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The application of caprylic acid in downstream processing of monoclonal antibodies
Accepted Manuscript The application of caprylic acid in downstream processing of monoclonal antibodies Yifeng Li PII:
S1046-5928(18)30451-0
DOI:
10.1016/j.pep.2018.09.003
Reference:
YPREP 5324
To appear in:
Protein Expression and Purification
Received Date: 18 August 2018 Accepted Date: 7 September 2018
Please cite this article as: Y. Li, The application of caprylic acid in downstream processing of monoclonal antibodies, Protein Expression and Purification (2018), doi: 10.1016/j.pep.2018.09.003. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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The application of caprylic acid in downstream processing of monoclonal antibodies
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Yifeng Li
Technology and Process Development (TPD), WuXi Biologics (A WuXi AppTec Affiliate),
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288 Fute Zhong Road, Waigaoqiao Free Trade Zone, Shanghai 200131, China; e-mail:
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[email protected]
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Abstract
Caprylic acid (CA), a naturally occurring eight-carbon fatty acid, has long been used and bactericidal
agent in
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as albumin stabilizer, non-IgG fraction precipitant
pharmaceutical industry. The mechanisms through which CA achieves its effects have been correlated with the molecule’s protein/lipid binding capacity conferred by its octyl
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moiety. This article, following an initial review of CA’s historical applications, introduces CA’s relatively new application in downstream processing of monoclonal antibodies
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(mAbs). By taking advantage of CA mediated impurity precipitation and virus inactivation, it might be possible to develop a two-column purification process in replacement of the standard three-column process without compromising product quality.
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Keywords: Caprylic acid (CA); Fatty acid; Host cell proteins (HCPs); Impurity
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precipitation; Two-column process; Virus inactivation
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Abbreviations: CA, caprylic acid; mAb, monoclonal antibody; HSA, human serum albumin; BSA, bovine serum albumin; HCP, host cell protein; CHO, Chinese hamster ovary; pI, isoelectric point; MuLV, Murine Leukemia Virus; AEX, anion exchange; CEX,
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cation exchange; MVM, Minute Virus of Mice.
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Introduction
Caprylic acid (CA), also known as octanoic acid, is an eight-carbon fatty acid
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naturally found in mammalian milk, palm oil and coconut oil [1,2]. It is an oily liquid with low water solubility (0.818 g per L/~5.7 mM at 18.8 °C) [3]. The pKa of CA is 4.89. The sodium salt of CA (sodium caprylate) displays increased solubility which decreases with
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pH (~50 mM and 10 mM at pH 7 and pH 5, respectively). CA/caprylate has found wide applications in pharmaceutical industry. This article focuses on its relatively new
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application in downstream processing of monoclonal antibodies (mAbs). In this section, CA’s role as an albumin stabilizer during pasteurization, a selective precipitant for IgG isolation from plasma/serum and an antimicrobial agent for treating infections will be briefly reviewed. An understanding of the working mechanisms behind these historical
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CA applications will provide useful insights on its potential application in new area.
An albumin stabilizer
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In the early 1940s Ballou and coworkers found that CA could stabilize albumin at elevated temperatures [4]. In the presence of 0.3 M sodium caprylate a 25% albumin
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solution did not show visible turbidity even at 100 °C, a condition would otherwise cause severe denaturation of albumin. CA has been widely used as a stabilizer of albumin solution ever since. In fact, CA starts to show remarkable albumin-stabilizing effect at relatively low concentration (e.g., 20-40 mM for a 25% albumin solution) [5,6], and in practice a similar low concentration of CA is used to stable albumin products [7,8]. This stabilizing method allows albumin to undergo pasteurization at 60 °C for 10 hours (a procedure required for reliable inactivation of hepatitis virus) without being denatured [5].
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A selective protein precipitant
In addition to being an albumin stabilizer, CA acts as a precipitating agent for
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globulins. A considerable portion of the globulins of unheated plasma can be precipitated at pH 4.2 in the presence of CA [9]. Subsequent studies showed that ᵞ-globulin is not precipitated as readily as α- and β-globulins [10]. It is possible to remove about one-half
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of the total globulin at relatively low CA concentration without affecting ᵞ-globulin content. Furthermore, it was learned while only small amount of albumin is removed from plasma
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at low concentration of CA, appreciable amount of albumin is precipitated at increased concentration of CA [9]. A number of medium-chain (C6-C12) fatty acids have shown similar effects [10].
In the late 1960s a procedure for isolating IgG from mammalian plasma was
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developed based on CA mediated selective precipitation [11]. Under optimal conditions CA precipitates the bulk of plasma proteins without affecting IgG. In a standard procedure, the pH is adjusted to 4.8 before adding CA for best selectivity. For 100 ml
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human plasma of about 5% protein concentration, approximately 6.8 g of CA is required for precipitating non-IgG proteins [11]. Extensive non-immunoglobulin precipitation is
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best obtained at slightly acidic pH, but not below pH 4.5. Following standard procedure the purity of IgG in the supernatant fluid can reach 90-94% [11]. The residual contaminants, mainly IgA and ceruloplasmin, can be removed by batch adsorption on DEAE-cellulose. This CA precipitation technique allows IgG to be isolated from mammalian plasma with good yield and purity in a single step. A slightly modified version of the above procedure had been successfully used to purity IgG from spent media of hybridoma cultures and ascites fluid from mice injected with hybridomas [12]. CA treated spent culture medium or ascites fluid may still contain certain amount of 5
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albumin in addition to the desired immunoglobulin. The remaining albumin can be removed by precipitation of immunoglobulin using ammonium sulfate [13,14]. This approach, by combining CA triggered precipitation of contaminating proteins (albumin
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and other non-IgG proteins) and ammonium sulfate triggered precipitation of immunoglobulin, allows immunoglobulin to be obtained in greater purity and
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concentration.
For CA mediated differential precipitation of serum proteins, the best result (i.e., little
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or no precipitation of IgG but complete precipitation of albumin and other non-IgG proteins) is obtained when the serum sample was diluted with 3 or 4 volumes of 60 mM acetate buffer prior to addition of the acid. Insufficient serum dilution resulted in the precipitation of IgG and incomplete precipitation of contaminating proteins [13]. In addition, differential precipitation of serum proteins was optimal at CA concentrations
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ranging from 20 to 30 µl/ml of diluted sample. This is relatively consistent with the previously proposed concentration (6.8%) for undiluted sample. CA fractionation has been widely used in the production of antivenom from animal-derived sera [15-17]. CA
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treatment can also be used to remove proteolytic enzymes and vasoactive substances
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from partially purified IgG solutions [18].
An antimicrobial agent
It has long been known that fatty acids possess antimicrobial properties [19-21]. In
general, the growth of Gram-positive bacteria is inhibited by fatty acids to a greater extent than that of Gram-negative bacteria [21,22]. It was suggested that the difference in the sensitivities between these two groups of bacteria results from the protecting effect of Gram-negative bacteria’s outer membrane [22,23]. CA has been shown to 6
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display inhibitory effects on various Gram-positive and Gram-negative bacteria as well as fungi [24-35]. CA can potentially be used to treat bacteria-induced digestive disorders and yeast infections. In addition, CA’s antibacterial activity may allow it to be used as an
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active antiseptic ingredient in personal care products [36].
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Mechanisms of actions
CA and other fatty acids can bind various proteins [37]. A particular example is
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albumin, one of whose principal functions is to transport fatty acids. It is generally believed that the binding between fatty acids and albumin is mainly through hydrophobic interactions [38]. Faroongsarng and Kongprasertkit studied the role of CA in stabilizing human serum albumin (HSA) using a model for protein denaturation under thermal stress [39]. They proposed that at low concentrations CA stabilizes HSA by decreasing
unfolding/refolding.
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the rate of reversible unfolding as it binds to domain that is prone to reversible
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Bernard et al. previously proposed that protein precipitation by CA should be interpreted as the partitioning of protein between two partially miscible solvents: water
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and CA [40]. They suggested that these phenomena needs to be described as liquidliquid equilibria, although one of the liquid phases is so viscous that it looks like a solid. Morais and Massaldi recently studied CA mediated protein precipitation using bovine serum albumin (BSA) as the model protein [41]. They learned that for BSA solution at a given concentration, there is a minimum amount of CA required to start precipitation (this is consistent with the fact that CA stabilizes albumin at low concentrations). Once passed this threshold, increasing the CA concentration increases the amount of precipitated protein and can eventually precipitate nearly all BSA. For CA at a given 7
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concentration, there is a limit on the maximum amount of BSA it can precipitate. For example, 2% CA can precipitate approximately 20 g BSA per liter at most (in other words, 20 g/L BSA is the full precipitating capacity of 2% CA). Thus, for BSA solutions
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below 20 g/L, 2% CA can precipitate most of the protein, and the maximum precipitation reaches at BSA concentration around 40 g/L. The amount of precipitated protein drops at further increased BSA concentration (i.e., 40-60 g/L). When BSA concentration
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reaches about 60 g/L, precipitate disappears (2% CA does not reach the threshold for triggering precipitation at this BSA concentration). Further studies revealed that the
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precipitating capacities of 3% and 4% CA are 30 g/L and 40 g/L BSA, respectively. Thus, it appears that the mass ratio between CA and BSA is close to 1 for maximal precipitation.
Based on these observations, the authors proposed that CA binds to specific sites of
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the precipitating protein, inducing partial unfolding of the protein, which in turn exposes additional binding sites. When binding of CA reaches certain threshold, it triggers protein denaturation and precipitation. For different proteins at a similar concentration or same
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protein at different concentrations, the threshold varies and this forms the basis of selectivity. For each protein, the threshold is determined by its own property. In general,
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proteins with a large number of CA binding sites have a lower threshold and are more readily precipitated than those with a small number of binding sites. Albumin has been show to exhibit relatively high level of surface hydrophobicity [42]. Binding of CA to albumin can be promoted by hydrophobic interactions between CA’s alkyl chain and hydrophobic region on albumin surface. Thus, in comparison with immunoglobulin albumin allows more CA to bind under the same conditions, and this is likely why it precipitates first when serum is treated with CA. Also, according to the above model and observations, low abundant proteins should be more readily precipitated than high 8
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abundant proteins. This makes CA precipitation a potential means for removing small amount of contaminating proteins from a relatively concentrated protein product.
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CA, which contains the hydrophobic alkyl chain and the hydrophilic carboxylate, is an amphipathic molecule and can function as a surfactant [43-45]. CA’s surfactant property allows it to interact with plasma membrane, thereby increasing membrane permeability.
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Disintegration of the cell membrane consequently leads to leakage of cell contents, which eventually results in loss of cellular function and cell death. This is believed to be
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at least one of the mechanisms through which CA kills bacteria/fungi. It is also well known that surfactants at low concentrations stabilize proteins. For example, surfactants like polysorbates 80 and 20 are widely used in protein formulations to protect protein against agitation induced aggregation [46]. However, at increased concentrations
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surfactants precipitate or denature proteins.
Putnam and Neurath reported that surfactant caused precipitation was influenced by the surfactant protein ratio, pH, temperature and ionic strength [47]. With other factors
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kept constant, the surfactant protein ratio was found to be a decisive factor in precipitation. The authors observed three separate regions: the region of protein excess,
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in which the protein was incompletely precipitated; the equivalent zone, in which complete protein precipitation occurred; and the region of detergent excess, in which precipitate initially formed was partially or completely disappeared. In the case of CA, region one and region two are observed, but region three was not observed in the study conducted by Morais and Massaldi [41]. The non-observation of region three in this case is likely due to CA’s low water solubility. At increased CA concentration the solution will become turbid regardless of the amount of protein precipitated. In a previous study where CA was used for differential precipitation of proteins from rabbit serum, it was 9
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noticed that adding CA less or more than the necessary amount both resulted in incomplete precipitation of albumin [13]. This suggests that region three exists for CA mediated protein precipitation. It is well known that detergents can compromise cell
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membrane integrity and facilitate cell lysis. Detergents were found to exert marked inhibiting and killing effects on Gram-positive bacteria but somewhat less pronounced action on Gram-negative bacteria [48,49]. Taken together, these facts imply that CA’s
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main effects and activities can likely be attributed to its surfactant property.
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Application in downstream processing of mAbs
For host cell protein (HCP) removal
HCPs are a major class of process-related impurities in mAb manufacturing and their
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removal is always a difficult task. CA, as a precipitating agent, has been added to clarified bulk or culture harvest to remove HCPs [50-52]. CA selectively precipitates HCPs while maintaining the target antibody in solution. The efficiency of CA mediated
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HCP precipitation depends on both CA concentration and pH [50-52]. In one study, it was shown that increasing the CA concentration from 0.1% to 0.5% (~7 and 35 mM,
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respectively) at both pH 5.0 and pH 4.5 results in a ~2 log reduction of HCP, and HCP reduction was more efficient at the lower pH level (i.e., 4.5) when the CA concentration was low (i.e., 0.2%) [50]. In another study, Glynn showed that for CHO-produced antibody at a fixed pH value HCP reduction continued to increase in line with the CA concentration [52]. Precipitation performed under optimal conditions achieved 650-fold HCP reduction with >90% product yield. Most Chinese hamster ovary (CHO) HCPs have an isoelectric point (pI) between 4.5 and 7.0, and become less soluble in this pH range
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[53]. Adjusting to acidic pH alone has been shown to reduce HCPs by 20% [50]. At low pH the hydrophobicity of the octyl moiety of CA dominates, which greatly promotes precipitation of acidic proteins. Antibodies with basic pIs are not affected and remains in
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solution as they have sufficient charge to counteract that hydrophobicity. Also, mAbs are relatively inert to CA. In addition, compared to HCPs mAbs are more abundant in the harvest. Both facts suggest that mAbs will only precipitate at high CA concentration. It is
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the difference in pI, CA binding potential and abundance all together forms the basis of selectivity. Another process parameter that may influence precipitation efficiency is the
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length of mixing time. Under optimal conditions (i.e., pH 4.5, 1% CA), prolonging the mixing time from 30 to 120 min has a small impact on HCP clearance but can significantly improve DNA removal [50].
Gagnon et al. recently proposed that chromatin heteroaggregates, which contain the
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DNA-histone core and associated non-histone host proteins, are a major source of HCPs copurified with target mAbs [54]. Adding CA to culture harvest likely achieves HCP clearance by precipitating chromatin heteroaggregates in addition to unassociated
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individual host proteins [54,55]. In early studies it was observed that CA precipitates form three layers after centrifugation (the top and bottom layers mainly contain HCPs
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and DNA, respectively) especially when higher concentration of CA was used [50,52]. Optimized depth filtration will be required to remove the fluffy top layer. Alternatively, a lower concentration of CA which avoids three-layer formation but maintains most of the HCP-removing capacity can be used as a process compromise.
As an alternative approach, CA precipitation can also be implemented post Protein A capture step [52,56,57]. The advantage of conducting CA precipitation post Protein A is reduced product volume and thus easy handling. Similarly, pH and CA concentration are 11
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the two major factors affecting HCP reduction in Protein A elution pool [56,57]. Whereas changing pH alone can reduce HCPs to a certain level, adding CA can significantly improve HCP clearance. The optimal precipitation condition is a balance between HCP
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level and product yield. In one published work, Brodsky et al. studied post-Protein A CA precipitation with six mAbs [56]. The pIs of the selected mAbs range from 6.9 to 9.1. It was found that pH<4.5 was ineffective in precipitating HCPs while pH>5.5 had adverse
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effect on product yield. In addition to the pI difference between HCPs and mAbs, the pKa of CA, which is 4.89, likely also plays a role in determining the optimal pH range for
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selective precipitation. Previous study showed that CA precipitation of non-IgG fraction from plasma was best obtained at pH 5.0 [41]. This is not a coincident. According to Bull and Breese, it is the nonionized form of CA that binds proteins [37]. At pH above pKa, the ionized form dominants, suggesting the amount of effective component decreases. At pH below pKa, the nonionized forms dominants but the soluble portion may remain
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the same due to this form’s low solubility. At pH around CA’s pKa, soluble nonionized form may have reached maximum. For all six antibodies studies the optimal pH fell within a range of 5.0-5.4 at a fixed CA concentration of 1%. HCP removal ranges from
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93 to 100%. In all cases, product yield was above 90% with one exception, which was 86%. In another work, Zheng et al. conducted similar studies with five mAbs [57]. The
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authors learned although the optimal condition varies slightly for different antibodies, an operational pH range (5.0-6.0) and CA concentration (0.5-1.0%) can be identified for all five mAbs [57]. Under optimal conditions, four antibodies had more than 99% HCP reduction and the fifth one had 79% reduction.
In addition to being used as a precipitating agent, CA/caprylate has also been used as a wash additive during Protein A chromatography to reduce HCPs [52,58,59]. Compared to the control wash, wash buffers containing this agent improved HCP 12
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clearance, suggesting it can disrupt mAb-HCP interactions. In one study it was found that at pH 7.2 in the range of 25 to 100 mM, the higher the concentration of caprylate the greater the reduction of HCP for two tested antibodies. In addition, HCP reduction was
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further improved when sodium caprylate was combined with 0.3 M sodium acetate [60].
For virus inactivation
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CA/caprylate has been shown to be capable of inactivating enveloped viruses [61,62]. In particular, it is the nonionized form of caprylate rather than the ionized form that is
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responsible for the virus kill [61,62]. Inactivation is believed to be achieved through a detergent action on the lipid membrane of enveloped virus. The nonionized form’s lipophilic nature enables it to partition into the viral membrane, which eventually leads to lipid coat disruption and virus kill; the ionized form, on the other hand, is nonlipophilic and thus unable to partition into the lipid membrane [61]. For effective virus inactivation,
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the nonionized form needs to reach certain levels (e.g., 0.001-0.07% w/w) [62]. At a given caprylate concentration, the amount of the nonionized form varies with pH (the equilibrium of the dissociation reaction shifts to the ionized form at increased pH). For
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example, at pH 4.5 and 7.0 the ratio of nonionized form to ionized form is approximately 2:1 and 1:130, respectively [63]. A constant amount of the nonionized form can be
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maintained over a wide pH range by adjusting the concentration of the ionized form [60]. For instance, 0.05% of the nonionized form can be maintained from pH 4.8 to 8.0 by varying the concentration of caprylate from 0.05% to 60%. However, high concentration of caprylate can trigger protein precipitation. Thus, acidic pH (i.e., <6.5) is preferred as levels of nonionized form effective for virus destruction can be reached at moderate caprylate concentration under this condition.
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As demonstrated by validation studies using model viruses, CA/caprylate treatment gives reliable inactivation of enveloped viruses at suitable conditions [64,65]. Dichtelmüller et al. showed that CA at a concentration of 7.45 g per kg (at pH 5.5 the
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corresponding concentration of nonionized form is 1.49 g/kg) of antibody solution completely inactivate four test enveloped viruses (>4.68-6.25 log10 reduction) within the first minutes of treatment [64]. In addition to high efficacy, this procedure is shown to be
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highly robust over a wide range of several parameters. At pH 5.5 antibody concentration (30-40 g/L) and temperature (0-26 °C) variation within the studied range showed no
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significant influence on kinetics of virus inactivation [63]. The reliability and robustness of this inactivation process has been confirmed by another study, which showed that virus inactivation capacity remains unaffected by small variations in several studied parameters including caprylate concentration and temperature [65]. This study also proves that caprylate-mediated inactivation of enveloped viruses is as robust as
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standard solvent-detergent treatment [64]. In a relevant patent application, the inactivation of Murine Leukemia Virus (MuLV) with CA was investigated. In the pH range of 5.0 to 6.0 complete inactivation was achieved after 5 minutes when the concentration
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of the nonionized form was maintained at ≥4 mM [66]. These studies have paved the way for CA/caprylate treatment to be used as an effective virus inactivation procedure
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for manufacturing of plasma derived and monoclonal antibodies [67].
Concluding remarks
CA, a naturally occurring fatty acid, has been historically used as albumin stabilizer, non-IgG fraction precipitant and antimicrobial agent. CA mediated effects have been linked to the capacity of its nonionized form in binding proteins and lipids. Recently several studies showed that CA precipitation can be used as an effective and robust 14
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means for HCP reduction in downstream purification of mAbs [50-52,54-57]. In addition, CA has been shown to be capable of inactivating enveloped viruses, which can further benefit mAb manufacturing. In standard downstream processing, culture harvest is first
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clarified by depth filtration or centrifugation, and product is subsequently captured using Protein A affinity chromatography. CA can be added to culture harvest/clarified bulk or Protein A elution pool to induce impurity precipitation. The advantages and
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disadvantages of each scenario are summarized in Table 1. As indicated in the text, best results (significant HCP reduction and high product yield) are usually achieved around
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pH 5.0, which is close to CA’s pKa. In standard procedure there is a low pH viral inaction step after Protein A chromatography and upon completion the pH of viral inactivated pool is usually adjusted to a value between 5.0 and 7.5 [59]. Thus, it should be natural and smooth to have CA precipitation integrated into the process at this stage.
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For over 50 year CA/caprylate has been used as a stabilizer during the manufacturing of human albumin and its amount in intravenously-injected albumin products ranges from 4 to 20 mM [7,8]. The safe administration of CA-containing
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albumin suggests that CA at the above concentrations is non-toxic to human. In addition, mice intravenous toxicity study showed that CA had an LD50 of 600 ± 24 mg per kg and
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was less toxic than acidic acid, whose LD50 was 525 ± 21 mg per kg [68]. For HCP precipitation and virus inactivation, generally less than 70 mM of CA/caprylate is required at optimal pH. A significant portion of the added CA/caprylate will coprecipitate with the contaminants and the remainder can be effectively removed by a bind-elute mode polishing chromatography step [40,55-57]. The above facts indicate that implementation of CA precipitation/viral-inactivation into mAb manufacturing process shall not pose a safety concern.
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In a typical three-column purification process, Protein A is followed by two polishing steps, which usually consist of a flow-through mode anion exchange (AEX) and a bindelute mode cation exchange (CEX) chromatography. The main function of AEX is to
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remove HCPs and viruses. CEX can further remove HCPs and viruses, and more importantly it has a stronger ability in removing aggregates than flow-through AEX. Previous studies have shown that when post low pH neutralization and its subsequent
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depth filtration is performed under optimal conditions, they can be very effective in removing HCPs that the subsequent AEX flow-through step may provide no additional
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clearance [69,70]. Thus, when the neutralization step is enhanced by CA precipitation, it should be able to undertake most of AEX’s HCP clearing responsibility. AEX also provides reliable clearance for enveloped, non-enveloped viruses and retrovirus-like particles. In general, virus clearance heavily relies on AEX, which along with Protein A, low pH treatment and nanofiltration combinely achieves the required numbers of log
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reduction [71]. CA can inactivate enveloped viruses but not non-enveloped viruses. Thus, while CA precipitation would be a viral inactivation step complementary to low pH treatment, it cannot replace the role of AEX in viral clearance. CA precipitation only has
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a moderate effect on aggregate removal [56], and therefore it cannot replace the role of CEX in aggregate reduction. Thus, CA treatment alone cannot replace either of the two
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polishing steps. A mixed-mode resin Capto adhere has been shown providing significant viral clearance. According to the manufacturer’s data file, this resin achieves 5.8 and 4.5 log10 reduction for Minute Virus of Mice (MVM) and MuLV, respectively in flow-through mode [72]. It achieves a 5 log reduction factor for both viruses when run in bind-elute mode [73]. In addition, Capto adhere can effectively reduce aggregates [74,75]. These data suggest that CA precipitation plus Capto adhere can take the duties normally been taken by AEX and CEX. Thus, their combination can result in a two-column process that is equally effective as the three-column process in removing impurities and viruses. 16
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Acknowledgements
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The author would like to thank Drs. Weichang Zhou, Peter (Keqiang) Shen and Ying Wang for lending their supports to this work. The author also thanks Dr. Ying Wang for critical reading of the manuscript.
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1294 (2013) 70-75.
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Table 1. Advantages and disadvantages of CA precipitation implemented prior to and post Protein A chromatography
Advantages
Disadvantages
Prior to Protein A
Improved Protein A performance and resin lifetime; low level of residual CA/caprylate
Large handling volume and potential operational challenges at scale up
[50-52,54,56]
Post Protein A
Small handling volume and easy integration
Relatively high level of residual CA/caprylate
[56,57]
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Reference
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CA precipitation
S1046-5928(18)30451-0
DOI:
10.1016/j.pep.2018.09.003
Reference:
YPREP 5324
To appear in:
Protein Expression and Purification
Received Date: 18 August 2018 Accepted Date: 7 September 2018
Please cite this article as: Y. Li, The application of caprylic acid in downstream processing of monoclonal antibodies, Protein Expression and Purification (2018), doi: 10.1016/j.pep.2018.09.003. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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The application of caprylic acid in downstream processing of monoclonal antibodies
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Yifeng Li
Technology and Process Development (TPD), WuXi Biologics (A WuXi AppTec Affiliate),
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288 Fute Zhong Road, Waigaoqiao Free Trade Zone, Shanghai 200131, China; e-mail:
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[email protected]
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Abstract
Caprylic acid (CA), a naturally occurring eight-carbon fatty acid, has long been used and bactericidal
agent in
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as albumin stabilizer, non-IgG fraction precipitant
pharmaceutical industry. The mechanisms through which CA achieves its effects have been correlated with the molecule’s protein/lipid binding capacity conferred by its octyl
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moiety. This article, following an initial review of CA’s historical applications, introduces CA’s relatively new application in downstream processing of monoclonal antibodies
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(mAbs). By taking advantage of CA mediated impurity precipitation and virus inactivation, it might be possible to develop a two-column purification process in replacement of the standard three-column process without compromising product quality.
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Keywords: Caprylic acid (CA); Fatty acid; Host cell proteins (HCPs); Impurity
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precipitation; Two-column process; Virus inactivation
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Abbreviations: CA, caprylic acid; mAb, monoclonal antibody; HSA, human serum albumin; BSA, bovine serum albumin; HCP, host cell protein; CHO, Chinese hamster ovary; pI, isoelectric point; MuLV, Murine Leukemia Virus; AEX, anion exchange; CEX,
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cation exchange; MVM, Minute Virus of Mice.
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Introduction
Caprylic acid (CA), also known as octanoic acid, is an eight-carbon fatty acid
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naturally found in mammalian milk, palm oil and coconut oil [1,2]. It is an oily liquid with low water solubility (0.818 g per L/~5.7 mM at 18.8 °C) [3]. The pKa of CA is 4.89. The sodium salt of CA (sodium caprylate) displays increased solubility which decreases with
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pH (~50 mM and 10 mM at pH 7 and pH 5, respectively). CA/caprylate has found wide applications in pharmaceutical industry. This article focuses on its relatively new
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application in downstream processing of monoclonal antibodies (mAbs). In this section, CA’s role as an albumin stabilizer during pasteurization, a selective precipitant for IgG isolation from plasma/serum and an antimicrobial agent for treating infections will be briefly reviewed. An understanding of the working mechanisms behind these historical
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CA applications will provide useful insights on its potential application in new area.
An albumin stabilizer
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In the early 1940s Ballou and coworkers found that CA could stabilize albumin at elevated temperatures [4]. In the presence of 0.3 M sodium caprylate a 25% albumin
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solution did not show visible turbidity even at 100 °C, a condition would otherwise cause severe denaturation of albumin. CA has been widely used as a stabilizer of albumin solution ever since. In fact, CA starts to show remarkable albumin-stabilizing effect at relatively low concentration (e.g., 20-40 mM for a 25% albumin solution) [5,6], and in practice a similar low concentration of CA is used to stable albumin products [7,8]. This stabilizing method allows albumin to undergo pasteurization at 60 °C for 10 hours (a procedure required for reliable inactivation of hepatitis virus) without being denatured [5].
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A selective protein precipitant
In addition to being an albumin stabilizer, CA acts as a precipitating agent for
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globulins. A considerable portion of the globulins of unheated plasma can be precipitated at pH 4.2 in the presence of CA [9]. Subsequent studies showed that ᵞ-globulin is not precipitated as readily as α- and β-globulins [10]. It is possible to remove about one-half
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of the total globulin at relatively low CA concentration without affecting ᵞ-globulin content. Furthermore, it was learned while only small amount of albumin is removed from plasma
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at low concentration of CA, appreciable amount of albumin is precipitated at increased concentration of CA [9]. A number of medium-chain (C6-C12) fatty acids have shown similar effects [10].
In the late 1960s a procedure for isolating IgG from mammalian plasma was
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developed based on CA mediated selective precipitation [11]. Under optimal conditions CA precipitates the bulk of plasma proteins without affecting IgG. In a standard procedure, the pH is adjusted to 4.8 before adding CA for best selectivity. For 100 ml
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human plasma of about 5% protein concentration, approximately 6.8 g of CA is required for precipitating non-IgG proteins [11]. Extensive non-immunoglobulin precipitation is
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best obtained at slightly acidic pH, but not below pH 4.5. Following standard procedure the purity of IgG in the supernatant fluid can reach 90-94% [11]. The residual contaminants, mainly IgA and ceruloplasmin, can be removed by batch adsorption on DEAE-cellulose. This CA precipitation technique allows IgG to be isolated from mammalian plasma with good yield and purity in a single step. A slightly modified version of the above procedure had been successfully used to purity IgG from spent media of hybridoma cultures and ascites fluid from mice injected with hybridomas [12]. CA treated spent culture medium or ascites fluid may still contain certain amount of 5
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albumin in addition to the desired immunoglobulin. The remaining albumin can be removed by precipitation of immunoglobulin using ammonium sulfate [13,14]. This approach, by combining CA triggered precipitation of contaminating proteins (albumin
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and other non-IgG proteins) and ammonium sulfate triggered precipitation of immunoglobulin, allows immunoglobulin to be obtained in greater purity and
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concentration.
For CA mediated differential precipitation of serum proteins, the best result (i.e., little
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or no precipitation of IgG but complete precipitation of albumin and other non-IgG proteins) is obtained when the serum sample was diluted with 3 or 4 volumes of 60 mM acetate buffer prior to addition of the acid. Insufficient serum dilution resulted in the precipitation of IgG and incomplete precipitation of contaminating proteins [13]. In addition, differential precipitation of serum proteins was optimal at CA concentrations
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ranging from 20 to 30 µl/ml of diluted sample. This is relatively consistent with the previously proposed concentration (6.8%) for undiluted sample. CA fractionation has been widely used in the production of antivenom from animal-derived sera [15-17]. CA
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treatment can also be used to remove proteolytic enzymes and vasoactive substances
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from partially purified IgG solutions [18].
An antimicrobial agent
It has long been known that fatty acids possess antimicrobial properties [19-21]. In
general, the growth of Gram-positive bacteria is inhibited by fatty acids to a greater extent than that of Gram-negative bacteria [21,22]. It was suggested that the difference in the sensitivities between these two groups of bacteria results from the protecting effect of Gram-negative bacteria’s outer membrane [22,23]. CA has been shown to 6
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display inhibitory effects on various Gram-positive and Gram-negative bacteria as well as fungi [24-35]. CA can potentially be used to treat bacteria-induced digestive disorders and yeast infections. In addition, CA’s antibacterial activity may allow it to be used as an
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active antiseptic ingredient in personal care products [36].
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Mechanisms of actions
CA and other fatty acids can bind various proteins [37]. A particular example is
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albumin, one of whose principal functions is to transport fatty acids. It is generally believed that the binding between fatty acids and albumin is mainly through hydrophobic interactions [38]. Faroongsarng and Kongprasertkit studied the role of CA in stabilizing human serum albumin (HSA) using a model for protein denaturation under thermal stress [39]. They proposed that at low concentrations CA stabilizes HSA by decreasing
unfolding/refolding.
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the rate of reversible unfolding as it binds to domain that is prone to reversible
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Bernard et al. previously proposed that protein precipitation by CA should be interpreted as the partitioning of protein between two partially miscible solvents: water
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and CA [40]. They suggested that these phenomena needs to be described as liquidliquid equilibria, although one of the liquid phases is so viscous that it looks like a solid. Morais and Massaldi recently studied CA mediated protein precipitation using bovine serum albumin (BSA) as the model protein [41]. They learned that for BSA solution at a given concentration, there is a minimum amount of CA required to start precipitation (this is consistent with the fact that CA stabilizes albumin at low concentrations). Once passed this threshold, increasing the CA concentration increases the amount of precipitated protein and can eventually precipitate nearly all BSA. For CA at a given 7
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concentration, there is a limit on the maximum amount of BSA it can precipitate. For example, 2% CA can precipitate approximately 20 g BSA per liter at most (in other words, 20 g/L BSA is the full precipitating capacity of 2% CA). Thus, for BSA solutions
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below 20 g/L, 2% CA can precipitate most of the protein, and the maximum precipitation reaches at BSA concentration around 40 g/L. The amount of precipitated protein drops at further increased BSA concentration (i.e., 40-60 g/L). When BSA concentration
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reaches about 60 g/L, precipitate disappears (2% CA does not reach the threshold for triggering precipitation at this BSA concentration). Further studies revealed that the
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precipitating capacities of 3% and 4% CA are 30 g/L and 40 g/L BSA, respectively. Thus, it appears that the mass ratio between CA and BSA is close to 1 for maximal precipitation.
Based on these observations, the authors proposed that CA binds to specific sites of
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the precipitating protein, inducing partial unfolding of the protein, which in turn exposes additional binding sites. When binding of CA reaches certain threshold, it triggers protein denaturation and precipitation. For different proteins at a similar concentration or same
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protein at different concentrations, the threshold varies and this forms the basis of selectivity. For each protein, the threshold is determined by its own property. In general,
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proteins with a large number of CA binding sites have a lower threshold and are more readily precipitated than those with a small number of binding sites. Albumin has been show to exhibit relatively high level of surface hydrophobicity [42]. Binding of CA to albumin can be promoted by hydrophobic interactions between CA’s alkyl chain and hydrophobic region on albumin surface. Thus, in comparison with immunoglobulin albumin allows more CA to bind under the same conditions, and this is likely why it precipitates first when serum is treated with CA. Also, according to the above model and observations, low abundant proteins should be more readily precipitated than high 8
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abundant proteins. This makes CA precipitation a potential means for removing small amount of contaminating proteins from a relatively concentrated protein product.
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CA, which contains the hydrophobic alkyl chain and the hydrophilic carboxylate, is an amphipathic molecule and can function as a surfactant [43-45]. CA’s surfactant property allows it to interact with plasma membrane, thereby increasing membrane permeability.
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Disintegration of the cell membrane consequently leads to leakage of cell contents, which eventually results in loss of cellular function and cell death. This is believed to be
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at least one of the mechanisms through which CA kills bacteria/fungi. It is also well known that surfactants at low concentrations stabilize proteins. For example, surfactants like polysorbates 80 and 20 are widely used in protein formulations to protect protein against agitation induced aggregation [46]. However, at increased concentrations
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surfactants precipitate or denature proteins.
Putnam and Neurath reported that surfactant caused precipitation was influenced by the surfactant protein ratio, pH, temperature and ionic strength [47]. With other factors
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kept constant, the surfactant protein ratio was found to be a decisive factor in precipitation. The authors observed three separate regions: the region of protein excess,
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in which the protein was incompletely precipitated; the equivalent zone, in which complete protein precipitation occurred; and the region of detergent excess, in which precipitate initially formed was partially or completely disappeared. In the case of CA, region one and region two are observed, but region three was not observed in the study conducted by Morais and Massaldi [41]. The non-observation of region three in this case is likely due to CA’s low water solubility. At increased CA concentration the solution will become turbid regardless of the amount of protein precipitated. In a previous study where CA was used for differential precipitation of proteins from rabbit serum, it was 9
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noticed that adding CA less or more than the necessary amount both resulted in incomplete precipitation of albumin [13]. This suggests that region three exists for CA mediated protein precipitation. It is well known that detergents can compromise cell
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membrane integrity and facilitate cell lysis. Detergents were found to exert marked inhibiting and killing effects on Gram-positive bacteria but somewhat less pronounced action on Gram-negative bacteria [48,49]. Taken together, these facts imply that CA’s
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main effects and activities can likely be attributed to its surfactant property.
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Application in downstream processing of mAbs
For host cell protein (HCP) removal
HCPs are a major class of process-related impurities in mAb manufacturing and their
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removal is always a difficult task. CA, as a precipitating agent, has been added to clarified bulk or culture harvest to remove HCPs [50-52]. CA selectively precipitates HCPs while maintaining the target antibody in solution. The efficiency of CA mediated
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HCP precipitation depends on both CA concentration and pH [50-52]. In one study, it was shown that increasing the CA concentration from 0.1% to 0.5% (~7 and 35 mM,
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respectively) at both pH 5.0 and pH 4.5 results in a ~2 log reduction of HCP, and HCP reduction was more efficient at the lower pH level (i.e., 4.5) when the CA concentration was low (i.e., 0.2%) [50]. In another study, Glynn showed that for CHO-produced antibody at a fixed pH value HCP reduction continued to increase in line with the CA concentration [52]. Precipitation performed under optimal conditions achieved 650-fold HCP reduction with >90% product yield. Most Chinese hamster ovary (CHO) HCPs have an isoelectric point (pI) between 4.5 and 7.0, and become less soluble in this pH range
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[53]. Adjusting to acidic pH alone has been shown to reduce HCPs by 20% [50]. At low pH the hydrophobicity of the octyl moiety of CA dominates, which greatly promotes precipitation of acidic proteins. Antibodies with basic pIs are not affected and remains in
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solution as they have sufficient charge to counteract that hydrophobicity. Also, mAbs are relatively inert to CA. In addition, compared to HCPs mAbs are more abundant in the harvest. Both facts suggest that mAbs will only precipitate at high CA concentration. It is
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the difference in pI, CA binding potential and abundance all together forms the basis of selectivity. Another process parameter that may influence precipitation efficiency is the
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length of mixing time. Under optimal conditions (i.e., pH 4.5, 1% CA), prolonging the mixing time from 30 to 120 min has a small impact on HCP clearance but can significantly improve DNA removal [50].
Gagnon et al. recently proposed that chromatin heteroaggregates, which contain the
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DNA-histone core and associated non-histone host proteins, are a major source of HCPs copurified with target mAbs [54]. Adding CA to culture harvest likely achieves HCP clearance by precipitating chromatin heteroaggregates in addition to unassociated
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individual host proteins [54,55]. In early studies it was observed that CA precipitates form three layers after centrifugation (the top and bottom layers mainly contain HCPs
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and DNA, respectively) especially when higher concentration of CA was used [50,52]. Optimized depth filtration will be required to remove the fluffy top layer. Alternatively, a lower concentration of CA which avoids three-layer formation but maintains most of the HCP-removing capacity can be used as a process compromise.
As an alternative approach, CA precipitation can also be implemented post Protein A capture step [52,56,57]. The advantage of conducting CA precipitation post Protein A is reduced product volume and thus easy handling. Similarly, pH and CA concentration are 11
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the two major factors affecting HCP reduction in Protein A elution pool [56,57]. Whereas changing pH alone can reduce HCPs to a certain level, adding CA can significantly improve HCP clearance. The optimal precipitation condition is a balance between HCP
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level and product yield. In one published work, Brodsky et al. studied post-Protein A CA precipitation with six mAbs [56]. The pIs of the selected mAbs range from 6.9 to 9.1. It was found that pH<4.5 was ineffective in precipitating HCPs while pH>5.5 had adverse
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effect on product yield. In addition to the pI difference between HCPs and mAbs, the pKa of CA, which is 4.89, likely also plays a role in determining the optimal pH range for
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selective precipitation. Previous study showed that CA precipitation of non-IgG fraction from plasma was best obtained at pH 5.0 [41]. This is not a coincident. According to Bull and Breese, it is the nonionized form of CA that binds proteins [37]. At pH above pKa, the ionized form dominants, suggesting the amount of effective component decreases. At pH below pKa, the nonionized forms dominants but the soluble portion may remain
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the same due to this form’s low solubility. At pH around CA’s pKa, soluble nonionized form may have reached maximum. For all six antibodies studies the optimal pH fell within a range of 5.0-5.4 at a fixed CA concentration of 1%. HCP removal ranges from
EP
93 to 100%. In all cases, product yield was above 90% with one exception, which was 86%. In another work, Zheng et al. conducted similar studies with five mAbs [57]. The
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authors learned although the optimal condition varies slightly for different antibodies, an operational pH range (5.0-6.0) and CA concentration (0.5-1.0%) can be identified for all five mAbs [57]. Under optimal conditions, four antibodies had more than 99% HCP reduction and the fifth one had 79% reduction.
In addition to being used as a precipitating agent, CA/caprylate has also been used as a wash additive during Protein A chromatography to reduce HCPs [52,58,59]. Compared to the control wash, wash buffers containing this agent improved HCP 12
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clearance, suggesting it can disrupt mAb-HCP interactions. In one study it was found that at pH 7.2 in the range of 25 to 100 mM, the higher the concentration of caprylate the greater the reduction of HCP for two tested antibodies. In addition, HCP reduction was
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further improved when sodium caprylate was combined with 0.3 M sodium acetate [60].
For virus inactivation
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CA/caprylate has been shown to be capable of inactivating enveloped viruses [61,62]. In particular, it is the nonionized form of caprylate rather than the ionized form that is
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responsible for the virus kill [61,62]. Inactivation is believed to be achieved through a detergent action on the lipid membrane of enveloped virus. The nonionized form’s lipophilic nature enables it to partition into the viral membrane, which eventually leads to lipid coat disruption and virus kill; the ionized form, on the other hand, is nonlipophilic and thus unable to partition into the lipid membrane [61]. For effective virus inactivation,
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the nonionized form needs to reach certain levels (e.g., 0.001-0.07% w/w) [62]. At a given caprylate concentration, the amount of the nonionized form varies with pH (the equilibrium of the dissociation reaction shifts to the ionized form at increased pH). For
EP
example, at pH 4.5 and 7.0 the ratio of nonionized form to ionized form is approximately 2:1 and 1:130, respectively [63]. A constant amount of the nonionized form can be
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maintained over a wide pH range by adjusting the concentration of the ionized form [60]. For instance, 0.05% of the nonionized form can be maintained from pH 4.8 to 8.0 by varying the concentration of caprylate from 0.05% to 60%. However, high concentration of caprylate can trigger protein precipitation. Thus, acidic pH (i.e., <6.5) is preferred as levels of nonionized form effective for virus destruction can be reached at moderate caprylate concentration under this condition.
13
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As demonstrated by validation studies using model viruses, CA/caprylate treatment gives reliable inactivation of enveloped viruses at suitable conditions [64,65]. Dichtelmüller et al. showed that CA at a concentration of 7.45 g per kg (at pH 5.5 the
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corresponding concentration of nonionized form is 1.49 g/kg) of antibody solution completely inactivate four test enveloped viruses (>4.68-6.25 log10 reduction) within the first minutes of treatment [64]. In addition to high efficacy, this procedure is shown to be
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highly robust over a wide range of several parameters. At pH 5.5 antibody concentration (30-40 g/L) and temperature (0-26 °C) variation within the studied range showed no
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significant influence on kinetics of virus inactivation [63]. The reliability and robustness of this inactivation process has been confirmed by another study, which showed that virus inactivation capacity remains unaffected by small variations in several studied parameters including caprylate concentration and temperature [65]. This study also proves that caprylate-mediated inactivation of enveloped viruses is as robust as
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standard solvent-detergent treatment [64]. In a relevant patent application, the inactivation of Murine Leukemia Virus (MuLV) with CA was investigated. In the pH range of 5.0 to 6.0 complete inactivation was achieved after 5 minutes when the concentration
EP
of the nonionized form was maintained at ≥4 mM [66]. These studies have paved the way for CA/caprylate treatment to be used as an effective virus inactivation procedure
AC C
for manufacturing of plasma derived and monoclonal antibodies [67].
Concluding remarks
CA, a naturally occurring fatty acid, has been historically used as albumin stabilizer, non-IgG fraction precipitant and antimicrobial agent. CA mediated effects have been linked to the capacity of its nonionized form in binding proteins and lipids. Recently several studies showed that CA precipitation can be used as an effective and robust 14
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means for HCP reduction in downstream purification of mAbs [50-52,54-57]. In addition, CA has been shown to be capable of inactivating enveloped viruses, which can further benefit mAb manufacturing. In standard downstream processing, culture harvest is first
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clarified by depth filtration or centrifugation, and product is subsequently captured using Protein A affinity chromatography. CA can be added to culture harvest/clarified bulk or Protein A elution pool to induce impurity precipitation. The advantages and
SC
disadvantages of each scenario are summarized in Table 1. As indicated in the text, best results (significant HCP reduction and high product yield) are usually achieved around
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pH 5.0, which is close to CA’s pKa. In standard procedure there is a low pH viral inaction step after Protein A chromatography and upon completion the pH of viral inactivated pool is usually adjusted to a value between 5.0 and 7.5 [59]. Thus, it should be natural and smooth to have CA precipitation integrated into the process at this stage.
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For over 50 year CA/caprylate has been used as a stabilizer during the manufacturing of human albumin and its amount in intravenously-injected albumin products ranges from 4 to 20 mM [7,8]. The safe administration of CA-containing
EP
albumin suggests that CA at the above concentrations is non-toxic to human. In addition, mice intravenous toxicity study showed that CA had an LD50 of 600 ± 24 mg per kg and
AC C
was less toxic than acidic acid, whose LD50 was 525 ± 21 mg per kg [68]. For HCP precipitation and virus inactivation, generally less than 70 mM of CA/caprylate is required at optimal pH. A significant portion of the added CA/caprylate will coprecipitate with the contaminants and the remainder can be effectively removed by a bind-elute mode polishing chromatography step [40,55-57]. The above facts indicate that implementation of CA precipitation/viral-inactivation into mAb manufacturing process shall not pose a safety concern.
15
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In a typical three-column purification process, Protein A is followed by two polishing steps, which usually consist of a flow-through mode anion exchange (AEX) and a bindelute mode cation exchange (CEX) chromatography. The main function of AEX is to
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remove HCPs and viruses. CEX can further remove HCPs and viruses, and more importantly it has a stronger ability in removing aggregates than flow-through AEX. Previous studies have shown that when post low pH neutralization and its subsequent
SC
depth filtration is performed under optimal conditions, they can be very effective in removing HCPs that the subsequent AEX flow-through step may provide no additional
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clearance [69,70]. Thus, when the neutralization step is enhanced by CA precipitation, it should be able to undertake most of AEX’s HCP clearing responsibility. AEX also provides reliable clearance for enveloped, non-enveloped viruses and retrovirus-like particles. In general, virus clearance heavily relies on AEX, which along with Protein A, low pH treatment and nanofiltration combinely achieves the required numbers of log
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reduction [71]. CA can inactivate enveloped viruses but not non-enveloped viruses. Thus, while CA precipitation would be a viral inactivation step complementary to low pH treatment, it cannot replace the role of AEX in viral clearance. CA precipitation only has
EP
a moderate effect on aggregate removal [56], and therefore it cannot replace the role of CEX in aggregate reduction. Thus, CA treatment alone cannot replace either of the two
AC C
polishing steps. A mixed-mode resin Capto adhere has been shown providing significant viral clearance. According to the manufacturer’s data file, this resin achieves 5.8 and 4.5 log10 reduction for Minute Virus of Mice (MVM) and MuLV, respectively in flow-through mode [72]. It achieves a 5 log reduction factor for both viruses when run in bind-elute mode [73]. In addition, Capto adhere can effectively reduce aggregates [74,75]. These data suggest that CA precipitation plus Capto adhere can take the duties normally been taken by AEX and CEX. Thus, their combination can result in a two-column process that is equally effective as the three-column process in removing impurities and viruses. 16
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Acknowledgements
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The author would like to thank Drs. Weichang Zhou, Peter (Keqiang) Shen and Ying Wang for lending their supports to this work. The author also thanks Dr. Ying Wang for critical reading of the manuscript.
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Table 1. Advantages and disadvantages of CA precipitation implemented prior to and post Protein A chromatography
Advantages
Disadvantages
Prior to Protein A
Improved Protein A performance and resin lifetime; low level of residual CA/caprylate
Large handling volume and potential operational challenges at scale up
[50-52,54,56]
Post Protein A
Small handling volume and easy integration
Relatively high level of residual CA/caprylate
[56,57]
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Reference
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CA precipitation