Biotechnology and Bioengineering, Vol. 108, No. 7, July, 2011

8 independent of back pressure. This result is interesting as peristaltic pumps are frequently advertised as a mechanism to minimize shear during processing. Thus, protein aggregation via a peristaltic pump is unlikely related to hydrodynamic shear mechanisms and requires further investigation (Thomas and Geer, 2011). Final Filling Product filling is the final step in the manufacturing process no further purification after the fill. It is critical, therefore, that the formation of aggregates during this step is minimized if not prevented completely. For this reason it is essential that compatibility of the protein formulation with the filling equipment is assessed before production. Fill-and-finish operations may use pumps that can mechanically denature the protein because of shear stress or introduce impurities that serve as nucleation sources of protein aggregates (Tyagi et al., 2009). Some pistondisplacement pumps, for example, can interact with protein drug product in a similar way that a car motor engine piston interacts with lubricant oil. The intimate contact between protein drug product and a piston rod can disrupt an otherwise stable drug product. This was the case for an antibody drug product that was found to have increased levels of aggregate particles with more pump passes, as determined by absorbance and light obscuration methods (Cromwell et al., 2006). Materials used during final filling operations also impact aggregates formation. Glass from vials, rubber from stoppers, silicone from stoppers, and syringes and tungsten from syringes are some of the foreign particles which will come into contact with drug product during final filling operations (Carpenter et al., 1999). Many of these foreign particles are electrostatically charged and, therefore, have the potential to interact with proteins, protein aggregates, and protein aggregate precursors to form heteronuclei. Such was the case with prefilled syringes containing tungsten particles shed during syringe barrel manufacturing, which served as nuclei for aggregate formation (Eckhardt et al., 1991). Syringes, stoppers, and other surfaces are treated with silicone oil for lubrication or to inhibit protein binding but silicone oil can actually induce protein aggregation (Jones et al., 2005; Thirumangalathu et al., 2009). Substituting silicone oil with other substances like Teflon 1 has proven to cause protein aggregation due to adsorption of protein molecules at the solid/liquid interface as well (Sluzky et al., 1991). Freeze, Thaw, and Storage Most therapeutic proteins are delivered to patients in vials that will be accidentally or deliberately shaken between filling and delivery. Kiese et al. (2010) showed that shaking vials cause IgG1 aggregation but that the insoluble aggregates caused by the former could reversibly dissociate into soluble aggregates. This aggregation was probably due to the air liquid interface in the vials. Therapeutic proteins are usually frozen for long-term storage of bulk drug substance prior to fill and finish operations. Frozen storage of drug products is generally preferred over liquid storage for several reasons including increased product stability and shelf life, decreased microbial growth, and elimination of foaming during transport (Webb et al., 2002). Freeze thawing can be considered as a combination of various stresses, like cold denaturation, by introducing ice liquid interfaces (Harding et al., 1999; Strambini and Gabellieri, 1996), and by freeze-concentration (cryo-concentration) of solutes when the water crystallizes (Franks, 1982). This stress can also lead to ph shifts if the buffer salts crystallize (Hawe et al., 2009; Van den Berg, 1996). Freezeconcentration can cause phase separation of excipients and loss of native protein structure during subsequent drying (Heller et al., 1997). All these stresses might cause protein aggregation as reported for antibody freeze thawing with decreasing ph, which correlated well with T m (protein melting temperature) values (Kohle and Badkar, 2010; Kueltzo et al., 2008; Manning et al., 2010). Partial unfolding of proteins at the ice freeze concentrate interface was found to be one mechanism of aggregates formation during freezing (Chang et al., 1996; Eckhardt et al., 1991; Strambini and Gabellieri, 1996). Studies performed with human growth hormone have also suggested that protein aggregation could be correlated with the area of the solid/liquid interface (Eckhardt et al., 1991). Low temperature can cause spontaneous unfolding of proteins. Refolding and aggregation pathways compete for unfolded protein molecules. Slow freezing of solutions can lead to elevated concentrations of macromolecules interacting over extended periods of time, increasing the likelihood that an unfolded molecule will aggregate (Strambini and Gabellieri, 1996). On the contrary rapid freezing may lead to greater concentration of solution polarization. Keeping in mind the content and type of salt, and the damaging effect of the ice surface area, an optimal freezing rate as function of protein, and temperature would balance against the total exposure time with ice surfaces present. Aggregation after freeze thawing was detected primarily by the formation of particles in the size range from 1 to 25 mm, as well as aggregates of about 0.5 mm (Kueltzo et al., 2008). Heating and freeze thawing of monoclonal IgG1 antibody formulations results in the formation of aggregates, which differ considerably in size and structure (Bhatnagar et al., 2007; Hawe et al., 2009; Vermeer and Norde, 2000). Heating stress is mainly evidenced by the formation of small soluble IgG aggregates in the size range up to 30 nm, which are structurally changed. Freeze thawing on the other hand mainly leads to the formation of particles in the size range from 1 to 25 mm and few aggregates in the size range of 500 nm (Hawe et al., 2009). The containers commonly used for storage of bulk solutions are polypropylene, polyethylene, glass vials, Teflon 1 and ethylene vinyl acetate (EVA) bioprocessing bags. Kueltzo et al. (2008) demonstrated that less soluble Vázquez-Rey and Lang: Aggregates in mab Manufacturing Processes 1501 Biotechnology and Bioengineering

9 aggregate was detected at low ph (4) in the EVA bioprocessing bag and Teflon 1 vials than in polypropylene tubes. The preparation of freeze-dried mab formulated without excipients resulted in reversible solid-state protein structural alteration. The mechanisms by which sugars/polyols can improve the stability of a protein during drying and storage are still incompletely understood. There are two main hypotheses advanced to rationalize the role of solutes in stabilizing proteins during drying and storage. One is the glass dynamics hypothesis and another is the water substitute hypothesis (Chang et al., 2005). The glass dynamics hypothesis states that a good stabilizer forms a rigid, inert matrix into which the protein is molecularly dispersed and can couple the motion of protein to the motion of matrix. Dilution of proteins in the glass matrix separates the protein molecules, and limited mobility in the glass minimizes bimolecular interactions. Stabilization is via a purely kinetic mechanism, and the stability of the protein would be expected to correlate with the molecular mobility in this rigid matrix (Chang et al., 2005; Franks et al., 1991; Slade and Levine, 1991). The water substitute hypotesis states that stabilizers can form hydrogen bonds at specific sites on the surface of the proteins and substitute for the thermodynamic stabilization function of water that is lost during drying (Carpenter and Crowe, 1989; Carpenter et al., 1993; Chang et al., 2005). Following this theory, Strambini and Gabellieri (1996) proposed that the removal of tightly bound water from the protein surface by excessive dehydration correlated with increased rates of protein aggregate formation during storage. The addition of the carbohydrate excipients sucrose or trehalose to the formulation provided a solid-state environment where complete coverage of protein surface-accessible hydrogen binding sites was achieved. This correlated with improved native-like solid-state protein structure and reduced protein aggregation during storage (Strambini and Gabellieri, 1996). Strategies for Aggregate Removal and Minimization During mab Manufacturing Processes During manufacturing, it may not be possible in all cases to completely prevent or suppress aggregation, so that effective removal methods are essential for overall protein aggregate management. Numerous strategies are employed to minimize the level of aggregates in bio-therapeutics including protein engineering, expression system selection, optimization, separation during downstream processing, and storage formulation buffer screening. Separation during downstream processing is of particular interest as it provides the biggest opportunity to remove aggregates once they have been generated. Thus, different purification strategies can be effectively used to deal with increases in aggregate levels due to process-driven modifications or other unexpected changes that may occur during technology transfer and scale-up. Strategies for removal of protein aggregates need to be evaluated on a case-by-case basis, however, in this section we intend to provide a summary of the available strategies to minimize or reduce aggregates levels across mabs manufacturing processes. Chromatography as a Tool to Reduce Aggregates Levels During downstream manufacturing, chromatography is typically the step that mostly contributes to aggregate removals (Table I). The choice of a particular resin and mode of operation should be guided by fit and compatibility with the overall process purification train as well as an appropriate balance of productivity, yield, and product quality. Protein A affinity chromatography is typically used as the first downstream step in purification of therapeutic mab. This purification step is not capable of removing aggregates because product aggregates might bind to the resin ligand as well as monomer forms of product. Although this step is unable to remove aggregates, there are several studies intended to decrease aggregates formation during the Protein A chromatography step. Sodium chloride ( M) has been mentioned as an elution buffer additive to increase the elution ph (Carpenter et al., 1999; Gagnon, 1995). Ethylene glycol has been used to weaken hydrophobic interactions and thus increase elution ph from Protein A columns (Bywater et al., 1983). Another approach is to reengineer the Protein A ligand to allow for milder elution conditions. This allowed IgGs to be eluted at a ph of 4.5 instead of ph 3.0 (Gulich et al., 2000). The use of anion- and cation-exchange chromatography has been demonstrated to be useful at production scale to Table I. Chromatography as a tool to decrease aggregates levels. Type of chromatography Objective How the objective is achieved Affinity (Protein A) Minimize aggregates formation The use of NaCl or ethylene glycol increase elution ph Re-engeneering protein A ligand will allow higher elution ph Anion exchange Decrease aggregates levels At protein PI, aggregates bind to resin and product flow-through Cation exchange Decrease aggregates levels Product elutes earlier than aggregates. Only elution peak fraction containing the product is collected SEC Decrease aggregates levels Monomer has a smaller size than aggregates and elutes earlier Hydrophobic interaction Decrease aggregates levels Aggregates are more hydrophobic than monomers and are more strongly bound to the resin. The use of gradients or more volume of buffer can be used to first elute the product and later the aggregates 1502 Biotechnology and Bioengineering, Vol. 108, No. 7, July, 2011

10 separate mab monomers from dimers and larger molecular weight species (Ansaldi and Lester, 2003). When the working ph is close to the product s pi (around 0.2 logs lower than the pi), the overall net charge of the product is low. Aggregates generally carry more charge than the product at this ph range and, therefore, if the sample is loaded on a Q Sepharose column (anion exchange resin) at these conditions, most of the charged aggregates will be bound to the column, resulting in removal of aggregates (Wan and Wang, 2001). A novel class of dextran-grafted agarose based ion exchangers has gained popularity for process scale bioseparations. These matrices have been shown to exhibit both high equilibrium binding capacity and rapid protein mass transport kinetics. This is of interest as dextran-grafting dramatically impacts apparent pore radius as determined using inverse size exclusion chromatography (Harinarayan et al., 2006; Yao and Lenhoff, 2006). At ph values that are not the pi of the protein, the charge properties of protein aggregates are expected to be similar to the monomer but the size will be quite different, it is reasonable to anticipate that dextran-grafting may influence the separation of these two forms (Suda et al., 2009). Aggregates bind more strongly to ion exchangers than the corresponding monomeric form. For separation of aggregates, similar monomer pool purity and yield could be achieved with SP Sepharose XL and SP Sepharose FF (Suda et al., 2009). Some antibody purification processes use size exclusion chromatography (SEC) to reduce the levels of aggregates in the final solution. By employing either Sephacryl 300 or Superdex 200, gel filtration resins that separate according to size, it is possible to achieve final product pools with <2% aggregates (Phillips et al., 2001; Wang et al., 2006). Although it is technically possible to reduce the level of aggregates in a protein solution using SEC, it is often not cost efficient to do so. Preparative SEC is typically inefficient because of the poor resolution of aggregates from monomer (Litzen et al., 1993; Wang et al., 2006). Alternatively, it is feasible to separate mab from aggregates based on differences in hydrophobicity, which has been mainly used for the removal of both aggregates and impurities such as HCP (Lu et al., 2009). Hydrophobicity of mab increases with aggregation, a fact that has significant theoretical as well as practical significance (Suda et al., 2009). In the market there are several resins with low to high hydrophobicity. Charged-hydrophobic mixed mode chromatography methods have been applied to antibody purification for decades and have focused more recently on aggregate removal capacity. They exploit various combinations of alkyl and aromatic hydrophobic groups with positively and/ or negatively charged residues (Gagnon, 2009a). Hydroxyapatite has also been reported to provide good capabilities for aggregate removal; aggregates levels greater than 60% have been reduced to less than 0.1% using this resin (Gagnon, 2009b). Phosphate gradients have also been proven to remove aggregates in IgG mab. Effective aggregate removal is obtained with most antibodies that can be eluted at 5 15 mm phosphate, but declines in parallel with removal of HCP, DNA, and endotoxin for antibodies that require higher phosphate concentrations (Gagnon, 2009b). Hydroxyapatite chromatography in the presence of polyethylene glycol (PEG) provides a valuable enabling method for removing aggregates from IgG and IgM mab (Gagnon, 2008). PEG preferentially enhances aggregate retention, thereby, increasing the degree of separation between aggregated and non-aggregated antibodies. How to Minimize Aggregates Formation During mab Processing Treatments with solvents and detergents had been successfully used to inactivate virus as an alternative to low ph inactivation. It has been shown that solvents and detergent treatments of a recombinant protein can completely and rapidly inactivate enveloped viruses (Horowitz et al., 1998). The application of these methods instead of the low ph exposure will reduce the level of aggregates in the product. Pumping solutions containing mabs is a common practice in large-scale manufacturing processes. This fact implies applying shear stress to mabs that cause aggregates formation. The use of lobular pumps is very extended at large-scale protein purification processes. When a protein solution is pumped using a lobular pump, the protein is exposed to shear stress forces ejected by the pump lobes. By reducing the distance of the pump heads, shear stress will be reduced and hence aggregates formation (Gomme et al., 2006a,b). UF has also been used to remove aggregates in mab processes (Wan et al., 2005). A minimal wall shear stress has to be applied to ensure adequate back transport of deposited solutes to the retentate but, at the same time, it has to be limited to avoid the generation of soluble and insoluble aggregates which adversely affect bulk quality. Rosenberg et al. (2009) reported that if flow and pressure values are adjusted depending on the prevailing retentate concentration, the resulting stage process shows an improved permeate flux and reduced concentration time, also showing reduced aggregation compared to other methods operating under constant pressure and flow conditions. Filtration is a common strategy used to remove insoluble aggregates. Different studies have shown that aggregates are precipitated better than monomers by ammonium sulphate, also indicating that aggregates bind more strongly to PDVF membranes (Wang et al., 2006). Agitation has been reported to be a source of aggregates formation, but some studies have demonstrated that the magnitude and duration of shear exposure, in the absence of the air water interface, does not cause protein aggregation (Harrison et al., 2003). Therefore, if solutions containing proteins are agitated gently (avoiding vortex formation), aggregation will be minimized. Also, it may be possible to inhibit aggregation induced by agitation by careful choice of excipients (Serno et al., 2010). Vázquez-Rey and Lang: Aggregates in mab Manufacturing Processes 1503 Biotechnology and Bioengineering

11 mabs are also exposed to gas liquid interface when they are transferred to hold tanks. Typically large-scale mabs manufacturing processes use hold tanks to store intermediate process pools. When intermediate pools are being collected into hold tanks, it is essential to avoid product splashing, therefore, pool hold tank should have dip tubes or product inlet pipes designed in such a way that product is directed toward tank walls. Another solution is to fill tanks from the bottom instead from the top, in order to avoid denaturing proteins when filling pool hold tanks. In the UF systems, it is not recommended to concentrate product to volumes that are below recirculation retentate dip tube in order to minimize product foaming and hence aggregates formation, especially if this step is located downstream aggregates reduction chromatography. Surfactants such as the non-ionic surfactants polysorbate (20 and 80) are the most widely used excipients to prevent protein aggregation induced by agitation, which has been explained through different mechanisms. The adsorption competition between the surfactant and the protein to the gas liquid or solid liquid interfaces has been suggested to minimize the exposure of protein to these interfaces and, therefore, protecting from surface induced denaturation and aggregation (Bam et al., 1998; Carpenter et al., 1999). Surfactant molecules may interact with the exposed hydrophobic regions of the protein and, therefore, cover such sites that cause aggregation (Mahler et al., 2005). The most likely mechanism for polysorbate inhibition of rhgh aggregation due to agitation is a steric effect caused by polysorbate blocking aggregation-prone hydrophobic sites on the protein surface (Bam et al., 1998). The protective nature of polysorbate 20 was clearly observed for samples subjected to shaking stress whilst stirring required much higher concentrations of polysorbate 20 in order to demonstrate a protective effect (Kiese et al., 2008). In order to avoid aggregates formation during freezing, an ideal strategy is to freeze the entire solution at the same time and rapidly to minimize thermal transitions (such as eutectic melting) and glass transitions. With this strategy solute molecules will be entrapped and prolonged freezeconcentration stress will be prevented (Webb et al., 2002). The exposure of non-polar surfaces reduces the entropy and enthalpy of the system, at high as well as at low temperatures. It is, therefore, important to store biopharmaceuticals well below their thermal unfolding temperature, typically at 2 88C in order to minimize aggregates during storage process (Fesinmeyer et al., 2009). The appropriate formulation becomes critical when bulk freezing and thawing is required, and excipients may serve as protein cryo-protectants (Pikal-Cleland and Carpenter, 2001). As discussed in Strategies for Aggregate Removal and Minimization During mab Manufacturing Processes Section, many of the mab are delivered to patients filled into vials. Brych et al. (2010) showed that eliminating the headspace prevented the aggregation of IgG A in shaken vials and this may be a better approach than a label saying Do not shake. Future Outlook Having discussed aggregate formation and its removal during bioprocessing the overall goal of any manufacturing process will always be the achievement of highest possible product yield and purity with minimal aggregate levels. Tables I and II shows a list of recommended strategies that can be used to minimize or reduce aggregates levels across mabs manufacturing process. These data can be used to aid process development phase and/or equipment selection. The concept of quality by design (QbD) was introduced in 2004 as a result of the cgmp for the 21st Century Initiative with the objective of achieving a desired state for pharmaceutical manufacturing (Rathore, 2009). QbD is defined in the ICH Q8 guideline as a systematic approach to development that begins with predefined objectives and emphasizes product and process understanding and process control, based on sound science and quality risk management (International Conference of Harmonization (2008) ICH Harmonized Tripartite Guideline: Q8(R1) Pharmaceutical Development ( media/media4986.pdf)). The main benefits of this QbD system are to (1) assure product quality through design and performance-based specifications, (2) facilitate continuous improvement and reduce chemical manufacturing control (CMC) supplements, (3) enhance the quality of CMC reviews through standardized review questions, and (4) reduce CMC review time when applicants submit a quality overall summary (QOS) that addresses the questions (Yu, 2007). Table II. How to minimize aggregates formation. Process step How to minimize aggregates formation Viral inactivation Use of solvents and detergents to inactivate virus Pumping Minimize the use of pumps if possible, for example, pressurizing tanks to transfer product When using lobular pumps, reduce the pump heads distance to minimize shear stress Tank design Avoid product splashing Ultrafiltration Adjust flow and pressure values depending on retentate concentration Agitation Avoid vortex formation and or use of surfactants like polysorbate 20 or 80 Freeze Use of cryo-protectors Freeze the entire solution at the same time and rapidly Storage Eliminate the headspace in vials and avoid shaking 1504 Biotechnology and Bioengineering, Vol. 108, No. 7, July, 2011

12 Question based review involves three primary components (Rathore and Winkle, 2010): (1) process knowledge that includes a thorough understanding of process inputs and their impact on performance, (2) the relationship between the process and a product s critical quality attributes (CQA), and (3) the association between CQA and a product s clinical properties. The application of the QbD during process development phase helps to develop a suitable process control strategy for those quality attributes that are considered critical. For aggregate removal, the application of QbD in the development of the hydrophobic interaction chromatography has been successfully proved by Jiang et al. (2010). The manufacturing process of a biotechnological product, however, is a complex multi-step process where aggregation can be caused during different stages of the process. Thus improved process understanding contributing to aggregation is essential and would allow for a better design of individual process steps broad design space needs to be established and opportunities for changes in scale, equipment, etc. without prior FDA approval. Referring to aggregate removal, it might be recommended to generate multi-dimensional design spaces for individual process steps as outlined in Tables I and II and amended by the design space investigation of upstream manufacturing conditions which have a strong impact on product quality, aggregate formation, as well. 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