Mixed-mode chromatography materials contain ligands of multimodal functionality that allow protein adsorption by a combination of ionic interactions, hydrogen bonds, and/or hydrophobic interactions. Complex mixtures like fermentation supernatants or cell lysates can be applied directly at relatively high conductivity, and elution is usually achieved by electrostatic charge repulsion. We used mixed-mode materials for capturing and intermediate purification of several recombinant therapeutic proteins from various expression systems like yeast, Escherichia coli, and mammalian cells. Product-related impurities as well as process-related impurities from fermentation media were efficiently removed while the desired product was bound with high selectivity. Because these purification protocols can be scaled up easily to production scale, mixed-mode materials are being considered as potential elements of a general purification platform for recombinant therapeutic proteins produced in various expression systems.

The impurity profile of a starting material for downstream processing depends mainly on the conditions of the fermentation process, such as the expression system, medium composition, fermentation regimen, time point of harvest, or shear forces during isolation. This leads to a variety of process- and product-related impurities which must be removed by an efficient, orthogonal, and robust purification process. Modern downstream processes often consist of only two or three separation steps, and usually avoid conditioning steps like ultrafiltration/diafiltration (UF/DF) for buffer exchange to reduce the total number of process steps. However, this strategy significantly increases the risk that contaminants are not completely removed during the purification process, especially if complex microbial feedstocks or 

 lysates are used as starting materials. In these cases, single-mode chromatography materials like ion exchange or hydrophobic resins often fail, in particular if the feedstock contains large amounts of complex colored impurities like melanoidins in combination with a relatively high conductivity of 15–30 mS/cm. Mixed-mode resins may help to overcome these problems because multimodal ligands offer excellent selectivity in combination with a salt-tolerant adsorption of the target protein.  

The ligands of mixed-mode materials typically contain a combination of multiple binding modes like ion exchange, hydrogen bonding, and hydrophobic interactions.

  The target protein itself is also a multimodal molecule. This situation results in a variety of protein–ligand interactions and often leads to unique selectivity, which in some cases includes pseudo-affinity. Furthermore, binding of the desired protein often is achieved at the conductivity of the feedstock without further dilution or addition of lyotropic salts. That feature makes mixed-mode resins an excellent choice for direct capture steps.  

Commercially available mixed-mode materials are based on several principles. Resins containing hydrocarbyl amine ligands (e.g., PPA Hypercel, HEA Hypercel, Pall Corporation) allow binding at neutral or slightly basic pH values by a combination of hydrophobic and electrostatic forces. Elution usually is achieved by electrostatic charge repulsion when the pH value is lowered below the isoelectric point (pI) of the target protein and the pKa of the ligand.

  Another ligand, 4-mercapto-ethyl-pyridine (MEP Hypercel, Pall), is based on a similar principle, but hydrophobic interaction is achieved by an aromatic residue and the sulphur atom facilitates binding of immunoglobulins by thiophilic interaction.

  Another group of mixed-mode materials (Capto MMC and Capto adhere, GE Healthcare) contains ligands with hydrogen bonding groups and aromatic residues in the proximity of ionic groups, which leads to the salt-tolerant adsorption of proteins at different conductivities.

  However, in contrast to electrostatic charge repulsion, which is driven mainly by changes in the pH value, the elution of proteins from Capto MMC usually requires an increase in the pH value and salt concentration. It also should be noted that many other chromatography materials that have been around for decades, like affinity resins with dye ligands, hydroxyapatite, and some ion-exchange resins, such as Amberlite CG 50 (Rohm & Haas) or Lewatit CNP 105 (Lanxess) often show a unique specificity that is based mainly on a multimodal protein–ligand interaction.

Mixed-mode materials were used in our laboratories to purify a variety of different therapeutic proteins, like cytokine muteins, protease inhibitors, and antibody fragments (Table 1). Although these resins are particularly useful for capture steps because of their selectivity and salt tolerance, they can be used for intermediate purification or polishing as well. In the following sections, we describe two case studies in which mixed-mode materials showed significant advantages compared with single-mode resins for removing a product-derived impurity containing a post-translational modification, and for purifying a therapeutic protein with challenging physicochemical properties.  

Table 1. The use of mixed-mode materials in the downstream processes of various therapeutic proteins 

Purifying Recombinant Protease Inhibitors from Yeast

Mixed-mode chromatography was used successfully to purify several recombinant Kunitz-type protease inhibitor muteins from yeast. Mutein I was expressed in 

 and secreted into the fermentation supernatant using a complex medium with a conductivity >20 mS/cm that contained a large amount of colored impurities (i.e., melanoidins). The protease inhibitor (pI ~6.3) showed a reasonable solubility of ~13 mg/mL at pH <3.2 that decreased significantly to <0.5 mg/mL when the pH value was increased only slightly (Figure 1). At a neutral or slightly basic pH value, an acceptable solubility could be achieved only at high-salt conditions. 

The design of a suitable purification strategy focused on the following objectives: 1) identifying  chromatography steps that keep the protein solution at a pH value below 3 or at high salt concentration and 2) using the low solubility of mutein I at neutral pH to find a suitable crystalline storage form for the bulk drug substance. Because the use of conventional ion exchange (IEX) resins was not possible given the unusual solubility profile of mutein I and the high conductivity of the starting material, the salt-tolerant properties of two mixed-mode resins (Capto MMC and HEA Hypercel), which are orthogonal with respect to their binding and elution pH levels, proved to be particularly useful. The purification process is shown schematically in Figure 2. The protein concentration of the feed stock was <0.5 mg/mL, and the clarified fermentation supernatant was applied at >20 mS/cm directly onto Capto MMC at a pH value of 5.5. Elution was achieved at a pH value of 8.5 under high-salt conditions (2 M NaCl), and, subsequently, the protein was applied directly onto HEA Hypercel without any conditioning steps. Elution at a pH value of 2.7 made it possible to keep the protein in solution under low-salt conditions. Lastly, increasing the pH value to more than 4 in a controlled mode of operation initiated crystallization, which led to a stable storage form of the bulk drug substance.  

Capturing mutein I with Capto MMC was the key chromatography step of the purification process (Tables 2 and 3). The clarified fermentation supernatant was applied directly at relatively high conductivity (>20 mS/cm) without further dilution. The maximum capacity of 17 mg/mL was only moderate but sufficient for an early-phase production process. Process- and product-derived impurities were removed efficiently, and the purity of mutein I was >90% after this chromatography step, which indicates that Capto MMC acts as a pseudo-affinity resin in this case. This was particularly possible by using stringent washing conditions (Tables 2 and 3). High-salt washing conditions at a pH value of 5 and a low-salt washing step at a pH value of 8.5 led to an almost complete removal of the colored impurities (melanoidin) from the fermentation medium, and the Capto MMC eluate was colorless and clear. De-colorization by Capto MMC is mainly a pH-driven process. During the development of a production process for another mutein of a protease inhibitor (mutein II, see Table 1), we were able to show that colored impurities from complex yeast media bind to Capto MMC at pH values from 3 to 6, but can be completely removed at a pH value of more than 6.5. Thus, the binding of the desired protein to the resin under these conditions is of particular importance for a successful de-colorization strategy using Capto MMC. 

Table 2. Experimental conditions for a typical Capto MMC capture run of the recombinant protease inhibitor mutein I 

Several approaches for intermediate purifications were tested during process development. Reversed-phase chromatography under strongly acidic conditions worked well for purification of research-grade material, but using high-pressure equipment was not considered suitable for the large-scale manufacturing process. Because mutein I was eluted from Capto MMC at 2 M NaCl, pH 8.5, we screened for a resin that allowed binding at high-salt conditions as well as elution at a low pH value under low-salt conditions. The mixed-mode Hypercel-based resins from Pall meet these requirements because they allow salt-tolerant binding at neutral or slightly basic pH value, and elution is usually achieved at a low pH value by electrostatic charge repulsion. When binding mutein I on the Pall mixed-mode resins was tested, HEA Hypercel showed the highest capacity (27 mg/mL) compared to PPA Hypercel (19 mg/mL) and MEP Hypercel (9 mg/mL). We decided, therefore, to use HEA Hypercel for intermediate purification. The Capto MMC eluate was applied directly without prior buffer exchange, and elution at a pH value of 2.7 enhanced the solubility of mutein I under low-salt conditions (Tables 4 and 5). Product purity was increased only marginally during this step; only traces of product-derived impurities were separated. However, HEA Hypercel enabled us to remove large amounts of salt from the Capto MMC eluate and to immediately lower the pH value, which was necessary to prepare the protein for the crystallization step. 

Table 3. Purification data from a typical Capto MMC capture run of the recombinant protease inhibitor mutein I 

All unit operations were successfully scaled up by a factor of 5 to 10 and manufacturing was performed using a 5-L Capto MMC and a 2-L HEA Hypercel column, respectively. Crystallization was carried out in a 5-L glass reactor. The overall process yield was 70%.  

Table 4. Experimental conditions of a typical HEA Hypercel intermediate step of the recombinant protease inhibitor mutein I 

In summary, chromatography resins with multimodal ligands enabled us to purify a recombinant mutein of a protease inhibitor with an unusual solubility profile in a three-step process. Ligands combining ionic interactions with hydrogen bonding and hydrophobic interactions showed a unique selectivity as well as salt-tolerant binding and allowed for efficient removal of major product- and process-derived impurities under conditions where classical chromatography methods like IEX or hydrophobic interaction chromatography (HIC) failed. 

Table 5. Purification data from a typical HEA Hypercel intermediate step of the recombinant protease inhibitor mutein I 

Purifying a Recombinant Cytokine Mutein from 

Ceramic hydroxyapatite (CHT, Bio-Rad Laboratories) is a crystalline mineral with multimodal functionalities that are significantly different from the mixed-mode ligands described above. The amino groups of proteins bind to the phosphate ions of the mineral by a classical cation exchange mechanism, and elution is typically achieved by increasing salt or phosphate buffer concentration. In contrast, carboxylic side chains of proteins bind to the Ca

  ions of the resin by a combination of calcium metal affinity and anion exchange. The combination of different binding modes leads to excellent selectivity, which allows for efficient removal of aggregates and multimers in production processes of antibodies or antibody fragments.

  We used CHT type I (particle size: 80 µm) to separate a closely related, product-derived impurity with a post-translational modification during clinical manufacturing of a recombinant cytokine mutein from 

The production process of the cytokine mutein is shown schematically in Figure 3. The protein was expressed in 

 as an inclusion body and refolded. After pH adjustment and depth filtration, reversed-phase chromatography was performed to reduce the volume of the protein solution after the refolding step and to decrease the salt concentration. CHT type I (80 µm) was used as intermediate step to remove an N-formylated product-derived impurity. Finally, polishing was performed by reversed phase chromatography to separate several product-derived disulfide isoforms. The overall process yield was 50%. 

Figure 3. Flow scheme of the cytokine mutein purification process 

Analytical characterization of the cytokine mutein intermediate after refolding and reversed-phase chromatography revealed an N-formylated product-derived impurity that had to be removed from the desired nonformylated isoform. A broad screening of commercially available ion exchange resins showed that a separation of the formylated and nonformylated species could be achieved only with a 15-µm Source 15S cation exchanger operating at a pressure of 1–2 MPa. To perform intermediate purification at low pressure, several CHT and ceramic fluorapatite (CFT) resins were tested. Purification with CHT type I (80 µm) resulted in a reasonable separation of the formylated and nonformylated species (Figure 4a) when a column length of 9.5 cm and a column load of 17.5 mg/mL were used. To further improve the removal of the formylated impurity, an optimization program using Design of Experiments (DoE) was performed. Standard conditions for binding were 5-mM K-phosphate at pH 6.5, and elution was achieved using a linear 5–500 mM K-phosphate gradient. Five process variables were selected as factors: column length (5–14 cm), column load (5–30 mg protein/mL resin), flow rate (10–60 column volumes/h), gradient steepness (1–2 % eluent B/column volume), and pH value (6.0–7.0). A chromatogram-response function (CRF) was selected as a response variable to reflect successful separation of the formylated and nonformylated species.

  Purity and yield of the cytokine mutein were defined as additional response variables. A linear d-optimal design with center points was used that was later augmented to a full response surface design (i-optimal design). Column length and column load showed the largest effects, and several two-factor interactions were identified. Calculating the optimal factor settings (column length: 14 cm, column load: 5 mg/mL) resulted in a significant improvement of the separation of formylated and nonformylated species (Figure 4b). The optimized unit operation was successfully scaled-up (to a 2.8-L column) and used to manufacture large quantities of material. The overall yield of the process step was 85%, and the purity of the cytokine mutein increased from 60 to 75%.  

Figure 4a-b. Separation of an N-formylated cytokine mutein species by CHT type I chromatography. The figure shows the elution profile of the native cytokine mutein (blue) and the N-formylated impurity (red) before (a) and after (b) optimization by Design of Experiments 

A Generic Strategy for Screening and Optimizing Mixed-Mode Resins

The interaction of mixed-mode ligands with the desired protein is often complex, and extensive optimization is required during process development. Many of the mixed-mode chromatography steps described in Table 1 were optimized successfully without using DoE. However, DoE offers considerable advantages for developing complex separation steps because it enables the identification of main effects as well as two-factor interactions and critical/noncritical parameters. Furthermore, the model makes it possible to calculate the impact of selected factors for each point in the design space, which offers an enormous flexibility for process optimization in both early and late stages of development. We decided, therefore, to develop a generic strategy for optimizing mixed-mode purification steps (Figure 5). This approach includes screening various resins (Capto MMC, Capto adhere, MEP Hypercel, PPA Hypercel, and HEA Hypercel) using batch binding conditions, which allows for a selection of suitable resins based on binding and elution of  the desired protein at different pH values and salt concentrations. Then, resins are selected and further optimized by DoE using small column chromatography (2 mL). We usually conduct a linear full-factorial design using four factors (loading pH value, elution pH value, salt concentration in the elution buffer, and column load), a design that can be augmented later to a full response surface design, if necessary. This approach was used successfully to optimize mixed-mode steps of an antibody fragment expressed in 

 and a monoclonal antibody expressed in mammalian cell culture. The complete experimental program takes approximately four to five weeks and the total amount of protein needed is usually 1 to 2 g, which is acceptable even in early stages of process development.   

Figure 5. Flow scheme of a generic approach for screening and optimization of mixed-mode chromatography steps 

Salt-tolerant adsorption and unique selectivity are the major advantages of mixed-mode materials over single-mode resins. These features enabled us to meet a variety of challenges in downstream processing, such as capturing proteins from complex feedstocks, de-colorizing feed streams, purifying difficult proteins, and removing special product- and process-derived impurities. Moreover, the overall number of unit operations was significantly lower in these cases when mixed-mode steps were implemented in the downstream process. Thus, mixed-mode materials offer considerable flexibility for designing efficient downstream processes, in particular if no generic affinity ligand such as Protein A is readily available. The complexity of interactions between the ligand and the desired protein might be considered as a limiting factor. This can, however, be overcome easily by using a combination of resin screening and statistical design of experiments, which makes it possible to optimize complex separation steps in a generic and systematic fashion. 

The contributions of Torsten Minuth and many other colleagues from Bayer Schering Pharma Biotech Development are gratefully acknowledged. Solubility experiments were performed by Dirk Havekost, Bayer Technology Services. We thank Hans-Dietrich Hoerlein for his support and many fruitful discussions. 

 is a senior scientist in downstream process development and 

 is the pilot plant manager for biotechnology, both in Biotech Development, Global Biologics, at Bayer Schering Pharma AG, Wuppertal, Germany, +49 202 36 7743, 

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 2. Brenac Brochier V, Schapman A, Santambien P, Britsch L. Fast purification process optimization using mixed-mode chromatography sorbents in pre-packed mini-columns. J Chromatogr A. 2008;1177:226–33 

 3. Burton SC, Harding DRK. Salt-independent adsorption chromatography: new broad-spectrum affinity methods for protein capture. J Biochem Biophys Methods. 2001;49: 275–87. 

 4. Lees A, Topping A, Razzaq A, Reiter K, Acosta A-L. Purifying a recalcitrant therapeutic recombinant protein with a mixed-mode chromatography sorbent. Bioprocess Int. 2009;7(2):42–8. 

 5. Guerrier L, Flayeux I, Boschetti E. A dual-mode approach to the selective separation of antibodies and their fragments. J Chromatogr B. 2001;755:37–46 

 6. Arakawa T, Kita Y, Sato H, Ejima D. MEP chromatography of antibody and Fc-fusion protein using aqueous arginine solution. Protein Expr Purif. 2009;63:158–63. 

 7. Chen J, Tetrault J, Zhang Y, Andy Wasserman A, Conley G, DiLeo M, et al. The distinctive separation attributes of mixed-mode resins and their application in monoclonal antibody downstream purification process. J Chromatogr A. 2009; doi:10.1016/j.chroma.2009.09.047 

 8. Kaleas KA, Schmelzer CH, Pizarro SA. Industrial case study: evaluation of a mixed-mode resin for selective capture of a human growth factor recombinantly expressed in E. coli. J Chromatogr A. 2009; doi:10.1016/j. chroma.2009.07.023 

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Articles

Salt-tolerant adsorption and unique selectivity are the major advantages of mixed-mode materials over single-mode resins.

Mixed-Mode Chromatography in Downstream Process Development

Various precipitation techniques have been used in the industrial purification of proteins for many years. Precipitation processes can be separated into two main categories: impurity precipitation and product precipitation. Impurity precipitation is operationally simpler but carryover of the precipitants can challenge subsequent unit operations. Product precipitation may have a higher risk of damage to the target molecule, but in addition to purifying the product, product precipitation also enables buffer exchange and concentration during the resolubilization step. An antibody precipitation step has been developed using a recombinant antibody produced in PER.C6 cells and statistical design of experiments to optimize product yield and host cell protein (HCP) removal. After appropriate precipitation conditions were developed, two methods to capture the antibody pellet were evaluated: depth filtration and microfiltration. A wash step was incorporated in both methods to reduce soluble impurities. The final process resulted in a product yield of 90% and HCP reduction of approximately 1 LRV.

The method of pellet capture was shown to have a significant impact on the purity of the redissolved product. The precipitation step is readily scalable and fits a fully disposable downstream process.  

Efforts are ongoing to identify alternatives to packed bed chromatography to reduce the time and cost of processing high-titer product streams. Although many efforts focus on membrane adsorbers that directly replace columns of the same or similar chemistries, some older technologies are beginning to gain ground in recombinant protein manufacturing. One attractive alternative to chromatography is precipitation, which has been used in the plasma protein industry for many years.

  A simple method of precipitation involves titrating the process fluid to the isoelectric point of the protein that is to be precipitated.

  Lyotropic salts, such as ammonium sulfate, also have a long history of use in precipitation processes.

 Short-chain fatty acids, such as caprylic acid, are well known for their ability to precipitate DNA and host cell proteins (HCPs).

  Polyionic species also are useful precipitants for capturing a product of interest or removing contaminating proteins.

Polyethylene glycol (PEG) has been used for product and impurity precipitation.

  It also can be combined with isoelectric precipitation to improve the efficiency of the separation.

  After precipitation, centrifugation or filtration can be used to perform solid–liquid separation.

  Although centrifugation is a well-established method to achieve this separation, washing the product pellet to remove impurities could be problematic, and it is not suited to a single-use process. Filtration—normal- or tangential-flow—requires more development, but washing the pellet is simpler, and it is readily adaptable to a single-use process. 

In the present work, a product precipitation step was developed using PEG to recover a monoclonal antibody (MAb) from clarified PER.C6 cell culture media. Appropriate precipitation conditions were identified through the use of full factorial experimental designs. Two filtration steps were evaluated for the capture and washing of the precipitated product, and the superior method was scaled-up 10-fold. The total precipitation process resulted in yields of approximately 90% and HCP reduction of 1 LRV with no significant increase in the aggregate level of the redissolved MAb. Finally, the impact of the precipitation step on the subsequent cation exchange (CEX) capture step was investigated. 

USP grade salts, Tween 20, hydrochloric acid, acetic acid, and sodium hydroxide were purchased from JT Baker (Phillipsburg, NJ). PEG was of reagent grade and purchased from JT Baker or EMD Chemicals (Gibbstown, NJ). All buffers were prepared using MilliQ-grade water (Millipore, Billerica, MA) and were filtered by 0.22-µm filtration before use.  

, pI = 8.3, 150 kDa) was produced at Percivia, LLC using a PER.C6 cell line. PER.C6 cells are human embryonic retinal cells immortalized by the adenovirus E1 gene, as described in US patent 5,994,128.

  The cells were cultured in a standard fed-batch process or the XD process, both using chemically defined media.

  The fed-batch media were clarified by sedimentation and depth filtration, and the XD media were clarified by the enhanced cell settling (ECS) method followed by depth filtration.

  During ECS, Silica-PEI resin was used to enhance cell settling and also reduce DNA and HCP. 

The conditions used to precipitate the MAb—PEG molecular weight, PEG concentration, and pH—were optimized by full factorial experimental designs using Minitab software (State College, PA). The pH of the clarified XD media was adjusted to the desired level with 2-M Tris in a 15-mL conical tube. The PEG was added as a 40% (w/w) stock solution to the desired final concentration. The tube was then centrifuged at 1,000

 and the supernatant decanted. Finally, the pellet was redissolved in phosphate-buffered saline (PBS). 

Pellet Capture by Depth Filtration or Microfiltration 

Depth filtration was performed with various grades of filter media. Millistak+HC D0HC, C0HC, and X0HC were purchased from Millipore Corp. (Billerica, MA), and ZetaPlus 60SP02A was purchased from Cuno (Meriden, CT). Precipitation was carried out using a 40% (w/w) stock solution of PEG-3350 and the precipitated media was loaded at a feed flux of 50 L/m

 /h until all of the material was loaded or the transmembrane pressure (TMP) was 15 psid. The filters were then washed with 20–30 L/m

  of 20 mM Tris pH 8.5 + 14.4% (w/w) PEG-3350. After washing, 80 L/m

  of resolubilization buffer was passed through the filters at 100 L/m

 /h, and the permeate was recirculated through the device at 600 L/m

 /h until the A280 of the permeate pool was stable indicating complete MAb dissolution. Finally, any held-up product was recovered with a 20 L/m

  buffer flush and air blowdown of the filter module. In some tests, the filter media was subsequently washed with 85-mM acetate pH 5.3 followed by 1-M NaCl. Pressure and flow data were collected using a custom engineered system from ARC Technology Services (Nashua, NH). 

Microfiltration was performed with a 0.22-µm hollow fiber membrane from GE Healthcare Life Sciences (Piscataway, NJ). The PEG-3350 was added as a 40% (w/w) stock solution for the small-scale experiment and in powder form for the scale-up work. The feed flux was 710 L/m

 /h and the retentate and permeate were unrestricted. The precipitate was first concentrated 10- to 14-fold and then washed with three diafiltration volumes of 20 mM Tris pH 8.5 plus 14.4% (w/w) PEG-3350. Finally, the precipitate was redissolved in 85 mM sodium acetate pH 5.3 or 20 mM Tris plus 50 mM NaCl pH 7.5. Pressure, flow, and conductivity data were collected using a Slice 200 benchtop system (Sartorius, Gottingen, Germany) for small-scale testing and a SciPro system (SciLog, Middleton, WI) for the scale-up experiment. 

Toyopearl GigaCap S-650 was procured from Tosoh Bioscience (Montgomeryville, PA) in the Toyoscreen 5-mL format. This resin has been previously demonstrated as a high capacity capture step for MAbs.

  The column was equilibrated with 74-mM sodium acetate pH 5.3 and loaded to 90–95 mg-MAb/mL-resin using either clarified media or clarified and PEG-treated material, each adjusted to the same pH and conductivity as the equilibration buffer. The column was then washed with equilibration buffer and the antibody eluted with 50 mM sodium acetate pH 5.3 plus 90 mM NaCl. 

The MAb concentration in media-containing samples was determined by analytical Protein A HPLC (Applied Biosystems, Foster City, CA). Aggregate levels were measured by size-exclusion chromatography (SEC) using a TSKgel G3000SWXL column from Tosoh Bioscience (Montgomeryville, PA), with peak detection by UV absorbance at 280 nm. HCP levels were quantified by a PER.C6-specific ELISA from Cygnus Technologies (Southport, NC). SDS-PAGE was performed with NuPAGE 4–12% Bis-Tris gels and staining was done with SimplyBlue SafeStain, both from Invitrogen (Carlsbad, CA). 

Results and Discussion Precipitation Conditions

For both molecular weights of PEG, 3,350 and 6,000 Da, the PEG concentration was the dominant factor in the recovery and purity of the redissolved MAb. Precipitation with PEG-3350 resulted in the highest recovery (Figure 1). However, the higher recovery came at the expense of higher HCP burden in the redissolved MAb. In the case of aggregated MAb, the levels were not significantly different from the starting material, but it should be noted that the particular MAb used in this work is not prone to forming aggregates. The improvement of the HCP reduction with the use of PEG-6000 was offset by the reduction in product yield. The final condition selected was 14% (w/v) PEG-3350 (equivalent to 14.4% w/w) and pH 8.5.  

In the first experiment, ECS-clarified XD media was precipitated and the pellet was captured by depth filtration with X0HC media. No substantial increase in the transmembrane pressure (TMP) was observed during the loading of 361 g-MAb/m

  (data not shown). After washing, resolubilization of the immobilized pellet was done with 85 mM acetate pH 5.3. Even after recirculation for 2 h, the antibody had not completely redissolved, so 0.1% v/v Tween 20 was added to the resolubilization buffer. After an additional 30 min of resolubilization, the MAb was fully dissolved.  

Table 1. Yield and impurity removal for the PEG precipitation operation using depth filtration with X0HC to recover the product 

Mass balance data are summarized in Table 1. The HCP reduction was lower than in the previous experiment (Figure 1), where only 5,000 ppm of HCP was in the final pool as compared to 8,300 ppm in this experiment. Some depth filters, however, are known to have hydrophobic and anion exchange (AEX) adsorptive characteristics.

  The precipitated media was loaded at a relatively high pH (8.5) and low conductivity (<10 mS/cm), which may have induced binding of acidic proteins on an AEX matrix. Furthermore, in the presence of PEG, the protein binding capacity of ion-exchange matrices has been shown to increase.

  Therefore, it is probable that some of the HCPs that remained in solution after precipitation bound to the depth filter media and eluted into the product during resolubilization. This hypothesis also is supported by the difference in the HCP content of the precipitation supernatant and the depth filter flow-through (9,900 versus 240 mg) which indicates HCP removal from the solution by the depth filter. Following immobilization of the antibody onto the depth filter, it was redissolved at a significantly lower pH (5.3) resulting in the elution of bound HCP, and reducing the purity of the final product. This phenomenon could be mitigated by using less adsorptive depth filter media, such as Millistak+HC D0HC/C0HC and ZetaPlus SP filters like 60SP02A, or by redissolving the product

in a low-salt, higher-pH buffer, like 20-mM Tris pH 7.5.  

These strategies were tested by loading precipitated fed-batch media onto D0HC, C0HC, X0HC, and 60SP02A filters. All filters were washed identically with the PEG/Tris buffer, but the resolubilization was done with 20 mM Tris pH 7.5 plus Tween 20 for the Millipore filters and 85 mM acetate pH 5.3 plus Tween 20 for the Cuno filter. The Millipore filters also were stripped with a low pH buffer (85 mM acetate pH 5.3) and high salt (1 M NaCl) after the product was recovered to determine if any bound HCPs could be eluted. Figure 2 shows that there was substantial fouling of all filters tested. Because the fed-batch media was not clarified by the ECS method, which has been shown to significantly reduce DNA levels in XD harvests, there may be more DNA present, which precipitates in high PEG concentrations.

  The higher DNA content in the precipitate may have reduced filter capacities, but this has not been investigated. Table 2 shows that each of the experiments resulted in better HCP reduction (84–88% versus 46%), and even though the starting HCP burden was higher (49,000 ppm versus 13,000 ppm), the redissolved MAb pools generally had lower HCP contents (6,000–7,800 ppm versus 8,300 ppm). The use of low-adsorptive filters and redissolving in a higher pH buffer appear to solve the problem of HCPs eluting from the depth filter media. SDS-PAGE of the strip fractions revealed that both HCPs and product were bound to the filters—including the less adsorptive D0HC and C0HC filters—and confirmed that X0HC media were more adsorptive than D0HC and C0HC media (Figure 3). 

Table 2. Yield and impurity removal for the PEG precipitation operation using depth filtration with various filter media to recover the product 

Although the HCP burden of the redissolved MAb was reduced by using a buffer with a higher pH and less adsorptive depth filter media, the capacities of the depth filters were low for fed-batch media (<400 g-MAb/m

 Microfiltration (tangential flow filtration, MF TFF) was tested with the aim of improving capacity with the added benefit of being able to use any buffer for resolubilization because of the low binding characteristic of the hollow fiber. The hydraulic performance of the MF TFF concentration and washing of fed-batch precipitate is shown in Figure 4. The permeate flux was about 100 L/m

 /h and the TMP was between 1.0 and 2.5 psid for the entire operation. The mass loading of 475 g-MAb/m

  was better than that achieved in any of the depth filtration operations. The product recovery and HCP reduction were both around 90%, slightly better than that achieved in depth filtration (Table 3).   

The resolubilization was much simpler and faster than for the depth filtration; rather than recirculating buffer through the device, the buffer was pumped through the inside of the lumens into the retentate vessel with the permeate closed and allowed to mix for about 60 min. This was sufficient to redissolve the antibody, and no excipients were needed. Because of the difficulty in resolubilization with depth filtration, the product was redissolved at or near the concentration in the feed media. The final pool from the MF TFF process was nearly two-fold more concentrated than the starting material. 

Because the retentate and permeate were open to atmospheric pressure, minimal instrumentation was required: a crossflow pump, one pressure sensor, and a transfer pump for the diafiltration. The combination of high capacity, high recovery and HCP clearance, and operational simplicity make MF TFF a preferable option for pellet capture as compared with depth filtration. The precipitation and MF TFF step was scaled-up 10-fold to a 0.12 m

  hollow fiber device, and the performance was comparable to the small scale (Table 4). 

Table 3. Yield and impurity removal for the PEG precipitation operation using microfiltration to recover the product at bench scale

High capacity cation exchange  (CEX) chromatography was evaluated with feeds pretreated with or without PEG precipitation. The precipitation step did not have any significant impact on the step yield or the percentage reduction of HCPs, but the eluate resulting from the precipitated load material had seven-fold less HCP (Table 5). 

Table 4. Yield and impurity removal for the PEG precipitation operation using microfiltration to recover the product at pilot scale 

Precipitation has long been used in the plasma protein industry to purify proteins at large scales. The technique has been adapted here to the initial MAb purification from clarified fed-batch and XD media in a scalable manner. Two single-use filtration steps have been developed to capture and wash the precipitated product, eliminating the need for centrifugation. It was shown that the precipitation operation did not negatively affect the yield of the CEX capture step, and it reduced the HCP content of the eluate by a factor of seven. 

Table 5. Yield and HCP removal for GigaCap S-650 loaded with precipitated (+PEG) and nonprecipitated (âPEG) antibody

The ability to reduce the impurity burden so far upstream in the purification train is key to truncating the downstream process or replacing traditional chromatography with other single-use technologies. Lower impurity burdens can improve the loading capacity of flow-through membrane adsorbers and possibly virus filters, which are generally very expensive items in a process. 

An added benefit is the ability to redissolve the antibody in a buffer that facilitates the subsequent unit operation. For example, cell culture media typically requires extensive titration and dilution or a UF–DF step to prepare for capture chromatography with a cation exchanger. Here, the precipitated antibody can be dissolved in equilibration buffer at high concentration, thus shortening the processing time. This can be important for products that do not tolerate long exposure to low pH/conductivity conditions. In the case of this particular antibody, the clarified media requires a more than two-fold dilution to be loaded onto a CEX column, whereas the redissolved MAb could be loaded directly at nearly two-fold the concentration of the unadjusted media. This is at least a four-fold reduction in the load volume, which can result in substantial time savings for modern, high-capacity CEX resins. 

This alternative purification method to chromatography is readily scalable and fits a fully disposable downstream process.

PEG Precipitation: A Powerful Tool for Monoclonal Antibody Purification

Over the years, much has been learned about the downstream purification of monoclonal antibodies (MAbs). Standard processes are well established and there is extensive literature that describes methods and approaches.

  At the same time, there is a need to improve the process. In recent years, the US Food and Drug Administration has been influential in steering process improvement efforts through its 21st Century, Quality by Design (QbD), and process analytical technology (PAT) initiatives. This has led to industry reports such as, "A-MAb: A Case Study in Bioprocess Development," and novel methods for defining the design space.

One driver for improvement is the desire to accelerate the process of getting products to market, which is a direct result of the speed of knowledge development. The speed of obtaining knowledge (especially quantitative knowledge) affects timing, cost, quality, and the ability to meet regulatory requirements (Figure 1). Costs go down with more knowledge to asymptote steady state cost. Speeding up knowledge acquisition means costs are lowered more quickly. So the time value of information grows fastest at the early stage of a project. 

Strategies that emphasize the highest payout in quantitative knowledge per development time segment provide a significant increase in the time to review findings. The result is better decisions and better product quality.  

Stephen Covey teaches, "Begin with the end in mind."

  Doing so will increase the likelihood that you end up where you want to be as quickly as possible. The building blocks of QbD (Figure 2) show us what needs to be done to get to the end result. QbD shows the sequence and linkage of steps in knowledge development, allowing the right data to be collected when you need them. 

Along the way, four critical aspects of the downstream development process must be addressed:  

1. development of process and product understanding (risk assessment, experimentation method development, defining the design space) 

2. development of the process control (process control strategy, change control strategy) 

The development of appropriate measurement systems is central to the QbD building blocks. Measurement can be thought of as part of the process modeling work shown in Figure 2, which cannot be done effectively without good inputs. QbD clearly is more than just creating the design space. 

Process Understanding: How Do We Know When We Have Sufficient Data?

A commonly asked question is, "How much data?" It can't be said early on whether QbD will require more or fewer data; the answer is situational and depends on the approach currently being used. In the early stages of QbD implementation, you will likely collect more data than needed. Also, regulatory agencies are reviewing QbD approaches and will become more comfortable with data collected in ways different than in past filings. Over time, the amount of data required will decrease as we learn how to effectively target the most critical data. The FDA, and indeed good science, stress that to produce quality products on a sustainable basis, you need to understand your process. Process understanding exists when you can accurately predict the performance of your process. This leads to the conclusion that there are sufficient data when performance can be accurately predicted. The required accuracy (quality) and precision of the prediction vary over the life of the development process; it is higher at the end of development than at the beginning.  

Process Control: Strategy for Speeding Up the Work at Each Step

Of course, it is critical to balance speed and risk. An effective mantra is to be bold but not reckless. At each step in the development process, be aware of what data will be needed at various times during process development and manufacturing operations. The most important data to collect at any time are those needed to satisfy the short- and long-term objectives of the program. Over time, all of the important data will be collected. Non-important, or low value, data and information may become too expensive to collect, with low payout. The goal is to minimize risk at each point in time. 

Winning Strategies Recognize the Experimental Environment

Experience has shown that effective experimental strategies are a function of the experimental environment at hand. The environment defines the appropriate designs to be used to collect the data.

  A major goal is process understanding, which is a function of knowing the variables that have a major effect on the process. There are three environments that are commonly encountered. 

1. Little is Known About the Critical Process Variables

At the beginning of many studies, the critical variables are not known. In such cases, it is prudent to start with a screening study to be followed by characterization and optimization experiments that develop more detailed data on the variables, with the large effects identified in the screening experiment.

The end product when using a prioritization matrix approach (i.e., a cause and effect matrix) is a ranked list of the variables and actions to be taken, including performing studies using designed experiments, refining the measurement system, and an assessment using a failure modes and effects analysis (FMEA).

In many cases, the subject matter experts already know the core relationships that have effects so large that they swamp the effects of the other factors. In such cases, the first step is to run a small set of experiments to understand the nature of this core response relationship.  

3. Generalizing the Core Relationship to Other Products

As we move up the development cycle, we may want to determine if the core relationship can be generalized across similar product types. The question here is whether we can spot key parameters that allow us to be more efficient in quickly identifying the experimental design space of interest, and understanding the nature of the underlying relationship. The objective is to be even more efficient with new pipeline products. 

An example that illustrates many of the points made above is determining the operating range for low pH viral inactivation, a critical step in downstream purification. The purpose of this process step is to achieve maximum viral inactivation at an acidic pH condition that still maintains product quality. Based on prior knowledge, product quality can be significantly affected by several process parameters in addition to pH, including temperature, concentration, and hold time. The objective is to find the process design space of these process parameters for normal operation and the worst conditions. 

A typical pH response curve is shown in Figure 3. A commonly used strategy is for a fixed set of process parameters to run several pH points to identify the knee of the curve, which is the critical pH range for viral inactivation. After the knee of the curve has been identified, the process parameters could be varied one factor at a time until the desired set of conditions is identified. This process requires a large number of tests and takes considerable time. There is a better strategy. 

First, we must quantitatively understand the core relationship illustrated in Figure 3. This is similar to how the commonly used strategy begins. The first step is to run experiments for a small number of points along the range of the core response relationship, in this case pH and the quality attribute (QA) in the expected target setting of the other process parameters. Figure 4 shows this relationship. Knowing the nature of the core relationship, we can create the experimental design space in the region of interest. 

In this case, the region of interest is where the derivative is changing, i.e., the "knee," where the curve is bending over. When the knee of the curve has been found, a designed experiment is set up in the region of interest to explore the effects and interactions of the factors of interest, as illustrated in Figure 4. The workhorse central composite design is centered at the knee. From a relatively small number of experiments we can gain a good quantitative calibration of the region of interest and can establish a reasonable design space (Figure 5). 

We are now ready to establish the acceptable region of process operation. It has been found that a logit function models the relationship between pH and the QA very well. Knowing the non-linear model form, we can determine the optimal design points and the most precise estimate of the acceptance region using the following equation: 

 is the point of inflection where the quality attribute (QA) is equal to 

 is the steepness of the curve close to the inflection point.  

The optimal design points to fit a logit model are known based on point of inflection and range. These are shown in Figure 6 for two different situations. 

Different products have different points of inflection. Working from these points, a sensible design is found that takes into account the features of the nonlinear model and provides minimum error of prediction in the region of interest. 

To speed up the downstream process, you must get the right data, in the right amount, at the right time. Here's how.

The Time Value of Information in Designing Downstream Purification Processes

Challenges in the purification of monoclonal antibodies (MAbs) include reducing production cost, developing robust processes for both product purity and viral clearance, and integrating upstream and downstream processes. Increases in titers for MAb feed streams and clinical doses have led to the need to develop new products and technologies or adapt existing ones to meet these challenges. This paper will discuss the status of some of these new technologies and products and their potential for improving platform processes, as well as issues related to their implementation. 

Purifying proteins for therapeutic use requires highly selective and robust technologies to achieve the very high purity required for biopharmaceuticals. Processing such valuable product streams at large-scale while manufacturing to such high standards requires careful technology design and execution. This is because feed stocks containing the target protein most often are obtained from the culture of live cells and are variable in both product content and composition. Since 2003, 26 of the 31 newly approved protein therapeutics are produced in either 

 microbial biosynthesis or mammalian cell culture.

  Because proteins degrade easily as a result of extremes of pH or temperature, or contact with common solvents, controling the variables during processing is especially important to avoid having to remove impurities formed during processing, because these impurities affect manufacturing yields and the activity profile of the therapeutic protein.

The lowest production cost is achieved not by increased process complexity but by the opposite: Operating faster and with the fewest and most efficient downstream steps reduces investments in validation and subsequent batch failures.

  Some high-dose monoclonal antibody (MAb) products require an annual production of metric tons of material to supply the market. Demand for quantities this large exceed current manufacturing capabilities requiring companies to invest in additional capability to meet demand. This delay can contribute to missed revenue. Current platforms for the large-scale production of MAbs, for example, show that it is essential to design and operate an integrated series of purification steps that are both high yielding and product molecule–specific.

Emerging strategies to improve process efficiency include high-throughput technologies, using disposable equipment, and using new filters and adsorbents that combine inexpensive substrates with greater functionalities, such as charge or lipid binding. In addition, increasing volumetric titers into downstream processing also has created technological bottlenecks that must be solved. How to take advantage of the multitude of emerging technologies while streamlining and improving a platform downstream process for MAbs remains one of the major challenges faced by practitioners of the art today. 

Although some types of disposables such as media and buffer bags have been in use for years, the use of purification-related disposables is relatively new. The advantages in their implementation include reductions in preparation and product changeover, ease of implementation and validation, elimination of cleaning procedures, and a reduction in capital outlay.

  New and improved disposable technologies include single-use chromatography membranes and ready-to-use columns, ultrafiltration cassettes, and chromatography and ultrafiltration flow paths containing single-use monitors and transducers. However, it is not always easy to incorporate into existing processes as a one-for-one swap without additional development work. In addition, manufacturing costs must be considered, taking into account the advantages of disposables discussed above compared to disadvantages such as increased material demand and validation of leachables.

The use of disposable membranes in MAb purification has shown potential to replace traditional resins, especially for anion exchange (AEX) membranes that are used in flow-through mode, in which the impurities bind to the membrane while the antibody flows through. The binding capacity of the target protein has previously been a problem for membranes because of their lower surface-to-bed volume ratio as compared to resins,

  so flow-through operations present an ideal situation for membrane use. The advantages of these membranes include improved mass flow properties because of the porous structure of the membrane hierarchy, and ease of preparation before use.

  The initial preparation time is significantly reduced because column preparation operations such as packing and qualifying, and post-use cleaning and storage are eliminated.  

Figure 1 shows data comparing the DNA removal properties of two commercially available membranes, Sartobind Q (Sartorius Stedim Biotech, Germany) and Mustang Q (Pall Corporation, New York), as compared to various AEX resins. The starting material was affinity-purified broth from a Chinese hamster ovary (CHO) cell line expressing a MAb, and the flow was comparable based on a linear flow rate of 200 cm/h. The data are shown as DNA fold reduction, which is the starting value in the load material divided by the final value in the flow-through pool. The graph shows that both membranes were better at removing DNA than the resins tested, possibly because of faster, and therefore, more efficient charge interactions between DNA and the functional groups on the nonporous surface of the membrane. Additional experimentation also has shown a six-fold difference in the total amount of DNA that can be removed by a membrane as compared to resin.  

However, host cell protein (HCP) removal by membranes can be a challenge. Side-by-side comparison of the Sartobind Q membrane to Q Sepharose Fast Flow resin has shown the membrane to be significantly less capable of removing HCP for some antibodies (Figure 2). However, new advances in salt tolerant membranes, such as the STIC from Sartorius Stedim and the Chromasorb from Millipore (Bedford, MA), which includes changes in the ligand type as well as the support and attachment chemistry, have shown significant improvements in HCP removal capability as compared with traditional membranes (Figure 2).  

The question that remains for these disposable membranes relates  to the cost of goods. Given the higher cost of membranes, their use makes sense during early-phase manufacturing when the number of batches is small, but how do they stack up in commercial manufacturing? The answer may lie in the ability of the membrane to be used multiple times. We have shown that reusing the Sartobind Q membrane up to 10 times does not affect its ability to remove DNA, but validation for reuse is an issue because it has yet to be proven.   

Another advance in disposable technology is the single-use ultrafiltration cassette, such as the SIUS from Novasep (Pompey, France). In a comparison with standard multi-use cassettes, the SIUS showed a significant increase in flux capacity and reduced processing time for both concentration and diafiltration unit operations (Figure 3). The starting material in this case was a purified MAb, concentrated and diafiltered on small (100 cm

  or equivalent) 50 kD cassettes using an AKTA Crossflow system from GE Healthcare (Uppsala, Sweden). Although Figure 3 shows the data for only a single antibody, the same trend was seen with other therapeutic antibodies as well. These single-use cassettes can be paired with new disposable flowpath technologies to produce a true single-use unit operation that eliminates post-use cleaning and storage. Again, we must determine the cost of goods for commercial manufacturing as opposed to early-phase use.  

One potential purification challenge related to increased titer is the lack of capacity for Protein A resins. Several attempts to address this by removing Protein A resin from MAb purification schemes have met with not much success. However, one promising technology adapted from other industries is multicolumn countercurrent solvent gradient purification (MCSGP), which is a hybrid between simulated moving bed technology used in the chemical industry and standard batch chromatography. The adaptation of this technology for MAb purification has been explained in detail in multiple articles.

This technology can be applied either to increase the capacity of standard affinity resins in antibody purification platforms, or to increase the resolving power of non-affinity unit operations. This method potentially could facilitate the removal of affinity chromatography from an antibody purification process. Using this technology in a nonaffinity two-chromatography step process, we achieved a seven-fold increase in productivity with no reduction in impurity removal as compared with a standard affinity three-column process (data not shown). Multiple companies are in the process of developing chromatography systems and columns to optimize this technology. 

A second type of technology used in other industries that has been adapted with some success is precipitation. This method can be of two types: positive precipitation, in which the product of interest is precipitated, leaving the impurities behind; and negative precipitation, in which impurities are precipitated, leaving the product of interest in the supernatant solution. Precipitation generally is used early in the purification process, to remove large quantities of impurities for increased column performance before polishing steps using resin chromatography. It has been classified as a technology with low resolution potential but high industrial maturity.

  Chemicals such as ammonium sulfate, polyethylene glycol, and other polyelectrolytes for positive precipitation of antibodies from crude broth have been used for several years. Although they have not gained a significant foothold against standard capture chromatography methods, lately there has been renewed interest in developing them as alternatives to the standard antibody purification process in which affinity resin is the workhorse.

Recently coming into more extensive use are negative precipitants, designed to leave the product of interest in solution. One of these is caprylic acid, a short-chain fatty acid that can be added directly to conditioned media out of the reactor to remove host cell impurities such as DNA and protein. A significant reduction of DNA and HCP can be achieved using caprylic acid at concentrations of 100–500 mM and pH values of 4.0–6.0.

  However, HCP removal was achieved only for CHO-produced antibodies, not for NS0-produced antibodies.

  Even with this limitation, the use of caprylic acid has been incorporated into manufacturing processes to facilitate the use of a two-chromatography step process without Protein A resin.

New advances in filtration, including both multilayer depth filters and nanofilters, have begun to address limitations in throughput and purification by providing new tools that increase the flexibility of order for current unit operations. These new filters also have the potential to replace standard chromatography unit operations. 

A new depth filter under development by 3M Purification (St. Paul, MN), is being designed to increase flexibility in MAb purification by enhancing the removal of DNA after centrifugation, and of both DNA and HCP removal after capture-step chromatography. This filter, designated ZPQ020, is a hybrid purifier that contains both a size exclusion component and an AEX (charged) component. 

Figure 4 shows the excellent DNA removal capability of this filter after centrifugation of harvest broth. The graph presents the log reduction values from five CHO-produced antibodies where a new filter was used after centrifugation. In two of the five cases, the DNA was removed completely, and in the other three, the log reduction was significant. Other filters tested showed minimal to no DNA reduction. Removing DNA before an initial Protein A capture chromatography step could potentially extend the resin life of a very expensive raw material, or even allow the resin to be eliminated altogether and substituted by a cheaper resin such as ion exchange.  

In addition, this filter also exhibits excellent impurity removal post-capture as compared with standard AEX technologies. The results from two CHO-produced MAb tested post-affinity columns are presented in Figure 5. In both cases, these antibodies exhibited higher than normal levels of DNA and HCP. The synthetic 3M filter exhibits better HCP removal for feed streams with higher than normal impurity levels. In terms of DNA removal, the 3M filter showed better DNA and HCP reduction for the two antibodies tested than the other AEX technologies evaluated. Future studies are needed to compare this filter with the new salt tolerant membranes.  

Advances in nanofilters also have provided flexibility in the purification process, allowing for greater throughput resulting in decreased membrane area, or reducing the processing time for comparably sized membranes. New nanofilters such as Asahi Kasei's BioEX or Millipore's VPro can significantly increase flux. Figure 6 shows the flux data for two CHO-produced antibodies comparing Asahi Kasei's Planova 20N to the new Planova BioEX. The flux for the BioEX is significantly higher than for the 20N, though with other antibodies we have seen greater flux decay for the BioEX (data not shown). The effect of this increased flux on viral clearance could be a significant concern, although initial assessments show no effect.   

However, as we push the envelope toward higher concentrations and salt conductivities (such as in conjunction with the use of salt-tolerant membranes), we may start to see a reduction in clearance values. Figure 7 shows the log reduction values for Mouse Minute Virus (MMV) for two purified CHO-produced MAbs at a low pH and high pH value, at two higher salt concentrations (>100 mM sodium chloride). The load antibody aggregate concentration for these antibodies was also relatively high (>5%). These starting materials were filtered across a Planova 20N filter at standard operating conditions per the manufacturer's recommendations. Complete removal of virus was only seen under one set of conditions—low pH and low salt (MAb 5). For all other conditions, virus was detected in the filtrate. These data indicate that under certain conditions, the virus is extruded through the nanofilter, a factor that must be considered when designing the purification process. 

Expression titers and dosing levels for monoclonal antibodies (MAbs) continue to increase as medical needs are successfully met with these products, placing pressure on both existing production capabilities and development decisions for downstream purification processes. This means rising production costs because extra unit operations are needed to meet the regulatory requirements for product purity and viral clearance. New technologies, such as disposable membranes and cassettes, along with new depth filters and nanofilters, or the adaptation of existing technologies from other industries, can be used to meet these production challenges. By providing flexibility not only in the order of unit operations, but in the design of the steps themselves, these new products and methods will enable purification processes that are both cost efficient and effective in producing the desired product. 

The authors would like to thank the Cell Line Development and Process Development Analytics groups at Pfizer St. Louis for their support, as well as Sartorius Stedim, 3M, and Asahi Kasei for providing product samples and technical expertise.  

 is an associate research fellow, all at Pfizer BioTherapeutics Research and Development, St. Louis, MO, 636.247.6519, 

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New technologies and adaptations of existing technologies can improve platform processes.

BioPharm International-03-02-2010

Mixed-Mode Chromatography in Downstream Process Development

PEG Precipitation: A Powerful Tool for Monoclonal Antibody Purification

The Time Value of Information in Designing Downstream Purification Processes

Advances in Monoclonal Antibody Purification