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Chapter 11

Alternative Separation Methods: Flocculation and Precipitation

James M. Van Alstine*,†, Günter Jagschies‡, Karol M. Łącki§ *JMVA Biotech AB, Stockholm, Sweden, †Royal Institute of Technology, Stockholm, Sweden, ‡GE Healthcare Life Sciences, Freiburg im Breisgau, Germany, §Karol Lacki Consulting AB, Höllviken, Sweden

11.1 INTRODUCTION Precipitation refers to phenomena where substances, which are dissolved in , come out of solution, often due to the actions of a precipitant. In flocculation, suspended in a liquid come out of due to a spontaneous pro- cess such as weak association and aggregation over time, or more rapid association promoted by the action of an operation (e.g., temperature increase), or addition of a flocculating agent such as a polymer that “bridges” between colloids, creat- ing larger aggregates. Table 11.1 provides a SWOT analysis of precipitation and flocculation related to their usefulness in bioprocesses. Flocculation may be used to enhance the efficiency of or steps to remove cell debris from a solution. Precipitation may also be used to remove contaminants. In some cases, that precipitate out of a solution may further associate and flocculate. This has led to a tendency in bioprocessing to use the terms interchangeably. Precipitation may be used to remove contaminants (e.g., caprylic acid precipitation in antibody processing) or to precipitate targets (e.g., cold-ethanol fractionation of plasma proteins). Precipitation can offer significant selectivity—though it may not be able to resolve challenges such as minor posttranslational, or bioprocess- related target modifications (e.g., deamida- tion, oxidation occurring during the process). may offer more effective target resolution due to its inherent large number of equilibrium separation events (theoretical plates); however, mass transport effects and nonspecific can offer their own challenges. Precipitation often involves rapid equilibrium events and can be run in continuous multiex- traction formats. Over the past two centuries, a very large number of methods have been explored for precipitating proteins, including salting-out, isoionic precipitation, organic co-solvent based precipitation, as well as the use of hydrophilic neutral polymers or osmolytic agents, use of di- and tri-valent , or , to enhance -protein association, or methods based on enhancing protein-protein interactions via the use of affinity or hydrophobic ligands.

11.2 CLARIFICATION AND PRIMARY RECOVERY CHALLENGES Advances in cell line development and culture have resulted in higher product titers (>5 g/L), cell culture densities (e.g., 20% packed cell volume), and contaminant levels (Table 11.2). This has placed significant challenges on existing “­workhorse” methods such as of disk-stack centrifugation and depth filtration in regard to isolating recombinant targets from cell debris [11–15]. The situation has been exacerbated by the larger scales of modern fermentation as well as the ad- vent of various culture and other methods that enhance cell densities beyond 107 cells/mL. The end result is larger amounts of more viscous and turbid (e.g., 1000 nephrelometric units or NTUs), feed that must be clarified. The tendency is to push technologies to their limit of throughput and clearance. Under such conditions classical clarification methods such as centrifugation and can result in poorly clarified supernatants, especially when taxed to process large fermentations in single work shifts. This can affect downstream filtration and chromatography column performance, cleaning requirements, and cycle lifetime. Use of more technically involved clarification approaches, (see Chapter 9 and Refs. [4,14,15]) may partially offset such challenges, as may the classic chemical engineering approach of adding “other” reagents to promote contaminant precipitation or flocculation. Fig. 11.1 shows a map of volumes, cell densi- ties, and possible clarification strategies, and is intended to be suggestive, not definitive, over a wide range of condi- tions. The non-horizontal nature of the lines representing filtration are intended to reflect existing economic challenges (which new filter designs may eliminate). Fig. 11.2 places flocculation-based clarification in context with other approaches.

Biopharmaceutical Processing. https://doi.org/10.1016/B978-0-08-100623-8.00011-6 © 2018 Elsevier Ltd. All rights reserved. 221 222 SECTION | III Recovery Processes, Principles, and Methods

TABLE 11.1 SWOT Summary for Precipitation and Flocculation

Traits Comments

Strengths • Reasonably well understood at molecular level (e.g., [1]) • Possible use in continuous processing • Ease of scalability and use in bioprocessing demonstrated • Robust, can be used for clarification (flocculation), target capture, contaminant capture • Caprylic (octanoic) acid precipitation can be used for viral and host cell protein (HCP) load reduction in antibody (Ab) processing • Biocompatible, may provide some storage stability • Ideal for high throughput screening methods • Wide range of targets (proteins, nucleic acids, viruses, cells) • Inexpensive liquid-solid (L-S) nature is ideal for single use • May take advantage of responsive polymers • Can employ neutral polymers or other polymers already used in bioprocessing as antifoaming agents or formulation excipients

Weaknesses • May introduce new impurities and quality control (QC) requirements to a process • Resulting complexes may require dilution or pH change to dissolve • May be strongly affected by contaminant ions such as divalent ions • No vendor specializing in technology or optimized single use devices (although see Ref. [2]) • Industry knowledge was weak, but improving significantly (e.g., [3,4])

Opportunities • Predictive modeling based on primary structure possible • Continuous processing possible • New systems offering less costly polymers • New systems with kosmotropic salts offer robust processing and easy to dissolve precipitates • Utilization of polymers approved for processing and drug delivery

Technological threats • Other novel L-S approaches to bioseparation (e.g., ) • Improved clarification by centrifugation and filtration

TABLE 11.2 Bioprocess Target and Contaminant Net Charge Considerationsa

Substance Typical pI or pI Range Net Charge at pH >6.5 Polyacid polymers 2–6 Negative

Polycationic polymers 8–10 Positive

Human IgGs and fabs (a,b) 6.5–9.5 Positive

Human IgAs or IgMs (b) 4.5–6.5 Negative

BSA and HSA 4.5–4.8 and 5.5 Negative

Transferrin and insulin 5.8 and 5.3 Negative

Protein A ligands ≈5 Negative b CHO HCPs 4–9, most <6 Often negative

E. coli HCPs 4–10, many <8 Often negative DNA, RNA 2–3, polyacid Negative

Endotoxin 1–4, polyacid Most negative

Virus polyacid colloid Most negative

Cell debris polyacid colloid Negative

a Table concept from U. Gottschalk [5] and Singh et al. [4] values abstracted from various sources including [6–9]. b Many rPlant and rMilk feeds are expected to show similar properties [10]. CHO, Chinese hamster ovary cells; HCPs, host cell proteins. Alternative Separation Methods: Flocculation and Precipitation Chapter | 11 223

40 EOPO single polymer or ATPE 30 system Filtration + other 20 Centrifugation + other

Cell mass (% v/v) 10 Centrifugation Filtration 0 0 2.5 5 10 +20 Fermenter volume (× 1000 L) FIG. 11.1 Approximate useful operating regions for bioreactor feed clarification methods. ATPE two- polymer systems are noted in Chapters 10 and 12. “Other” refers to the use of flocculation or related methods to enhance clarification efficiency. The x-axis scale is not linear.

Bioreactor

Primary clarification (Centrifugation, TFF, depth filtration, Flocculation or ATPE)

Depth filtration

Bioburden reduction (0.2 or 0.45 µm)

Purification FIG. 11.2 Options for clarification of mammalian cell culture. Alternative separation methods are underlined. For more detail see Ref. [16].

The clarification ability of aqueous polymer two-phase systems (see Chapter 12) relies on interfacial tension rather than specific chemical interactions to associate materials and promote their flocculation. As such, it is not dependent on match- ing phase forming polymers to cell mass and can be a robust methodology (Section 10.2.4 below). Table 11.3 summarizes current experience in regard to the use of different flocculation reagents for clarification, as well as for primary target recovery from a clarified feed. If one considers the general net charge of bioprocess targets and typical process feed contaminants, many (but not all) contaminants will be net negatively charged under the conditions of clarification, recovery, and primary purification (Table 11.2). When target proteins are net positively charged, this creates an opportunity to use cationic or poly-cationic flocculating agents to aid removal of net negatively charged cellular debris and nontarget molecular entities. In the case of negatively charged proteins, it may be possible to choose pH ranges where the target protein is net positive, or flocculating agents which may otherwise selectively interact with contaminants. Industry initially considered enhancing the efficiency of clarification processes via the use of classic flocculants (see Table 11.3, and [4,66]) including polyethyleneimine (PEI) or (which may have some monomer toxicity issues, [14,17,67]). More natural flocculation agents such as chitosan or charge-modified dextrans may also be attractive (see [34], as well as [4,14]). It is possible to coat silica particles with PEI or use anion exchange beads to effect “Enhanced Cell ” with less concern for flocculant contamination of feed streams [67], but this also adds another level of com- plexity and cost. For these reasons, several authors including Coffman et al. [13,17], and Glynn et al. [23] suggested use of small molecular weight flocculation agents such as a binary mixture of calcium chloride and potassium . Of course any flocculation method based on charge-charge interaction may have to be optimized in terms of operating pH and conductivity, which again may add another level of complexity. This suggested the use of neutral biocompatible polymers such as polyethylene glycol (PEG) may also be of interest [25,26]. Approaches directed to removing net negatively charged cell debris (Table 11.2) offer three benefits in regard to pro- cessing of monoclonal antibodies, and other proteins net positively charged under process conditions [15]. First, they offer reduction in cellular debris that must be cleared by downstream filtration, resulting in more cost effective (e.g., smaller membrane area) filtration [4,14,18]. Second, some removal of other negatively charged contaminants, including many host cell proteins, nucleic acids, endotoxins, and viruses (Table 11.2 and Refs. [14,68]). Third, they may enhance downstream chromatographic performance in regard to reduced fouling and longer column bed life. 224 SECTION | III Recovery Processes, Principles, and Methods Ref. [ 17–20 ] [ 17 , 23 24 ] [ 46–55 ] [ 21 , 22 ] [ 25–28 ] [ 35 , 36 56–58 ] [ 23 , 29–33 ] [ 59–63 ] [ 23 , 34–45 ] [ 64 , 65 ] b Challenges Comment Scalability Stability Tox. Low concern Resolubilize Residue Tox. Often < 0°C Time for Selectivity HMW Residue Tox. Concern Low Tox. Low Antiviral Cost Resolubilize Residue Tox. Up to 40% cell mass Reagent Reagent Removal pH adjust, chrom. TFF, pH adjust, chrom. TFF, Filtration Filtration chrom. Varies TFF Chrom. Filtration, Filtration, chrom. TFF Chrom. Recycle Filtration Filtration chrom. Flow through Flow DNA Reduce 1–3 Logs < 3 Log > 3 Logs < 1 Log < 3 Logs > 5 Logs Varies > 3 Logs 5–6 Logs < 1 Log a HCP Reduce < 1 Log < 0.5 Log 1 Log < 1 Log < 1 Log < 1 Log > 1 Log < 3 Logs > 0.3 Log 0.3 Log > 85% > 80% > 90% Target Target Yield > 70% > 90% > 90% > 80% > 90% > 80% > 90 mM

4–5.5 5–6 5–8

pH or % Dosage pH 2–200 pH pH < 10% pH to pI, 10%–40% pH 5–8 5%–15% 0.1%–0.6% (w/v) 0.5%–1.5% (v/v) 0.25 × (target) 0.01%–1.0% (w/v) < 10% Main Mechanisms Charge Neutralization Solubility Solubility Co-precipitate Electrostatic hydrophobic Co-precipitate Charge neutralization Affinity Mixed mode Electrostatic and H-bond Phase separation Electrostatic Cell Clearance Possible Maybe Maybe Possible Maybe No Costly Yes Yes No ]. BPA: benzylated poly(allylamine); Chrom, chromatography; CMD, carboxymethyldextran; EOPO, poly(ethylene oxide, propylene oxide); NiPAAM, NiPAAM, oxide); propylene oxide, poly(ethylene EOPO, carboxymethyldextran; CMD, chromatography; Chrom, benzylated poly(allylamine); BPA: 4 , 23 64 66 ]. a Low pH Low Example Reagent Organic solvent PEG-metal NiPAAM, BPA NiPAAM, Metal cation Caprylate (Octanoate) ELP-protein pDADMAC PEI, PolyAA EOPO PVS CMD PAA, Comparison of Precipitants and Flocculants Used in ClarificationComparison of Precipitants and Primary Recovery

4 , 14 66 ] regarding high molecular weight aggregate (HMW) and viral reduction. See Refs. [ See Refs.

Table concept taken from Refs. [ from concept taken Refs. Table

TABLE 11.3 Type Precipitation a b N-isopropyl-; PAA, polyacrylic acid; PDADMAC, polydiallyldimethylammonium chloride; PEI, polyethylenimine;. PolyAA, poly(amino acid) e.g., polyhistidine; PVS, polyvinylsulfonate; polyvinylsulfonate; PVS, polyhistidine; poly(amino acid) e.g., PolyAA, polyethylenimine;. PEI, chloride; polydiallyldimethylammonium PDADMAC, polyacrylic acid; PAA, N-isopropyl-polyacrylamide; filtration. tangential flow TFF, Solution responsive (temp and other) Cationic polymer Anionic polymer Alternative Separation Methods: Flocculation and Precipitation Chapter | 11 225

7 Westoby et al. [18] investigated the effects of fermentation solution environment on high density (>10 cells/mL) mam- malian CHO cell properties as regards clarification and impurity removal. Their work indicates that small-scale experi- ments can identify large-scale bioreactor conditions aiding clarification efficiency and high product recovery. They found, as have other authors (Table 11.3) that pH adjustment from 7 to 4 resulted in significant reduction in particles of size <3 μm, as well as in the levels of host cell protein (HCP) and DNA in the cell-free supernatant (see also Fig. 11.8). “The data dem- onstrate significant drop in supernatant turbidity below pH 5.0 irrespective of ionic conditions.” In some cases the “target” to be precipitated may not be contaminants but product. This might be done with clarified feed in order to isolate the target from contaminants and also concentrate product (and perhaps transiently store it) as a precipitate, prior to re-suspension and further chromatographic or other processing (Fig. 11.3). It is difficult to isolate precipitation and flocculation from filtration [14], which is discussed elsewhere in this book in Chapters 9, 14, and 15. In addition to the reviews noted previously (e.g., [4,14]. Pegel et al. [69] presented results related to evaluating several different disposable depth filtration platforms from different vendors for mAb harvest clarification at both small and large scale. Dave, Dizon-Maspat, and Cano of Genentech presented on screening of dual layer trains from several vendors, and on evaluation and implementation of a single stage multimedia harvest depth filter for large-scale mAb processing [70]. Yigzaw et al. [71] have discussed exploitation of the adsorptive properties of depth filters for HCP removal. Sing et al. [4] have summarized and tabulated some results from these and other authors. Many groups including Gottschalk et al. [5] have reported on the use of membrane absorbers (i.e., cationic-ligand modified membranes) to aid contaminant removal, which is discussed elsewhere in this book. More references and examples of the use of alter- native separation methods to reduce contaminant load, such as in clarified feed, are provided in following sections of this chapter. In many cases, contaminants have the same net charge as cell debris components (Table 11.2) and some charged flocculants may find use in removing both types of contaminants (Table 11.3).

11.2.1 Self-Associating and Nonassociating Flocculation/Precipitation Agents Before moving on to specific examples, it is important to note that molecular-level events related to polymer-protein com- plex interactions are well studied and this knowledge can be used to enhance the use of polymer induced flocculation of targets or contaminants in bioprocessing (e.g., [4,14,46–52,56,57]). For example, in their modeling of polyanion complex formation with a basic protein (modeled after lysozyme) Carlson, Linse, and Malmsten noted that the interaction was not just electrostatic, but appeared to have nonelectrostatic interactional components [1]. This may allow for more complicated flocculating agent-target interaction, as well as agent self-association behavior. Fig. 11.4 provides a simple comparison of the target precipitation behavior for two different types of precipitating/flocculating agents. In the first case (A) an agent that does not readily self-associate interacts with a target. This could be a polyanion, such as polyvinylsulfonate, interacting with a basic protein, or even a mAb interacting with a protein or colloid for which it has an affinity. As agent concentra- tion increases relative to the target, flocculation will increase, until, at a higher concentration, bridging associations will be eliminated and flocculation will diminish. This is classically seen in hemagglutination, as well as in basic protein precipita- tion by polyvinylsulfone or polyacrylic acid [1,46–48,51,52]. As in the case of exchange chromatography, such interac- tions will be affected by solution pH and conductivity, as well as agent-target concentration or mass ratios. The second case

Cationic polyelectrolytes (e.g., Poly (arginine)* Anionic polyelectrolytes (e.g., Polyallylamine)* Caprylic acid

Product in Removal of Chromato- solution precipitate graphy

Precipitation Utilize unique product feature for best selectivity

Impurities in Recovery Chromato- solution of product graphy

Anionic polyelectrolytes (e.g., Polyvinylsulfonic acid, PVS)* EOPO, (e.g., Polyacrylic acid, PAA)* Ammonium sulphate Polyethylene glykol, PEG** Affinity precipitation FIG. 11.3 Precipitation or flocculation of clarified feed to remove soluble contaminants or target. *Charged polymer, **Uncharged neutral polymer, See Table 11.3. 226 SECTION | III Recovery Processes, Principles, and Methods

Non self-associating Self-associating or 1.0 agent or osmotic agent

0.5 target in solution Relative amount of 0 (A) (B)

Relative amount of precipitant or flocculant FIG. 11.4 Concentration-dependent behavior of precipitants or flocculants [72]. “Real data” figures similar to (A) appear in Ref. [48].

(B) relates to agents, such as ethanol or neutral polymers like PEG, which tend to bind water molecules and osmotically promote target-association, as well as responsive polymer or other flocculating agents, for example, elastin-like proteins (ELPs), which interact with targets and can be self-associating. In this case, increasing the concentration of agent past the level required for optimal flocculation has little primary effect on the amount of “floc” formed. One advantage of situation B is that conditions can be chosen with some excess of agent so the operation becomes much less affected by variation in target concentration. Another is that while electrostatic or hydrophobic interactions occur over broad ranges of conditions, the self-association behavior of responsive polymers can often be tailored to a narrow range of pH, temperature, or other conditions [4,56–61].

11.3 CELL DEBRIS REDUCTION AND CLARIFICATION Table 11.3 identifies some cationic or responsive polymer reagents that can be used for cell and cell debris reduction, often by flocculation. Generally such reagents will be cationic, polycationic, mixed-mode polycationic, and may be responsive polymers which become more hydrophobic and self-associating in response to increases in temperature, conductivity, ­divalent ion concentration, etc.

11.3.1 PEI, Chitosan, or CaCl2 Plus K2PO4-Based Cell Flocculation Most cells and cell debris are negatively charged and amenable to flocculation by polycations or similar bridging sub- stances including polyethleneimine, chitosan, and mixtures of multivalent salts [17,34,67]. In one example, Coffman et al. [17] provided methods to flocculate cellular debris using calcium chloride and potassium phosphate. of over 30 nephelometric turbidity units (NTUs) in controls could be reduced to 3 NTUs in 60 min at unit gravity. Experiments with five mAb feed samples indicated that target recoveries of 95 + % were possible, even over extended (3 h) times.

11.3.2 Cell Flocculation Using Poly (Diallyldimethylammonium Chloride) (pDADMAC) pDADMAC is a water-soluble flocculant that is used in waste [4] and has been shown to be of possible use 7 in initial clarification of high-density (>10 cells/mL) mAb feeds. Its effects appear to be enhanced if used together with PEG or similar neutral polymers with recoveries of 90 + % target and significant reduction in other contaminants such as HCP and DNA [4,35,66]. MerckMillipore has developed its possible use with Clarisolve depth filters, and their studies suggest 1 ppm residual polymer is not a QC concern. The 1 ppm level can be quantitated using surface plasmon resonance [2]. The polymer can be added at reasonably low concentrations (i.e., <0.1% w/w) and one test resulted in an 8× increase 2 in clarification filter throughput (to >700 L/m ) as well as 50%–70% reduced HCP levels in post-Protein A affinity chroma- tography target pools. One challenge with the polymer is that “excess” pDADMAC results in a decrease in performance of the subsequent chromatography steps with a loss in process yield and increase in residual polymer concentration [2]. This suggests careful dosing may be required to achieve optimal results with different feeds. Fig. 11.5 shows how the addition of ~0.04% (w/w) pDADMAC significantly increased the particle size distribution in a CHO cell culture feed stream, when compared with untreated or low-pH treated culture feed [2]. For comparison, Fig. 11.5 also shows low-pH treatment control results, which are in keeping with Westoby et al. [18]. Alternative Separation Methods: Flocculation and Precipitation Chapter | 11 227

Particle size distribution (PSD) 0.06 Low pH treated 0.05

0.04 Untreated 0.03 pDADMAC treated 0.02 Particle count (%) 0.01

0 1 10 100 Particle size (µm) FIG. 11.5 Particle size distribution (PSD) representative CHO cell culture feed stream. Data are shown for unadjusted (pH 7.0), acid treated (pH 4.8), and pDADMAC (0.0375%, w/w) treated cell culture feed streams. Reproduced from pDADMAC flocculant reagent for use with Clarisolve® depth filters, MerckMillipore Application Note Lit # AN33330000 Rev. C PS-14-09928 08/2014, © 2014 EMD Millipore Corporation, Billerica, MA USA. Copyright MerckMillipore, used by permission.

11.3.3 Cell Flocculation Using Benzylated Poly(Allylamine) Phosphate Responsive Polymer 6 Kang et al. [36] studied clarification of relatively high-density (4–6 × 10 cells/mL) CHO cell feeds, with mAb titers of 0.4–5.0 g/L with benzylated poly(allylamine) (BPA) polymers. The polymers, which can interact with cells and cell debris electrostatically via their amine groups, also interact with HCPs and nucleic acids via mixed-mode (hydrophobic, aro- matic, and electrostatic) based interactions. The polymers tend to be “responsive” to divalent anions such as . As such, they offer the possibility to reduce cell debris as well as soluble contaminants [57]. Results were compared with those obtained using PEI, as well as PDADMAC (see the preceding). In general, the BPA polymers performed well; however, all three polymers allowed for good target yields (>90%). The authors noted that results improved as pH was lowered, although at pH < 6 residual polymer concentrations increased. When flocculation was performed at pH 6–7, using 2 0.1%–0.4% polymer and 10–40 mM phosphate, the treated feed (of approx. 50 NTU) offered filter loadings of <250 L/m . Filtrate turbidities of 1–3 NTU and a 1.2–2.0 fold decrease in HCP with significant (>5 log) DNA clearance. The authors concluded that the methodology achieved residual levels of impurities in the Protein A eluate that might allow for streamlin- ing of further purification steps [36].

11.3.4 Cell Flocculation Using EOPO Temperature-Responsive Polymer GE Healthcare has preliminarily investigated the use of “EOPO” polymers to effect primary clarification of high-density cell fermentation feeds [73]. The method contains aspects of both polymer-induced flocculation and liquid-liquid (L-L) interfacial . EOPO polymers are based on ethylene oxide (EO) and propylene oxide (PO). They have both hy- drophillic (EO) and hydrophobic (PO) components. Common EOPO polymers are Pluronic, Breox, Tergitol, and Ucon. They are available in a variety of molecular weights and EO/PO ratios and as block copolymer and copolymer formats, and some are produced to be compatible with food processing. They often exhibit thermal responsiveness and critical solution temperatures (Tc′s), whereby if the temperature increases above Tc, the polymers self-associate (i.e., become more hydro- phobic). Many of these polymers are used as antifoaming agents (e.g., in food processing) and others are used as surfactants in cell culture media formulation, or as stabilizers in biopharmaceutical formulations. They are often used at low concentra- tions, for example, <1% (w/w); however, when added to various at higher concentrations (e.g., 8%) at T > Tc they rapidly form a polymer-poor (<1%) phase floating on top of a denser polymer-rich phase [64,65,73] (Fig. 11.6). They are relatively inexpensive (similar in cost to PEG) and available in a variety of Tc′s covering temperatures of bioprocess inter- est, (i.e., 4–40°C). The polymer-rich phase excludes target proteins, of which >95% can often be found in the cell-depleted phase. The neutral charge nature of the polymers means that at T < Tc they tend to flow through chromatography columns. EOPO polymers possess the normal ability of EO polymers such as PEG to enhance flocculation (see the preceding). When they phase separate, the resulting phase droplet structures offer L-L interfaces—which can localize cells, organelles, and other colloids by interfacial tension. As such dense droplets sediment, it allows rapid transport of such debris to the bulk phase interface. The end result is that the polymers are able to rapidly clear cellular debris—even from feeds where cell volume exceeds 30% and where mAb target protein exceeds 30 g/L (Fig. 11.7). While an EOPO polymer of suitable Tc 228 SECTION | III Recovery Processes, Principles, and Methods

Increase in cell-free liquid feed volume over time 2500

2000

1500

phase (mL) 1000

Clarified phase and cell rich 500

0 0 10 20 30 40 50 60 Clarification time (min) FIG. 11.6 Result of adding 8% (w/w) Breox 50A1000 polymer (Cognis Corp.) to 2 L of CHO cell feed at 40°C. The volume of feed enriched in cells (solid line, square symbols) is rapidly reduced as the volume of cell depleted feed (dashed line, round symbols) increases—the ratio of these two volumes varies linearly with time. Data from GE Healthcare, used with permission.

95% mAb recovered in top phase

7 FIG. 11.7 Appearance of a cell-dense phase 18 min after adding 10% (w/w) Breox to a 10 L fermentation (2.4 × 10 cells/mL and 5 g/L mAb) in a 20 L Wave bioreactor at 40°C. The bioreactor container was placed vertical after polymer addition and mixing. Used with permission from GE Healthcare. may have to be chosen for a specific application, the method does not appear very sensitive to cell or protein concentration. In some cases >50 mM of a kosmotropic salt such as phosphate or citrate may need to be added to the solution to enhance performance. The method does not offer the HCP and DNA clarification of pH and conductivity optimized feeds contain- ing added polycationic polymers (see below). However, it works with a wide variety of targets (mAbs, Fabs, etc.) and feeds (CHO-cell, E coli, blood, and rMilk) and does not significantly increase solution conductivity [64,65,73]. Figs. 11.6 and 11.7 show results related to trials involving Breox clarification of high cell-density CHO fermentations. Clarification time varies with reactor solution height, not volume [73]. The partially clarified feed solution was readily depth filtered and sterile filtered before being applied to a Protein A step, which showed normal target recovery and concentration, as well as reduction in HCP, DNA, and other contaminants. HMW and dimeric mAb levels did not increase [3]. In studies performed by a major biopharmaceutical company, 7.5 L of 20% cell-dense feed was clarified using 8% Breox and added citrate with >95% of mAb target in the clarified aqueous solution. Target-containing solution matched that of the original feed, as the added polymer stock solution volume was similar to that of the aggre- gated cell-containing liquid region. A further 50 L scale up yielded only 90% of product (as the recovery method was not Alternative Separation Methods: Flocculation and Precipitation Chapter | 11 229

­optimized), but indicated a 10% reduction in HMW contaminants. A 1.8 dilution reduced target-containing phase turbidity to 47 NTU (similar to the case of pDADMAC noted previously) before further processing. Product quality did not appear to be affected [65].

11.4 PRECIPITATION AND FLOCCULATION OF TARGET 11.4.1 Ammonium Precipitation of Clarified NS0 Cell mAb Feed

Fig. 11.8 shows results by J. Glynn et al. that indicate what one might expect from simple ammonium sulfate (AmSO4) precipitation of a clarified NS0 cell feed containing mAb [23]. It can be seen that variations in pH from 6 to 7 and ammo- nium sulfate (AmSO4) concentration at 1.8–2 M can have significant effect; however, in the cases shown, the target yield in the pelleted precipitate is ~90%. In addition, there was a notable reduction in DNA (40–80 fold) and also in HCP (10–25 fold). Such results are promising as regards a possible to replace a chromatographic step; however, pellet re- suspension may require significant time, dilution, container costs, and result in additional target loss (for further discussion see the next section). Even so, if the pellet can be resuspended at high-target concentration in a buffer suitable for loading directly onto a follow-on column, the method may be attractive for some applications. It is included here to emphasize that impressive results can often be obtained with simple reagents. It should be noted, however, that the use of high-salt concentrations and other precipitants for large-volume processes may be regulated under the environmental legislation of the plant location.

11.4.2 Protein A Chromatography Versus Polyacid Target Precipitation in an mAb Process McDonald et al. investigated selective antibody precipitation using polyanions such as polyvinylsulfonic acid (PVS), poly- acrylic acid (PAA), and carboxymethyl (CM) dextran [48]. They found that under conditions of pH and conductivity similar to cation exchange, the polymers would selectively form target-rich precipitates. The concentration of precipitating polymer had to be stoichiometrically matched to the target protein concentration due to behavior such as that illustrated in Fig. 11.4A (see also [1,46,47,52]). In addition to the need to optimize pH, conductivity, and concentration when forming precipitate, there was also a need (much as in ion exchange) to alter pH, and conductivity via dilution when re-solubilizing target-containing precipitates. The latter could significantly add to process volumes. McDonald et al. performed a com- parison of six different mAb purification “processes” (Table 11.4), including classic, and well optimized, Protein A affinity followed by cation exchange (CEX) and then anion exchange chromatography (Process 1) and other processes where PVS (which we must assume was not as thoroughly optimized) was used as a possible replacement for either Protein A affinity chromatography (Process 4) or CEX. Table 11.4 indicates that the PVS offered 88% yield, 60% CHO cell protein (CHOP) reduction, and 99% DNA reduction. Protein A affinity chromatography which offered two logs better CHOP reduction and one log better DNA reduction. However, the PVS unit operation showed promise to possibly replace an ion exchange chro- matography step. In hindsight, it might have been good to also test using PVS prior to Protein A, as an IEX column replace- ment might be possible if the precipitation allowed for added benefits related to viral removal, transient target storage, etc.

AmSulfate precipitation of clarified NS0 cell mAb feed 100 pH 6, 1.8 M AmSO4 pH 6, 2.0 M AmSO 80 4 pH 7, 1.8 M AmSO4 pH 7, 2.0 M AmSO4 60

40 Relative results 20

N D 0 Yield in pellet DNA fold reduction HCP fold reduction FIG. 11.8 Ammonium sulfate precipitation of clarified NS0 cell mAb feed. Data from J. Glynn, Process scale precipitation of impurities in mammalian cell culture broth, in: U. Gottschalk (Ed.), Chapter 15 in Process Scale Purification of Antibodies, John Wiley and Sons, New York, 2009. 230 SECTION | III Recovery Processes, Principles, and Methods

TABLE 11.4 Comparing Protein A Chromatography and PVS-Based Precipitation

Step/Process 1 2 3 4 5 6

Step 1 PrA PrA PrA PVS pH 5 PVS pH 5 PrA

Step 2 SPFF PVS pH 7 QFF

Step 3 QFF QFF QFF SPFF

Results

Step 1 yield (%) 95 95 95 88 88 95

a b CHOP (ng/mg) <1 4 1 18 9200 1105 DNA (pg/mg) 0.2 5 1.3 4 4484c 198d

PrA L (ng/mg) <2 23 2 NA NA Aggregate (%) 0.8 0.8 0.8 0.8 1.8 0.8

Monomer (%) 99 99 99 99 96 99

Fragment (%) 0.2 0.2 0.2 0.2 0.2

a 60% CHOP reduction. b 99.5% CHOP reduction. c 99% DNA reduction. d >99.9% DNA reduction. In all cases clarified CHO cell feed was used. CHO, Chinese hamster ovary; NA, not applicable; PrA, ProSep-vA protein A chromatography resin; SPFF, SP Sepharose Fast Flow chromatography resin, QFF, Q Sepharose Fast Flow chromatography resin, PVS, polyvinylsulfonic acid; CHOP, CHO host cell proteins; PrA L, (leeched residual) Protein A ligand. Data from P. McDonald, C. Victa, J. N. Carter-Franklin, R. Fahrner, Selective antibody precipitation using polyelectrolytes: A novel approach to the purification of monoclonal antibodies, Biotechnol. Bioeng. 102 (2009) 1141–1151, US 20080193981 A1, US 20080193981 A1, used with permission.

11.4.3 Selective Precipitation of Polyclonal Ig by Polyacrylic Acid and Kosmotropic Salts The PAA precipitation results of MacDonald et al. are in agreement with those found earlier by several authors in regard to polyacid precipitation of other target proteins [1,46–48,51,52]. Shanagar, Van Alstine et al. [54,55,72] investigated the abil- ity of kosmotropic salts such as >50 mM NaCitrate or NaPhosphate to eliminate such challenges and enhance protein pre- cipitation by polyacrylic acid (PAA). PAA was chosen due to its relatively low cost and relative biocompatibility. In these studies, PAA was used in the NaPAA form and in all cases >50 mM of a kosmotropic salt such as NaPhosphate or NaCitrate was added. Addition of the kosmotropic salt promotes PAA association with the protein as well as self-interaction and thus alters the PAA precipitation behavior from that of Fig. 11.4A to that of Fig. 11.4B. The reason for this may be that PAA is insoluble at approximately pH 4 and so can be thought of as a pH-responsive polymer. The kosmotropic salt appears to enhance self-association of protein and polymer groups as well as polymer-protein complexes [54,55,72]. Fig. 11.9 shows results of a design of experiments (DOE) study where the effect of NaPAA 15000 and NaCitrate concentration were inves- tigated in regard to selective precipitation of polyclonal IgG and human serum albumin (HSA). It can be seen that at 12% PAA (w/w) and 100 mM NaCitrate, pH 7 and room temperature that >90% of the p-IgG precipitates, while 75% of the HSA remains in solution. The precipitates appear to hold protein at >100 g/L and therefore contain little polymer with most of the PAA remaining in the supernatant. The more target protein available, the more PAA that is precipitated with target. The kosmotropic salt-influenced Fig. 11.4B behavior means that, as with solvent precipitation, one process can be used with a variety of protein concentrations (it has been tested to 20 g/L IgG) and also with both mAb and antibody fragment (Fab) targets. The precipitated aggregates are mechanically stable and micron sized. Interestingly, the PAA-based fractionation appears complementary to the EOPO-based clarification method noted herein. In studies involving 10% (w/w) NaPAA 5000 added to clarified CHO feed, >90% mAb was found in the precipitate, while 95% of HCP and DNA were in the supernatant, which also contained most of the polymer. The “kosmotropic” methodology also worked with carboxymethyl dextran and some other polycarboxylic acids. The precipitates may provide some temporary target storage options [50]. Fig. 11.10 reflects the behavior expected from the Cohn Equation [74,75]. Figs. 11.9 and 11.10 suggest that it should be relatively easy to scout pH and conductivity conditions, allowing for even more impressive selectivity. The precipitates formed in the presence of the kosmotropic salt are readily resuspended at high protein concentration in a variety of standard affinity or ion exchange column loading buffers [54]. Ideally, the next step might be filtration on a Q membrane or AEX Alternative Separation Methods: Flocculation and Precipitation Chapter | 11 231

HSA in Solution Ig in precipitate 12 12 72.8 11 76.5 11 90 80.2 10 83.9 10 80

9 87.6 9 70 8 91.3 8 60 7 7 50 6 6 NaPAA 15000 (% w/w) 95 40 30 5 5 20 98.7 10 4 4 60 80 100 120 140 160 180 200 220 240 60 80 100120 140160 180200 220240 NaCitrate (mM) NaCitrate (mM) FIG. 11.9 Comparision of effect of varying NaPolyacrylate 15000 polymer concentration (%, w/w) and NaCitrate salt (mM) at pH 7 and RT on the percentage of human serum albumin remaining in solution and the percentage of polyclonal human immunoglobulin (GammaNorm) precipitating. Data from GE Healthcare, used with permission.

3

2 Ln K 1

0 0 510152025 Conductivity (mS/cm) FIG. 11.10 Plot of K (ratio of polyclonal IgG precipitated to IgG not precipitated) and Ln K versus conductivity (mS/cm) for 10% NaPAA 8000 and added salt (100 mM NaPhosphate and NaCl) related experiments at RT and pH 7, at an IgG concentration 5 g/L. Conductivity does not include the ~40 mS/cm from the NaPAA [46]. Data from GE Healthcare, used with permission. hybrid filter such as Emphaze. The relatively low MW of the PAA reduces its effects on solution viscosity and also makes it easier to clear residual PAA. Follow-on use of Protein A or CEX or mixed-mode (e.g., Capto ImpRes MMC) would allow for flow-through of residual polymer. Protein A affinity chromatograpy would also be an option as it appears particularly good at clearing precipitating polymers (e.g., [36]).

11.4.4 Sequential Precipitation of Protein Mixture Components With NaPAA Plasma is the source of many important biotherapeutic products such as albumin, immunoglobulins, coagulation factors, and inhibitors [76]. Every year about 500 metric tons of human serum albumin (HSA) are produced from pooled human donated blood [77]. The initial separation of HSA and other valuable plasma proteins is often carried out by a sequence of increasing ethanol (15%–40% v/v)-based precipitation steps carried out (on plasma initially cryoprecipitated to recover Factor VIII) at varied pH and subzero temperatures in operations relatively unchanged from the cold ethanol fractionation (CEF) developed by E. J. Cohn et al., over 70 years ago [74,75]. The market value of this HSA is ~3–5$/g [10], underscor- ing the ability of precipitation to help deliver low cost-of-goods products. The requirement for subzero temperatures and ethanol at high concentration is a major disadvantage of these procedures. These solvent-rich, low- temperature processes require very large stainless steel tank-based facilities. As such, they are less amenable to flexible manufacturing. Based on earlier GE Healthcare research [55], GE Healthcare and CLS-Behring have undertaken studies to evalute if the NaPAA kosmotropic salt precipitation method has potential as an alternative to cold ethanol fractionation of plasma pro- teins (CEF), especially given that the precipitation steps can be conducted at 4°C or room temperature [50]. Polyacrylic acid (NaPAA 8000) was added with 50 mM NaCl and 50 mM Na3Citrate to cryoprecipitated plasma at 7%, 12%, and 20% (w/w) to sequentially precipitate fibrinogen-, immunoglobulin- and albumin-rich fractions at target yields of >80% at 5°C (Fig. 11.11). 232 SECTION | III Recovery Processes, Principles, and Methods

The precipitates were readily recovered by (4700 rpm, 10 min, 4°C) centrifugation. Thirteen plasma proteins were quan- tified by ELISA and other methods during processing (Fig. 11.12). Fibrinogen (64% w/w), IgG (14%) and IgM (3%) were major constituents of the 7% PAA pellet. Major constituents of the 12% PAA pellet were IgG (55%) and IgA (8%) (Fig. 11.12). The 20% pellet contained albumin (82%), transferrin (7%,) and α1-antitrypsin (3%). PAA-based protein fractions were comparable in yield and purity to those obtained from Cohn-type fractionation, and amenable to similar using commercially established methods [50]. The IgG-rich precipitate could be further processed using caprylic acid (OA) precipitation at 14 g/kg of suspension followed by anion exchange polish- ing to a purity of >99% (Fig. 11.12). In addition to enrichment of the IgG, the OA treatment resulted in enrichment of the

Component % of total Plasma Albumin 56.6 IgG 16.4 Fibrinogen 5.3

7% NaPAA 7% PAA pellet Result (resolubilised) % Fibrinogen purity 64 Fibrinogen recovery 81 Pellet (fibrinogen) Supernatant

12% NaPAA 127% PAA pellet Result (resolubilised) % lgG purity 55 lgG recovery 89 Pellet (lgG)Supernatant

20% NaPAA 20% PAA pellet Result (resolubilised) % Albumin purity 82 Albumin rocovery 81 Pellet (lgG)Supernatant

FIG. 11.11 Flow chart of trial fractionation of cryoprecipitated plasma using polyacrylic acid at sequential concentrations of 7%, 12%, and 20% (w/w). From K.B. McCann, J. Van Alstine, J. Martinez, J. Shanagar, J.Bertolini, Fractionation of IgG and Human Serum Albumin From Plasma Using Polyacrylic Acid.Manuscript to be submitted (2018).

100 90 IgG IgA 80 Albumin 70 α2-macroglobulin 60 Apolipoprotein B 50 Fibrinogen 40 Haptoglobin

% Composition 30 Other proteins* 20 10 0 12% PAA precipitate OA treated lgG IEX purified lgG

(* Transferrin, ceruloplasmin, apolipoprotein A1, α1-glycoprotein, α1-antitrypsin) FIG. 11.12 Composition of the 12% NaPAA precipitated IgG-rich fraction, the same fraction following treatment with caprylic acid (octanoic acid, OC), and ion exchange which increased IgG purity levels to >99% (see text). Data from author-submitted manuscript K.B. McCann, J. Van Alstine, J. Martinez, J. Bertolini, Fractionation of IgG and Human Serum Albumin From Plasma Using Polyacrylic Acid. Manuscript to be submitted to Biotechnol. Bioengineering (2017). Alternative Separation Methods: Flocculation and Precipitation Chapter | 11 233

other immunoglobulins, IgA and IgM. The major impurities, including albumin, α2-macroglobulin, apolipoprotein B, and fibrinogen were significantly depleted by the OA precipitation. The albumin-rich precipitate could be further processed via two-column bind and elute ion exchange as is sometimes used in industry to yield a final product with a purity of >99%. The authors believe it should be easy to improve target yields and purities, which already rival those of CEF [50].

11.5 EXAMPLES OF CONTAMINANT PRECIPITATION In general, contaminant precipitation might be undertaken to reduce (fouling) contaminant load on downstream processing columns, and if possible, reduce one column in a multicolumn process.

11.5.1 Contaminant Precipitation With Polyelectrolytes The advantage of contaminant precipitation is that it allows a target to stay in solution, often at high concentration, and be filtered and then subjected to chromatography or another follow-on operation. Many of the polycationic polymers noted herein in regard to clarification of negatively-charged cells and cell debris also demonstrated interaction with host cell proteins (HCPs) and nucleic acids, and as such offered significant HCP and DNA reduction (Table 11.3). This includes pDADMAC, polyamines, chitosan, and polyacrylamides. Such compounds should also interact electrostatically with (non-HCP) process contaminants such as insulin (Table 11.2). Peram et al. [43] and Ma et al. [45] screened various processes where polyamine precipitation was used for removing HCP impurities from mAb cell culture fluid, and tested incorporation of polyamine pre- cipitation into a downstream process to replace protein A. Results were similar to those shown by MacDonald et al. in regard to a study involving polyanionic polymers [48]. Poly(arginine) was favorably evaluated as possible flocculant to both reduce cell and cell debris as well as replace a anion exchange step in antibody purification. The reagent worked well; however, its relatively high cost meant that 100 L scale-up studies, involving centrifugal clarification of polymer treated cell culture fluid (CCF) were performed using less-expensive poly-(vinylamine) (PVA). Although the mammalian CCF was not very turbid (NTU 40), the PVA appeared to perform well, resulting in a 95% mAb yield, 84% HCP reduction, >2 log DNA clearance, and a turbidity reduction to 3 NTU. Peram et al., noted that, “residual solids tend to stick to the walls of the and further optimization is required to improve the pellet consistency to ensure simple and efficient centrifuge cleaning. Alternatively, depth filtration may possibly be used for the capture of the flocculated cell debris and precipitated impurities.” [43].

11.5.2 Impurity Precipitation With Caprylic Acid An example of results related to caprylic (octanoic) acid precipitation of a mixture of plasma proteins enriched (by PAA precipitation) in IgG is given in Fig. 11.12 [50]. Caprylic acid has been studied and used in research and the plasma frac- tionation industry for some time and over the past decade has been studied in regard to mAb bioprocessing [23,29–33]. One reason for this is the ability of caprylic acid to inactivate enveloped viruses [32,78]. The solubility of caprylic acid is ~0.7 g/L (5 mM); however, it can be added as a suspension to a protein mixture and will interact with proteins in solution (lowering its solution concentration) and promoting protein precipitation. As expected, its solubility is affected by pH as it influences the charge state of its carboxylic acid group (pKa approx. 5). OC exhibits less tendency to interact with mAbs and related proteins, so can be used to reduce contaminant proteins in plasma Ig preparations (Fig. 11.12) or mAb feeds. Its use would appear to be limited to antibodies or related proteins [33]. One drawback to its use is that the optimal amount of OC used may vary with the target, target concentration, and pH (which tends to be fixed in plasma processing). In addition, OC-induced contaminant precipitates may have a density <1, and residual OC may have toxicity issues [4,17,32]. Reagent cost may also be an issue, although as noted it is used in plasma protein processing. As indicated by Fig. 11.12, OC can effect a significant reduction in contaminant proteins. In regard to mAb processing, Brodsky et al. [32] reported a 90% reduction in HCPs. Judy Glynn studied effects of OC concentration (30–500 mM) at pH (5–7) for both NS0- and CHO-cell produced mAbs [23]. For CHO-produced mAb preparations, both HCP and DNA were reduced at pH 5–7, with HCP clearance increasing with OC concentration. However, NS0-produced mAb prepara- tions showed good DNA clearance at the lower pH, but little effective HCP clearance under the conditions studied. Glynn reported CHO-cell related mAb yields of >90% with 500 mM OC at pH 6 also yielding a 650-fold reduction in HCP (to 160 ng/mg Ab) and 6500 fold reduction in DNA to 700 pg/mg Ab (compare these numbers with Tables 11.3 and 11.4) [23]. At OC concentrations above 300 mM, the precipitation yields a three-phase system with a pellet of density >1, an ­aqueous supernatant-containing target, and a fluffy precipitate (density <1) on top. Having to separate such a complex mixture would be challenging. The cost of OC reagent (Glynn noted $4 a gram) suggests use of OC to replace an affinity chroma- tography step may not be cost effective unless the OC reagent also allows for streamlining of a viral removal step. 234 SECTION | III Recovery Processes, Principles, and Methods

11.6 SEQUENTIAL AND CONTINUOUS PRECIPITATION 11.6.1 Sequential Precipitation An example of sequential precipitation is given in Section 11.4.4. It should be appreciated that it will be much easier to ef- fect sequential precipitation if the concentration of the reagent used does not have to be matched to the precipitation target (products or contaminants) during each step. As a result, a choice of reagent and conditions (as in Section 11.4.4) to pro- mote the behavior noted in Fig. 11.4B are to be desired. This possibly includes osmotic agents such as PEG as well as the use of ethanol and pH close to the target pI originally advocated by Cohn et al. [74,75]. It is interesting to note that at larger scales, such as in plasma fractionation, sequential, but batch, operations may be preferable for some applications [77] as they offer some timing flexibility. Combining selective precipitation with controlled cross-flow membrane microfiltration has been noted by Belfort et al. and may also be of interest [79].

11.6.2 Batch Versus Continuous Precipitation

Jaquez, Gronke, and Przybycien presented a scalable, continuous precipitation process using PEG and ZnCl2 precipitation of monoclonal antibodies [80]. A process optimized in stirred batch configuration at low target titer did not scale well, in terms of precipitate yield, morphology, and particle size distribution when target concentration exceeded 2 g/L. Nor did it readily lend itself to continuous disc stack centrifugation. They therefore developed a continuous precipitation process using static mixers with in-line addition of reagents. This resulted in predictable performance. The methodology was inte- grated into a purification process involving precipitate generation, isolation, storage, and further target purification via two flow-through chromatography steps. Jungbauer et al. [21,22] have experimented with use of cold ethanol fractionation (CEF) and CaCl2-based precipitation for the purification of antibodies (and related proteins) from CHO cell supernatant. Hammerschmidt et al. [22] presented continuous CEF using a tubular bioreactor. The continuous mode performed in a manner similar to batch mode experi- ments. Results were compared in a detailed analysis with those expected from Protein A chromatography (see below). Similar methodology has been applied by the same group to IgM purification with a two-step precipitation and IEX process yielding target purities of 95% and up to 85% yield [81]. The economics of the process have been evaluated and are noted in Section 11.7 [82] (Table 11.5).

TABLE 11.5 High Titer Precipitation in Batch and Continuous Modesa

Sample Result Batch Continuous

Clarified cell culture IgG conc (g/L) 7.76 7.76 supernatant DNA (ppm) 359 359

HCP (ppm) 42777 42777

CaCl2 flocculation IgG yield (%) 95.4 ± 0.9 97.3 ± 0.7

DNA (ppm) 6 ± 1 7 ± 0

HCP (ppm) 21842 ± 2106 23143 ± 1509

Cold ethanol precipitation IgG yield (%) 94.7 ± 0.7 94.0 ± 4.0

HMW (%) 0.58 ± 0.02 0.52 ± 0.01

HCP (ppm) 9626 ± 530 8449 ± 2650

Overall Yield (%) 90.3 ± 0.5 91.5 ± 1.9 HCP reduction (fold) 4.4 5.1

DNA reduction (fold) 59.8 51.3

a Average ± standard deviation of triplicate analyses. Low titer (2.4 g/L) feed results matched those shown. Data from N. Hammerschmidt, B.N. Hintersteiner, A. Jungbauer, Continuous precipitation of IgG from CHO cell culture supernatant in a tubular reactor, Biotechnol. J. 10 (2015) 1196–1205, doi:10.1002/biot.201400608, used with permission. Alternative Separation Methods: Flocculation and Precipitation Chapter | 11 235

11.7 PROCESS ECONOMICS NOTES 11.7.1 Introduction The preceding chapter presented some comments in regard to process economic studies as applied to alternative separa- tions. In general, one of the great benefits of chromatography is the ability to amortize resin and related apparatus costs over many runs. While it is difficult to draw a general conclusion on economic benefits of one technology over the other, it can be noted that any economic analysis needs to account for a cost of all the preceding and subsequent steps, through yield and purification cost ($/g impurities), respectively.

11.7.2 Simple Cost of Goods Comparison of Caprylic Acid and Protein A A simple comparison of using caprylic acid precipitation instead of Protein A chromatography was presented in 2009 by Judy Glynn of Pfizer [23]. The key assumptions included a 20,000 L reactor with mAb target at 2 g/L and a Protein A resin with DBC of 30 g/L. At three cycles per purification run, the chromatography bed would be 1.8 m in diameter, with a bed height of 18 cm and a bed volume of 460 L. In this comparison, Protein A chromatography seemed more cost effective (i.e., assuming 90% recovery Protein A use cost 3$/g of mAb, whereas caprylic acid costs $4–$10). However, the compari- son only looked at basic reagent costs and did not take into account resin cleaning in place (CIP), buffer usage related to chromatography versus precipitation, residual caprylic acid removal, QC, and other costs. In addition, the conditions were related to reducing HCP and DNA, but not necessarily to viral load or high molecular weight target reduction. Today the caprylic acid step might be done using single-use containers at possibly reduced reagent cost/L, and modern Protein A resin (at ~1.5× the cost noted in the table) might offer more than double the dynamic binding capacity, reduced column bed vol- umes and buffer usage, faster cycle times, reduced CIP costs and lifetimes of >100 cycles (see the following). We provide Table 11.6 as it is illustrative that a simple analysis can provide insight when considering alternative separation methods. For other comparative examples see Ref. [82−84].

11.7.3 Precipitation Versus Protein A—Hammerschmidt, Jungbauer Hammerschmidt et al. [82] studied the economics of recombinant antibody production processes at various scales compar- ing industry-standard Protein A affinity chromatography-based processing compared with continuous precipitation involv- ing a series of precipitation steps—caprylic acid, followed by PEG-based precipitation, followed by CaCl2 flocculation, followed by cold ethanol fractionation. Several interesting conclusions came from this work. Ongoing from the 2 to 10 g/L bioreactor feed, the downstream processing costs became a more significant fraction of the total CoGs of bulk drug sub- stance, which they estimated at ~300 USD/g. If the Protein A lifecycle increased from 100 to 200 cycles, the CoGs only decreased by 8% (Table 11.7). Increasing affinity resin capacity from 50 to 60 g/L only decreased CoGs 6% (~18$/g—roughly in keeping with Franzreb et al. [84]). The precipitation-based process eliminated three chromatography operations and was amenable to continuous processing. Their analysis suggested that the precipitation-based process could be cost competitive in clinical phases and in full commercial production. Perhaps more importantly, and in keeping with plasma fractionation [50], savings were also possible if a “hybrid” chromatography and precipitation process was used.

TABLE 11.6 Protein A Chromatography Vs Caprylic Acid Precipitation

Lifetime Reagent Total Volume Costa Raw Material (Cycles) Cost ($/L) Required (L) ($/lot)

rProtein A Sepharose Fast Flow (GE Healthcare) 100 7770 460 per lifecycle 108,000

Caprylic Acid 500 mM n/a 250 1440 per lot 360,000

200 mM n/a 250 577 per lot 144,000

a Assuming 90% recovery (36,000 g/lot) costs per g of mAb vary from $3 to $10. This analysis assumed a then state of the art Protein A resin which offered low loads (see text). Data from J. Glynn, Process scale precipitation of impurities in mammalian cell culturebroth, in: U. Gottschalk (Ed.), Chapter 15 in Process Scale Purification of Antibodies, JohnWiley and Sons, New York, 2009. 236 SECTION | III Recovery Processes, Principles, and Methods

TABLE 11.7 Percent Change in CoGs Over Base Case Purification Process Costs

Fed-Batch + Chromatography-Based DSP % Δ Base Case 1-Reactor % Δ Base Case 6-Reactors Protein A lifespan reduced to 50 cycles +15 +14

Protein A lifespan increased to 200 cycles −8 −9

Protein A capacity increased to 60 g/L −6 −8 a Protein A lifespan and capacity: combined increase −14 −17

AEX bind-elute step: capacity reduced to 50 g/L +3 +2

Fed-batch + Precipitation Based DSP % Δ Base Case 1-Reactor % Δ Base Case 6-Reactors Caprylic acid price doubled to 182 US$/L +2 +2 +3

PEG price doubled to 6 US$/L +4 +4 +4

CaCl2 price doubled to 62 US$/kg +2 +2 +2

Ethanol price doubled to 2.8 US$/L +1 +1 +2

Filter capacity for precipitate recovery halved +4 +4 +3

Facility staff doubled (40/20/10/10) +22 +22 +21

a 200 cycles and 60 g/L are achievable with modern resins. Redrawn from Table 3 in N. Hammerschmidt, A. Tscheliessnig, R. Sommer, B. Helk, A. Jungbauer, Economicsof recombinant antibody production processes at various scales: Industry-standard compared to continuous precipitation, Biotechnol. J. 9 (2014) 766–775, doi: 10.1002/biot.201300480, used with permission.

11.8 CONCLUSIONS This chapter only provides a brief overview of precipitation and flocculation and how it might be used to clarify cell- containing culture media, isolate target, or remove contaminants. In many cases, several operations can be performed by the same alternative unit operation. As noted in the references supplied, and the summary in Table 11.3, there is now a very large body of theory, methods, and experience on how to use such methods in bioprocessing. Classical methods pioneered by scientists such as Hofmeister improved by others such as Cohn et al. [74,75] were modified and expanded by scientists such as Sternberg and Hershberger [52] and Glatz et al. [46]. This work has continued both in regard to practical studies related to solving real problems in bioprocessing, as well as in developing better methods to screen conditions and model the results. Theoretical understanding is also increasing, in part due to the wide range of protein structures that have been solved and our understanding of protein-polymer, protein-ligand, protein-surface, and protein-ion interactions [1,59,72,83–87]. Precipitation and flocculation by a growing range of available polymeric biocompatible reagents completes the opportunity. In addition to modern studies such as those of Hammerschmidt (see the preceding) and McCann et al. [50], many historical examples from the plasma and industrial enzyme industries indicate the feasibility of using these methods in bioprocessing, and their ease of optimization [88–90]. Can they effectively replace an affinity capture step such as Protein A, which has been proven in hundreds of scaled processes to effectively capture, concentrate, and purify its target in a cost-effective manner? In our opinion—probably not. However they can be used together with chromatography in hybrid processes, particularly up- stream of chromatography to handle the high cell mass and contaminant loads that industry now faces. They are well enough understood that they can be chosen to solve specific problems, such as chromatin or DNA [27,91,92], or otherwise provide enhanced return on investment by extending filter and column performance life, reducing viral loads, etc.

ACKNOWLEDGMENTS, NOTICES, AND DISCLAIMERS The authors work for, or have previously worked for, General Electric Healthcare. Bioprocessing operations in general, and alternative separation technologies in particular, have enjoyed active patenting, and the reader is advised to undertake some related “due diligence” before embarking on using any particular technology for commercial purposes. The authors would like to thank Karl McCann of CSL-Behring for discussion. ProSep-vA and Clarisolve are trademark of EMD-Millipore. Emphaze is a trademark of 3M Corporation. Sepharose is a trademark of GE Healthcare, GammaNorm is a trademark of OctaPharma. MacroPrep is a trademark of BioRad Laboratories. Pluronic is tradename of BASF Corp., Breox is a tradename of Cognis, Tergitoland Ucon are tradenames of Dow Chemical Corp. Alternative Separation Methods: Flocculation and Precipitation Chapter | 11 237

REFERENCES [1] F. Carlsson, P. Linse, M. Malmsten, Monte Carlo simulations of polyelectrolyte–protein complexation, J. Phys. Chem. B 105 (2001) 12189–12195. [2] pDADMAC flocculant reagent for use with Clarisolve® depth filters, MerckMillipore Application Note Lit # AN33330000 Rev. C PS-14-09928 08/2014, © 2014 EMD Millipore Corporation, Billerica, MA USA. [3] J. Shanagar, R. Hjorth, B. Bengt Westerlund, J.M. Van Alstine, GE Healthcare Poster, Polymer Aided Bioprocessing. Recovery of Biological Products XIV, Lake Tahoe, Nevada, August 2010. [4] N. Singh, A. Arunkumar, S. Chollangi, Z.G. Tan, M. Borys, Z.J. Li, Clarification technologies for monoclonal antibody manufacturing processes: current state and future perspectives. Biotechnol. Bioeng. 113 (2016) 698–716, https://doi.org/10.1002/bit.25810. [5] U. Gottschalk, Process Scale Purification of Antibodies, John Wiley and Sons, NY, ISBN: 978-0-470-20962-2, 2017. [6] L.S. Hanna, P. Pine, G. Reuzinsky, S. Nigan, D.R. Omstead, Removing specific cell culture contaminants in a Mab purification process, BioPharm. Int. 9 (1991) 33–47. [7] F. Chiod, Å. Sidén, E. Ösby, Isoelectric focusing of monoclonal immunoglobulin G, A and M followed by detection with the avidin-biotin system, Electrophoresis 6 (1985) 124–128. [8] D.G. Bracewell, R. Francis, C.M. Smales, The future of host cell protein (HCP) identification during process development and manufacturing linked to a risk-based management for their control. Biotechnol. Bioeng. 112 (2015) 1727–1737, https://doi.org/10.1002/bit.25628. [9] D. Blankenhorn, J. Phillips, J.L. Slonczewski, Acid- and base-induced proteins during aerobic and anaerobic growth of Escherichia coli revealed by two-dimensional gel electrophoresis, J. Bacteriol. 181 (1999) 2209–2216. [10] S. Moghaddassi, W. Eyestone, C.E. Bishop, The large-scale production of recombinant human serum albumin in the milk of transgenic cattle: strat- egy & implications, J. Sci. Appl. Biomed. 2 (2014) 6–19. http://inter-use.com/uploads/soft/140320/1-140320205040.pdf. [11] D. Low, R. O’Leary, N.S. Pujar, Future of antibody purification. J. Chrom. B 848 (2007) 48–63, https://doi.org/10.1016/j.jchromb.2006.10.033. [12] B. Kelley, Industrialization of mAb production technology: the bioprocessing industry at a crossroads, MAbs 1 (2009) 443–452. [13] J. Thömmes, M. Etzel, Alternatives to chromatographic separations. Biotechnol. Prog. 23 (2007) 42–45, https://doi.org/10.1021/bp0603661. [14] D.J. Roush, Y. Lu, Advances in primary recovery: centrifugation and membrane technology, Biotechnol. Prog. 24 (2008) 488–495. [15] H.F. Liu, J. Ma, C. Winter, R. Bayer, Recovery and purification process development for monoclonal antibody production. MAbs 2 (2010) 480–499, https://doi.org/10.4161/mabs.2.512645. [16] T.P. O’Brien, L.A. Brown, D.G. Battersby, A.S. Rudolph, L.P. Raman, Large-scale, single-use depth filtration systems for mammalian cell culture clarification, BioProcess Int. 10 (5) (2012) 50–57. [17] R. Shpritzer, S. Vicik, S. Orlando, H. Acharya, J. Coffman, Calcium phosphate flocculation of antibody-producing mammalian cells at pilot scale. 232nd American Chemical Society National Meeting, Sept 10-14, 2006, San Francisco, CA, BIOT division, paper 80. See also US Patent US7855280 B2. [18] M. Westoby, J. Chrostowski, P. de Vilmorin, J.P. Smelko, J.K. Romero, Effects of solution environment on mammalian cell fermentation broth properties: enhanced impurity removal and clarification performance. Biotechnol. Bioeng. 108 (2011) 50–58, https://doi.org/10.1002/bit.22923. [19] S. Hove, B. Cacace, M. Felo, K. Chefer, Development of a robust clarification process for high density mammalian cell culture processes, in: Presented at: Recovery of Biological Products XIV. CA, USA, 1–6 August, 2010. [20] B. Lydersen, T. Brehm-Gibson, A. Murel, Acid precipitation of mammalian cell fermentation broth, Ann. N. Y. Acad. Sci. 745 (1994) 222–231. [21] A. Tscheliessnig, P. Satzer, N. Hammerschmidt, H. Schulz, B. Helk, A. Jungbauer, Ethanol precipitation for purification of recombinant antibodies, J. Biotechnol. 188 (2014) 17–28. [22] N. Hammerschmidt, B.N. Hintersteiner, A. Jungbauer, Continuous precipitation of IgG from CHO cell culture supernatant in a tubular reactor. Biotechnol. J. 10 (2015) 1196–1205, https://doi.org/10.1002/biot.201400608. [23] J. Glynn, Process scale precipitation of impurities in mammalian cell culture broth, in: U. Gottschalk (Ed.), Chapter 15 in Process Scale Purification of Antibodies, John Wiley and Sons, New York, 2009. [24] J. Romero, J. Chrostowski, P. De Vilmorin, J. Case, US 2010/0145022 A1. (2010/0145022 A1), 2010. [25] G. Giese, A. Myrold, J. Gorrell, J. Persson, Purification of antibodies by precipitating impurities using Polyethylene Glycol to enable a two chroma- tography step process, J. Chromatogr. B 2013 (938) (2013) 14–21. [26] A. Arunakumari, G.M. Ferreira, Protein Purification by Citrate Precipitation, US Patent 8,063,189 B2, 2011. [27] R. Sommer, P. Satzer, A. Tscheliessnig, H. Schulz, B. Helk, A. Jungbauer, Combined polyethylene glycol and CaCl2 precipitation for the capture and purification of recombinant antibodies, Process Biochem. 49 (2014) 2001–2009. [28] M. Page, R. Thorpe, Purification of IgG by precipitation with PEG, in: J. Walker (Ed.), The Protein Protocols Handbook, Humana Press, New York, 2002, pp. 991. [29] M. Mckinney, A. Parkinson, A simple, non-chromatographic procedure to purify immunoglobulins from serum and ascites fluid, J. Immunol. Methods 96 (1987) 271–278. [30] C. Russo, L. Callegaro, E. Lanza, S. Ferrone, Purification of IgG monoclonal antibody by caprylic acid precipitation, J. Immunol. Methods 65 (1983) 269–271. [31] A. Johnston, E. Uren, D. Johnstone, J. Wu, Low pH, caprylate incubation as a second viral inactivation step in the manufacture of albumin. Parametric and validation studies, Biologicals 31 (2003) 213–221. [32] Y. Brodsky, C. Zhang, Y. Yigzaw, G. Vedantham, Caprylic acid precipitation method for impurity reduction: an alternative to conventional chroma- tography for monoclonal antibody purification, Biotechnol. Bioeng. 109 (2012) 2589–2598. 238 SECTION | III Recovery Processes, Principles, and Methods

[33] S. Herzer, A. Bhangale, G. Barker, I. Chowdhary, M. Conover, B.W. O’Mara, L. Tsang, S.-Y. Wang, S.R. Krystek, Y. Yao, Development and scale- up of the recovery and purification of a domain antibody Fc fusion protein-comparison of a two and three-step approach, Biotechnol. Bioeng. 2015 (112) (2015) 1417–1428. [34] F. Riske, J. Schroeder, J. Belliveau, X. Kang, et al., The use of chitosan as a flocculant in mammalian cell culture dramatically improves clarification throughput without adversely impacting monoclonal antibody recovery, J. Biotechnol. 128 (2007) 813–823. [35] S. Tomic, L. Besnard, B. Furst, R. Reithmeier, R. Wichmann, P. Schelling, C. Hakemeyer, Complete clarification solution for processing high den- sity cell culture harvests. Sep. Purif. Technol. 141 (2015) 269–275, https://doi.org/10.1016/j.seppur.2014.12.002. [36] Y.K. Kang, J. Hamzik, M. Felo, B. Qi, J. Lee, S. Ng, G. Liebisch, B. Shanehsaz, N. Singh, K. Persaud, D.L. Ludwig, P. Balderes, Development of a novel and efficient cell culture flocculation process using a stimulus responsive polymer to streamline antibody purification processes. Biotechnol. Bioeng. 110 (2013) 2928–2937, https://doi.org/10.1002/bit.24969. [37] J. Shan, J. Xia, Y. Guo, X. Zhang, Flocculation of cell, cell debris and soluble protein with methacryloyloxyethyl trimethylammonium chloride–­ acrylonitrile copolymer, J. Biotechnol. 49 (1996) 173–178. [38] S. Moghimi, P. Symonds, J. Murray, A. Hunter, G. Debska, A. Szewczyk, A two-stage poly(ethylenimine)-mediated cytotoxicity: implications for gene transfer/therapy, Mol. Ther. 11 (2005) 990–995. [39] D. Fischer, Y. Li, B. Ahlemeyer, J. Krieglstein, T. Kissel, In vitro cytotoxicity testing of polycations: influence of polymer structure on cell viability and hemolysis, Biomaterials 24 (2003) 1121–1131. [40] D. Salt, S. Hay, O. Thomas, M. Hoare, P. Dunnill, Selective flocculation of cellular contaminants from soluble proteins using polyethyleneimine: a study of several organisms and polymer molecular weights, Enzyme Microb. Technol. 17 (1995) 7–12. [41] N. Singh, K. Pizzelli, J. Romero, et al., Clarification of recombinant proteins from high cell density mammalian cell culture systems using new improved depth filters, Biotechnol. Bioeng. 110 (2013) 1964–1972. [42] T. Kean, M. Thanou, Biodegradation, biodistribution and toxicity of chitosan, Adv. Drug Deliv. Rev. 62 (2010) 3–11. [43] T. Peram, P. Mcdonald, J. Carter-Franklin, R. Fahrner, Monoclonal antibody purification using cationic polyelectrolytes: an alternative to column chromatography, Biotechnol. Prog. 26 (2010) 1322–1331. [44] J. Aunins, D. Wang, Induced flocculation of animal cells in suspension culture, Biotechnol. Bioeng. 34 (1989) 629–638. [45] J. Ma, H. Hoang, T. Myint, T. Peram, R. Fahrner, J.H. Chou, Using precipitation by polyamines as an alternative to chromatographic separation in antibody purification processes, J. Chromatogr. B. 878 (2010) 798–806. [46] K.M. Clark, C.E. Glatz, Polymer dosage considerations in polyelectrolyte precipitation of protein, Biotechnol. Prog. 3 (1987) 241–247. [47] C.L. Cooper, P.L. Dubin, A.B. Kayitmazer, S. Turksen, Polyelectrolyte-protein complexes, Curr. Opin. Colloid Interface Sci. 10 (2005) 52–78. [48] P. McDonald, C. Victa, J.N. Carter-Franklin, R. Fahrner, Selective antibody precipitation using polyelectrolytes: a novel approach to the purification of monoclonal antibodies, Biotechnol. Bioeng. 102 (2009) 1141–1151. US 20080193981 A1US 20080193981 A1. [49] J. Van Alstine, M. Berg, J. Kjorning, J. Shanagar, Plasma Protein Fractionation by Sequential Polyacid Precipitation. Patents US 20140343253A1 and WO2013039449A1. [50] K.B. McCann, J. Van Alstine, J. Martinez, J. Shanagar, J. Bertolini, Fractionation of IgG and Human Serum Albumin From Plasma Using Polyacrylic Acid. Manuscript to be submitted to Biotechnol. Bioengineering (2018). [51] R.R. Fisher, Polyelectrolyte precipitation of proteins: II. Models of the particle size distributions, Biotechnol. Bioeng. 32 (1988) 786–796. [52] M. Sternberg, D. Hershberger, Separation of proteins with polyacrylic acids, Biochim. Biophys. Acta 342 (1974) 195–206. [53] Th. Wieland von, H. Goldmann, W. Kern, H.E. Schultze, H.D. Matheka, Versuche zur fraktionierung von proteingemischen mit polyacrylsäure, Die Makromolekulare Chemie 10 (1953) 136–146. [54] J. Van Alstine, J. Shanagar, R. Hjorth, K. Lacki, Precipitation of Biomolecules With Negatively Charged Polymers, WO2010082894A1. [55] J.M. Van Alstine, M. Berg, J. Kjörning, J. Shanagar, Plasma Protein Fractionation By Sequential Polyacid Precipitation. WO2013039449A1. [56] V.A. Izumrudov, I. Galaev, B. Mattiasson, Polycomplexes-potential for bioseparation, Bioseparation 7 (1998) 207–220. [57] J. Jaber, W. Moya, J. Hamzik, A. Boudif, Y. Zhang, N. Soice, et al., Stimulus Responsive Polymers for the Purification of Biomolecules Patent US 8691918 B2, 2011. [58] F. Capito, J. Bauer, A. Rapp, C. Schroter, H. Kolmar, B. Stanislawski, Feasibility study of semi-selective protein precipitation with salt-tolerant copolymers for industrial purification of therapeutic antibodies, Biotechnol. Bioeng. 110 (2013) 2915–2927. [59] S.M. Cramer, M. Holstein, Downstream processing: recent advances and future promise. Curr. Opin. Biotechnol. 1 (2011) 27–37, https://doi. org/10.1016/j.coche.2011.08.008. [60] B.A. Fong, A.R. Gillies, I. Ghazi, G. LeRoy, K.C. Lee, L.F. Westblade, D.W. Wood, Purification of Escherichia coli RNA polymerase using a self- cleaving elastin-like polypeptide tag. Protein Sci. 19 (2010) 1243–1252, https://doi.org/10.1002/pro.403. [61] R.D. Sheth, M. Jin, B.V. Bhut, Z. Li, W. Chen, S.M. Cramer, Affinity precipitation of a monoclonal antibody from an industrial harvest feed-stock using an ELP-Z stimuli responsive biopolymer, Biotechnol. Bioeng. 111 (2014) 1595–1603. [62] D.E. Meyer, A. Chilkoti, Purification of recombinant proteins by fusion with thermally-responsive polypeptides, Nat. Biotechnol. 17 (1999) 1112–1115. [63] T. Kowalczyk, K. Hnatuszko-Konka, A. Gerszberg, A.K. Kononowicz, Elastin-like polypeptides as a promising family of genetically-engineered protein based polymers. World J. Microbiol. Biotechnol. 30 (2014) 2141–2152, https://doi.org/10.1007/s11274-014-1649-5. [64] R.R. Soares, A.M. Azevedo, J.M. Van Alstine, M.R. Aires-Barros, Partitioning in aqueous two-phase systems: Analysis of strengths, weaknesses, opportunities and threats. Biotechnol. J. 10 (2015) 1158–1169, https://doi.org/10.1002/biot.201400532. [65] R. Piper, G. Vedantham, A. Grilo, K. Lacki, J.M. Van Alstine, Alternative separation technologies for the harvest of CHO cell culture, in: 17th International Biopartitioning and Purification Conference, Newport, Rhode Island, October 2013, 2013. Alternative Separation Methods: Flocculation and Precipitation Chapter | 11 239

[66] M. Felo, Y.K. Kenneth, J. Hamzik, P. Balderes, D.L. Ludwig, L. Dale, Industrial application of impurity flocculation to streamline antibody purifica- tion processes. Pharm. Bioprocess. 3 (2015) 115–125, https://doi.org/10.4155/PBP.15.2. [67] E.B. Schirmer, M. Kuczewski, K. Golden, B. Lain, C. Bragg, J. Chon, M. Cacciuttolo, G. Zarbis-Papastoitsis, Primary clarification of very high- density cell culture harvests by enhanced cell settling, BioProcess Int. (2010) 32–39. [68] R. Tran, K. Lacki, E. Grund, A. Davidson, B. Sharma, N. Titchener-Hooker, Changing manufacturing paradigms in downstream processing and the role of alternative technologies. J. Chem. Technol. Biotechnol. 89 (2013) 1534–1544, https://doi.org/10.1002/jctb.4234. [69] A. Pegel, S. Reiser, M. Steurenthaler, S. Klein, Evaluating disposable depth filtration platforms for mAb harvest clarification, BioProcess Int. 9 (2011) 52–55. [70] P. Dave, J. Dizon-Maspat, T. Cano, Evaluation and implementation of a single stage multimedia harvest depth filter for a large-scale antibody pro- cess, Bioprocess Int. (June Suppl.) (2009) 8–17. [71] Y. Yigzaw, R. Piper, M. Tran, A. Shukla, Exploitation of the adsorptive properties of depth filters for host cell protein removal during monoclonal antibody purification, Biotechnol. Prog. 22 (2006) 288–296. [72] J. M. Van Alstine, et al., GE Healthcare Presentation, Towards Solvent-Free Plasma Protein Precipitation. Recovery of Biological Products XV, Vermont, July 2012. [73] J. Van Alstine, J. Shanagar, R. Hjorth, M. Hall, C. Estmer Nilsson, Separation Method Using Single Polymer Phase Systems. Patents, US 9115181 B2 and WO2010080062A1. [74] E.J. Cohn, The physical of the proteins, Physiol. Rev. 5 (1925) 349–437. [75] E.J. Cohn, L.E. Strong, W.L. Hughes Jr., D.J. Mulford, J.N. Ashworth, M. Melin, H.L. Taylor, Preparation and properties of serum and plasma pro- teins. IV. A system for the separation into fractions of the protein and lipoprotein components of biological of biological tissues and fluids. J. Am. Chem. Soc. 68 (1946) 459–475, https://doi.org/10.1021/ja01207a034. [76] J. Bertolini, N. Goss, J. Curling, Production of plasma proteins for therapeutic use, Wiley, New York, 2013. [77] T. Burnouf, Modern plasma fractionation, Transfus. Med. Rev. 21 (2007) 101–117. [78] J. Parkkinen, A. Rahola, L. von Bonsdorff, E. Törmä, A modified caprylic acid method for manufacturing immunoglobulin G from human plasma with high yield and efficient virus clearance, Vox Sang. 90 (2006) 97–104. [79] A. Venkitschwaran, P. Heider, L. Teysseyre, G. Belfort, Selective precipitation-assisted recovery of immunoglobulins from bovine serum using controlled-fouling crossflow membrane microfiltration. Biotechnol. Bioeng. 101 (2008) 957–966, https://doi.org/10.1002/bit.21964. [80] O.A. Jaquez, R.S. Gronke, T.M. Przybycien, Design of a scalable, continuous precipitation process for the high throughput capture and purification of high titer monoclonal antibodies, in: AIChE Conference, November, 2010. Paper 191335. https://aiche.confex.com/aiche/2010/webprogram/ Paper191335.html. [81] A. Tscheliessnig, D. Ong, J. Lee, S. Pan, G. Satianegara, K. Schriebl, A. Choo, A. Jungbauer, Engineering of a two-step purification strategy for a panel of monoclonal immunoglobulin M directed against undifferentiated human embryonic stem cells. J. Chromatogr. A 1216 (2009) 7851–7864, https://doi.org/10.1016/j.chroma.2009.09.059. [82] N. Hammerschmidt, A. Tscheliessnig, R. Sommer, B. Helk, A. Jungbauer, Economics of recombinant antibody production processes at various scales: industry-standard compared to continuous precipitation. Biotechnol. J. 9 (2014) 766–775, https://doi.org/10.1002/biot.201300480. [83] R. Tran, Y. Zhou, K.M. Lacki, N.J. Titchener-Hooker, A methodology for the comparative evaluation of alternative bioseparation technologies. Biotechnol. Prog. 24 (2008) 1007–1025, https://doi.org/10.1021/bp.20. [84] M. Franzreb, E. Müller, J. Vajda, Cost estimation for protein A chromatography, Bioprocess Int. 12 (2014) 44–52. [85] R.A. Lewus, P.A. Darcy, A.M. Lenhoff, S.I. Sandler, Interactions and phase behavior of a monoclonal antibody. Biotechnol. Prog. 27 (2011) 280–289, https://doi.org/10.1002/btpr.536. [86] L.A. Moreira, M. Boström, B.W. Ninham, E.C. Biscaia, F.W. Tavares, Hofmeister effects: why protein charge, pH and protein precipitation depend on the choice of background salt solution, Colloid. Surf. A 282–283 (2006) 457–463. [87] A. Mahn, G. Zapata-Torres, J.A. Asenjo, A theory of protein–resin interaction in hydrophobic interaction chromatography. J. Chromatogr. A 1066 (2005) 81–88, https://doi.org/10.1016/j.chroma.2005.01.016. [88] B.K. Nfor, N.N. Hylkema, K.R. Wiedhaup, P.D.E.M. Verhaert, L.A.M. van der Wielen, M. Ottens, High-throughput protein precipitation and hydro- phobic interaction chromatography: salt effects and thermodynamic interrelation, J. Chromatogr. A 1218 (2011) 8958–8973. [89] P. Diederich, M. Hoffmann, J. Hubbuch, High-throughput process development of purification alternatives for the protein avidin. Biotechnol. Prog. 31 (2015) 957–973, https://doi.org/10.1002/btpr.2104. [90] M. Buddha, S. Rauniyar, S. Qais, D. Goudar, S.S. Kandukuri, S. Mahajan, S. Siddik, P. Hazra, Precipitation as an alternative to chromatography in the insulin manufacturing process, BioPharm. Int. 29 (2016) 30–35. [91] P. Gagnon, Emerging challenges to protein A: chromatin-directed clarification enables new purification options, BioProcess. Int. 11 (2013) 44–52. [92] J. Persson, P. Lester, Purification of antibody and antibody-fragment from E. coli homogenate using 6,9-diamino-2-ethoxyacridine lactate as precipi- tation agent, Biotechnol. Bioeng. 87 (2004) 424–434. 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