Bio
`a“
`Protein A
`
`’
`
`- O
`
`The Life of a Disruptive Technology
`
`by Blanca Loin
`
`
`
`
`
`
`
`WiKiMEDiACOMMONS(HTTP//(OMMONSWWMED‘AORG)
`
`he number of blockbuster monoclonal antibody
`(MAb) drugs continues to grow. In 2008, MAbs
`generated revenues in excess of US$15 billion (1),
`making them the highest—earning category of all
`biotherapeutics. The world MAb market will reach
`$62.3 billion in 2015, with next—generation therapeutic
`antibody revenues reaching $2.3 billion in 2015 according
`to Visiongain reports published in September and
`November 2011 (2, 3). Biosimilar antibodies will also begin
`to enter established markets as regulatory authorities clear
`approval pathways for them. Most antibody drugs treat
`cancer and autoimmune diseases, and many of the rest are
`used to treat orphan and infectious diseases. Unfortunately,
`antibodies are complex proteins in many ways, which
`complicates their purification and characterization,
`making it difficult for their developers to meet the rigid
`requirements for therapeutics.
`Because of the inherent and engineered variations in
`therapeutic antibody structures, there is no “one—size—fits-
`all” when it comes to techniques for MAb purification.
`The closest anything has come to this is evidenced by the
`success of protein A affinity chromatography, which has
`become the workhorse for antibody production. However,
`protein A is expensive (with costs an order of magnitude
`over conventional chromatography resins), susceptible to
`degradation by proteases (cleaved domains can adhere to a
`MAb product, complicating separations) (4), and not fully
`stable to column washing and elution conditions. It can
`generate an immunomodulation response and has limited
`capacity to accommodate the increasingly high titers found
`in modern upstream feeds.
`Although the antibody purification field is advanced,
`among companies involved there has been some reluctance
`to invest in and introduce new technologies and/or further
`advance purification technologies. Some articles have
`referred to new alternatives as “disruptive” (5) while
`predicting that protein A will continue to be used for
`commercial-scale MAb purification throughout the
`
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`

`
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`
`industry needs to lower
`production costs and pass along
`those savings by making medications
`more affordable for patients. The
`emergence of biosimilars (or follow—on
`biologics) and a growing number of companies
`seeking to capitalize on such products may create a perfect
`climate to facilitate innovation in purification (7).
`
`
`
`
`foreseeable future (5, 6). But the
`protein A sorbents are based on controlled porous glass,
`
`coated porous polymer gel-filled mineral materials, and
`other supports (22). Such materials are rigid enough to
`allow for column operation at high flow rates. Table 1 lists
`some of these products.
`State of the Art: Since the first reports over 40 years ago
`involving use of immobilized protein A for affinity
`purification of antibodies (23,24), it has become the
`industrial standard for purification of clinical-grade MAbs
`(2S). Janssen Biotech’s Muronomab (brand—name Orthoclone
`OKT3) is a CD3-specific MAb that was approved by the
`US Food and Drug Administration (FDA) in 1986 (26) for
`use in treatment of acute transplant rejection. It was the first
`approved product made using protein A as a capture step in
`its manufacturing process. That serves as the key volume-
`reduction step in antibody downstream processing.
`The purification scheme for protein A chromatography
`is simple: Bind at neutral pH and elute at acidic pH.
`Method development is easy, which is probably the main
`reason why protein A affinity chromatography has been
`universally adopted in large—scale manufacturing processes.
`Its almost universal applicability and the overall scheme of
`associated operating conditions lend themselves readily to a
`platform format (6).
`
`How DID IT ALL START?
`
`Protein A is a 42—kDa protein anchored in the cell wall of
`Staphylococcus aurcus (8) with the ability to selectively
`interact with immunoglobulins (IgGs) (9). It binds strongly
`to all classes of human IgGs except for IgG3 (9). Full—
`length protein A consists of five homologous domains
`(referred to as E, D, A, B, and C, in order of their
`arrangement from the N—terminus) and one cell—wall—
`associated domain (10, 11). At first, protein A was produced
`by culturing the Cowan strain of S. aurcus and extracting
`the protein from the bacterial cell walls (12). Later, a strain
`of S. aurcus was discovered that secreted protein A into its
`culture supernatant (13). As recombinant DNA technology
`advanced, protein A could be expressed as a fragment
`without its cell-wall domain using Escherichia coli as an
`expression host (14—17).
`IgG binds to protein A at its Fc region (18, 19). The
`interaction is very specific and hydrophobic in nature. It
`involves some hydrogen bonds and two salt bridges. The
`high specificity enables protein A affinity chromatography
`to remove >98% of impurities from complex solutions such
`as cell harvest media in a single purification step (20). One
`drawback of the well-known specificity of protein A’s
`interaction with IgGs is that it usually necessitates the use
`of harsh conditions such as low pH for elution. That can be
`problematic for some antibodies that are either unstable or
`tend to aggregate at low pH levels. In general, only a small
`amount of impurities — e.g., aggregates, residual host-cell
`proteins, DNA, and leached protein A — will remain after
`this single starting unit of downstream process operation.
`They usually can be removed in either one or two
`additional chromatography steps.
`Affinity Supports: Protein A has been immobilized to
`all supports suited for liquid chromatography of proteins
`(21). Initially, the most popular product was protein A
`immobilized on CNBr—activated Sepharose CL 4B from
`Amersham (now GE Healthcare) in Sweden. The medium
`
`exhibited high selectivity and low nonspecific adsorption,
`but due to the nature of the agarose-based support, a
`packed bed would be too soft to allow for high flow rates.
`So that medium has been largely replaced by more highly
`cross-linked Sepharose for large-scale applications. Modern
`
`30 BioProcessInternational
`
`11(8)
`
`SEPTEMBER 2013
`
`ADDRESSING THE DIFFICULTIES
`
`Multiple studies have compared the performance of different
`sorbents that are commercially available. Hahn and
`coworkers studied and compared 15 different protein A
`sorbents (22,27, 28). Ghose et al (29) evaluated the dynamic
`binding capacity (DBC) of several resins for antibodies and
`Fc fusion proteins. Swinnen et al (30) studied the effect of
`load concentration on the DEC and used mathematic models
`
`to evaluate process robustness and volumetric productivity.
`You can divide protein A resins into those suitable for
`preparative scale and those that are not. Rated by DBC, the
`top four brands are MabSelect Xtra and MabSelect SuRe
`LX (higher ligand density) from GE Healthcare (31), Prosep
`Ultra Plus (which does not allow for cleaning with sodium
`hydroxide, NaOH) from EMD/Merck Millipore, and Poros
`MabCaptureA (which allows only 0.1N NaOH) from Life
`Technologies. The capacity of protein A resins at a residence
`time of three minutes ranges from 30 to 45 mg/mL. LX
`gives MabSelect a 20—40% increase in capacity over what
`SuRE media can offer, but only at residence times of six
`minutes or longer. Such long residence times would extend
`the length of this unit operation and adversely affect cost.
`Efficiencies: To address some pitfalls and limitations of
`protein A affinity resins, some vendors have made efforts to
`improve them. Most efforts have applied recombinant DNA
`techniques to increase the ligands tolerance to cleaning with
`NaOH, lower its binding affinity to enable milder elution
`
`

`

`Table 1: Some commercially available protein A sorbents for affinity chromatography; DBC : dynamic binding capacity
`
`Sorbent
`CaptivA
`
`MabSelect
`Xtra
`MabSelect
`SuRe
`
`MabSelect
`SuRe LX
`
`Prosep
`Ultra Plus
`Poros Mab—
`Capture A
`
`Ligand
`Recombinant
`native protein A
`Recombinant
`protein A
`Tetramer
`alkali—stabilized
`Z—domain
`
`Tetramer
`alkali—stabilized
`Z—domain
`
`Manufacturer Bead Matrix
`Repligen
`4% agarose
`4FF
`GE Healthcare Highly cross—
`linked agarose
`GE Healthcare Highly cross—
`linked agarose
`
`GE Healthcare Highly cross-
`linked agarose
`
`Recombinant
`native protein A
`Life
`Recombint
`native protein A Technologies
`
`EMD Millipore Controlled
`pore glass
`Polystyrene
`divenyl—
`benzene
`
`Mean Particle DBC at 3 min
`Diameter
`Residence
`90 um
`~38 mg/mL
`
`Working
`Flow Rate
`300 cm/h
`
`Caustic
`Tolerance
`0.1 N NaOH
`
`Estimated
`Price*
`$5,800/L
`
`75 pm
`
`35 mg/mL
`
`100—300 cm/h
`
`50 mM
`
`$12,803/L
`
`85 pm
`
`230 mg/mL
`
`100—500 cm/h
`
`85 um
`
`45 mg/mL
`
`100—500 cm/h
`
`0.1—0.5N
`NaOH
`
`0.1—0.5N
`NaOH
`
`$15,850/L
`
`$17,157/L
`
`60 um
`
`~48 mg/mL
`
`800 cm/h
`
`None
`
`$14,440/L
`
`45 um
`
`>45 mg/mL
`
`700 cm/h
`
`0.1 N NaOH
`
`$13,750/L
`
`TOYOPEARL
`AF—rProtein
`A—650F
`
`Tosoh
`Tetramer
`alkali—stabilized Bioscience
`C domain
`
`Polymeth—
`acrylate
`
`45 um
`
`>30 mg/mL
`
`51,000 cm/h
`
`0.1—0.5N
`NaOH
`
`$12,240/L
`
`* 2013 list prices in US dollars (from websites or direct sales inquiries) listed as fair comparison without discounts (e.g., for large—volume orders)
`
`conditions, and allow chemoselective-oriented
`
`immobilization to the base matrix through stable linkage
`chemistries (5,32). For example, the SuRe resin was
`developed to withstand stronger alkaline conditions.
`Using protein—engineering techniques, a number of
`asparagine residues (the most alkali-sensitive amino acids,
`making proteins prone to deamidation) were replaced in
`the Z domain (a functional analog and energy-minimized
`version of the B domain) of protein A, and a new ligand
`was created as a tetramer of four identical modified Z
`
`domains (29). Under the MabSelect brand, a recombinant
`
`protein A was engineered to include a C—terminal cysteine
`so it would allow oriented coupling by means of a thioether
`linkage through a stable 12-atom epoxide spacer arm to the
`base matrix. That ensures very low ligand leakage, and the
`selective orientation provides for a higher DBC. The Xtra
`version has the same backbone as the MabSelect brand, but
`
`its wider pores further increase binding capacity.
`Because protein A is expensive, most manufacturing
`processes run it in a smaller column for several cycles to
`purify a single batch rather than using a large column in a
`single cycle. Cycling increases total purification time and
`thereby decreases production rate. Antibody expression
`titers average 8—10 g/L and continue to increase. With
`current protein A capacities, processing times can be very
`long and throughputs very low. It is easy to see how a
`protein A resin with a DBC 250 g/L even at higher flow
`rates (lower residence times) is needed to accommodate
`
`such expression levels and improve operations. That would
`be very welcome, but how can it be achieved?
`Some options for next-generation adsorbent
`development could include higher ligand density (e.g., Sure
`LX brand), alternative ligand orientation and accessibility,
`bead and pore size changes, and modified support matrices
`with increased size and potential mass transfer (6). The
`theoretical limit for protein A in an agarose support is
`70 g/L, which may have been overestimated based on some
`
`32 BioProcesslnternational
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`11(8)
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`SEPTEMBER 2013
`
`simplistic assumptions (6). However, current reported
`capacities do show room for improvement.
`Economics: Cost is another very important factor. Some
`protein A resins cost as much as $15,000/L. Even with
`larger numbers of players and competition as time has gone
`by, prices continue to be quite high, although Repligen
`resins cost about $6,000/L (but they will tolerate only 0.1N
`NaOH). Some people argue that protein A’s main
`limitation is capacity or productivity rather than cost (7)
`and that capacity issues come from using porous particles
`in a fixed bed. Protein A itself occupies a large amount of
`intrapore space in porous media because of its size.
`A recent and possibly revolutionary approach to
`increasing protein A capacity was developed by Polybatics
`Ltd. in New Zealand (33). The company developed protein-
`A—based ligands that are already cross—linked to polyester
`beads. E. coli has been successfully engineered to
`manufacture those beads displaying multiple copies of the
`antibody-binding Z domain derived from protein A
`(PolyBind—Z beads). This technology harnesses a natural
`polyester storage granule formation process inside the
`bacteria for production of ligand-coated polyester beads.
`The specific lgG binding functionality of PolyBind—Z
`beads is 100 mg per gram of drained beads. That represents
`at least a doubling of the published binding capacities for
`most commercial protein A resins. Homogenous functional
`orientation of the ligand and elimination of the need for
`cross—linker removal represent further advantages of this
`new polyester bead-based technology. For commercial-scale
`applications, the company has investigated use of its beads
`in a cross-flow filtration system and obtained promising
`results. Polybatics proposed that these beads could be used
`as a disposable resin to reduce the overall cost of
`downstream processing for antibodies. It is not clear
`whether the capacity would remain that high if the beads
`were attached to a support. Other issues to be addressed
`are availability of the beads as good manufacturing practice
`
`

`

`(GMP)—compliant material and the potential for excessive
`compression when they are packed in a column. But these
`beads could be ideal for processing systems without packed
`beds (e.g., using fluidized beds or continuous
`countercurrent tangential chromatography).
`
`ALTERNATIVES
`
`Some very good reviews have been published on the matter
`of alternatives to protein A capture (5, 7, 32). Those
`alternatives come in nonchromatographic and
`chromatographic forms. Chromatography is often portrayed
`as a “necessary evil” of downstream processing due to its
`associated costs, batch operations, throughput, and
`complexity of scale-up. So nonchromatographic separation
`techniques for antibody purification are becoming attractive
`to many developers. The pharmaceutical industry is looking
`back now to methods that have been used for some time in
`
`the nutraceutical and enzyme industries: e.g., precipitation,
`crystallization, and aqueous two-phase systems (ATPS).
`Although none of those methods is especially amenable to
`platform use, they are under consideration because of their
`relative affordability and scalability. And they could offer
`benefits in capital avoidance.
`MAb crystallization early in downstream processing
`would be very difficult. Impurity levels are high in clarified
`harvest, and scalability of crystallization processes can be
`problematic. Even at small scales, such a process would be
`difficult because antibodies are large and glycosylated, and
`MAb molecules have a high degree of structural flexibility.
`PEG Precipitation: Precipitation with polyethylene glycol
`(PEG) can serve as a concentration step. Such a step can be
`designed either to precipitate antibody and leave impurities
`in the media (34, 35) or to precipitate the impurities and leave
`the antibody in solution (36). In the first case, precipitated
`antibody can be resuspended in a buffer of choice to be ready
`for a subsequent unit operation (without diafiltration or
`dilution). Removing supernatant by microfiltration or dead—
`end filtration then eliminates the need of bulky, expensive
`hardware for centrifugation. It also allows for washing the
`precipitated antibody, which further increases its purity.
`The only concerns in regard to precipitation are that
`some antibodies can denature during a precipitation
`process. Afterward, the precipitating agent must be
`completely removed from the final product. An assay
`capable of measuring residual precipitating agents would
`need to be developed and implemented. Some other
`reagents used for precipitation are ammonium sulfate and
`caprylic (octanoic) acid.
`ATPSs are formed by mixing a polymer (usually PEG)
`and a salt (e.g., phosphate, sulphate, or citrate), or two
`polymers, with water. These systems can be effectively used
`for separation and purification of proteins. The two-phase
`technique has been applied to antibodies that tend to
`transfer spontaneously from aqueous formulations into
`more hydrophobic PEG-rich solutions (37). Advantages of
`this separation method include the potential for relatively
`easy scale-up and continuous operation, ease of process
`integration, low toxicity of the phase-forming chemicals,
`
`SURVEY SAYS . . .
`
`BY CHERYL SCOTT
`
`In March of 2013, BPI conducted an informal, self-select survey
`of about a hundred industry participants in the United States,
`Canada, and Europe. We asked them a few questions about
`some industry, regulatory, and cultural trends related to
`protein A and its competitors.
`
`How long do you believe protein A chromatography as currently
`practiced can meet the biologics industry’s needs, including
`pipeline products and biosimilars?
`
`protein A replacements?
`
`10 yea rs
`
`Do you believe the high cost of
`protein A is a barrier to making
`top-quality healthcare
`available to the third world?
`
`For which ofthe following
`products would you consider
`Protein A replacement?
`
`Licensed
`' roducts
`
`Pipeline
`
`Biosimilars
`
` Is your company actively seeking
`
`Products
`
`1—2 yea rs
`
`Do you believe any
`packed-column technology
`can replace protein A?
`
`lfyou considered a technology
`qualified to replace protein A, what
`would be the transition time for its
`implementation in your company?
`
`and biocompatibility. Some ATPS issues that remain
`unresolved are performance variation among different
`antibodies and system capabilities in relation to virus and
`DNA reduction. It remains to be seen which intermediate
`
`and final purification steps would follow to deliver a
`product of high-enough quality.
`Other Chromatographic Methods: A significant amount
`of effort has gone into further developing one type of
`fluidized chromatography technique known as expanded-
`bed absorption (EBA). It involves the use of adsorbent
`particles dispersed in a liquid media. One main benefit of
`EBA is a reduction in the number of steps required for
`antibody recovery due to direct capture of product from a
`cell suspension. For antibodies, however, this application
`
`SEPTEMBER 2013
`
`11(8) BioProcesslnternatior-al 33
`
`

`

`BY CHERYL SCOTT
`SURVEY SAYs . . .
`
`In our March 2013 survey, we asked
`about a hundred industry participants a
`few questions about the benefits and
`liabilities of protein A and its competitors.
`Clearly it is a subject of much debate, as
`evidenced by a healthy representation in
`our results of both very positive and very
`negative outlooks.
`
`Assessing the Benefits: In order of
`importance (and based on percentages of
`responses for each), participants ranked
`the benefits of protein A as follows.
`
`. Most Important — high single-step
`purity; ease of use; capacity;
`recovery; broad, predictable
`applicability to lgGs (platform use);
`and familiarity to process
`developers, manufacturing
`operators, and regulators
`
`. Somewhat Important — minimal
`well-characterized optimization
`pathway; large selection of available
`products, and multiple suppliers
`
`Assessing the Drawbacks: In order of
`importance, participants ranked material
`cost as the main drawback of protein A,
`far and above other concerns such as
`
`capacity limitations, availability,
`
`immunotoxicity, low productivity,
`multiple cycling, and validation costs.
`
`As expected, lower cost ranked a clear
`first among features a candidate would
`need to demonstrate for replacing
`protein A. Other important features were
`equivalent (or broader) applicability and
`higher capacity, productivity, recovery,
`and purity — all ranked as"very
`important" alongside compatibility with
`existing facilities and a capability of
`producing highly concentrated lgGs.
`Single-cycle, single-shift batch processing
`was deemed "somewhat important," as
`were a familiar operating format and
`well-established vendor, single-use
`capability, similar or greater ease of use,
`and less toxic leachables.
`
`Why Does It Persist? Survey
`respondents listed competition among
`protein A vendors and increasing
`development of biosimilars almost
`equally as trends allowing for the
`persistance of protein A in the bioprocess
`arena. Coming in a close third place was
`a trend among regulators toward more
`stringent purity requirements (e.g., lower
`cell host protein, aggregates, DNA, virus).
`
`Supporting Data: Respondents most
`want to see a clear description of method
`development variables and ranges, along
`with a clear explanation of the fractionation
`method, for any potential replacment of
`protein A. They would also look for detailed
`examples, including results with similar
`products, and expect to see those
`published in a peer-reviewed format.
`
`The most likely candidates for success
`were bioaffinity chromatography using
`protein A analogues and synthetic
`protein A analogues (e.g., multimodal
`ligands). Less likely winners (in order of
`preference) included cation-exchange
`chromatography, aqueous two-phase
`separations, and precipitation methods.
`
`What Happens Next? Ifa highly
`superior replacement emerged, most
`respondents (53.8%) believe that protein
`A might be phased out as a
`manufacturing method within 10 years. A
`skeptical third (30%) thought it would
`persist as a manufacturing method
`indefinitely, and about half as many were
`optimistic enough to say that it could get
`phased out completely within five years.
`
`could not replace protein A, which in a form bonded to
`agarose is used to capture the antibody in EBA.
`The first generation of EBA adsorbers was plagued with
`technical problems. A second—generation version (38) has
`addressed some of those issues by development of high—
`density adsorbents coupled with improved column designs.
`Heavier particles have been synthesized by encasing a dense
`tungsten—carbide core with cross-linked agarose. Those
`particles are retained in a column by their own weight, even
`at the high flow rates required for commercial processing.
`The capacity of settled particles is low compared with
`traditional protein A packed resins, but that limitation is
`compensated for by eliminating the need for clarification.
`Simulated moving-bed (SMB) chromatography is a
`continuous, multicolumn system that increases throughput
`while reducing chromatography bed volume and buffer
`consumption (39). Some regulatory issues still must be
`addressed — e.g., in-process documentation, pooling, and
`batch definition — and it could be difficult to validate and
`
`maintain aseptic SMB operations. To a certain extent, it
`could be applicable to current manufacturing processes in
`which, to economize the high cost of protein A, companies
`pack smaller columns and cycle them multiple times to
`process a single batch. In most SMB examples, protein A is
`still the preferred method for antibody capture. And in a
`recent BPI industry survey (see the “Survey Says” boxes for
`more detail), this technology ranked above fluidized bed
`and monolith options as the most likely to significantly
`improve or prolong the performance of protein A.
`
`34 BioProcesslnternational
`
`11(8)
`
`SEPTEMBER 2013
`
`Warikoo et al. reported a very recent application of
`periodic countercurrent chromatography (PCC) as part of
`an integrated and continuous processing of recombinant
`proteins (40). Their study showed that such a system
`coupling a high-density perfusion cell culture with a
`continuous capture step could be used as a universal
`biomanufacturing platform. Another novel physical format
`(still in the experimental stage) merges the principles of
`SMB with EBA for continuous countercurrent tangential
`chromatography (CCTC), as reported by Shinkazh et al.
`(41). They pumped slurries of porous particle media
`through a series of static mixers and tangential-flow
`membrane modules to separate a model protein mixture
`containing bovine serum albumin and myoglobin using a
`commercially available anion-exchange resin.
`Membrane adsorbers (“membrane chromatography”)
`offer clear advantages over conventional resins, both in
`terms of disposability (which eliminates the need for
`cleaning and validation) and the ability to operate at high
`flow rates. Because of their lower surface area, however,
`
`most membrane adsorbers suffer from low binding capacity
`compared with an equivalent volume of porous particles. An
`exception to that limitation is membrane technology
`developed by Natrix Bioseparations (35). The company’s
`membranes consist of a polymeric hydrogel formed within a
`flexible porous support matrix. That macroporous hydrogel
`polymer structure provides high binding-site density and a
`large surface area for binding and rapid mass transfer.
`Kuczweski et al. developed a membrane-based, high-
`
`

`

`BY CHERYL SCOTT
`SURVEY RESPONDENTS SAY . . .
`
`In our March 2013 survey, we also asked
`for some general commentary, and here
`are some of the most interesting remarks
`we received.
`
`Defending Protein A
`"As more vendors start to offer protein A
`resins, I am wondering about a trend of
`decreasing the resin cost to alleviate the
`competition pressure from each other
`and from other technologies."
`
`"Protein A is a well—known, proven
`reagent that does not require an
`experienced scientist to handle it."
`
`"I think protein A will probably continue
`to be used extensively for some time.
`Continuous processing will enable
`significant improvements in productivity
`and investment required for use."
`
`"Improved stability to caustic sanitization
`solutions is good, but currently available
`stable resins are so much more costly
`that they offer no real benefit to
`biomanufacturers. It’s possible to double
`the lifetime of a more stable resin, but if it
`costs twice as much there is no incentive
`
`to change. Protein A costs are
`
`acceptable only with long media lifetimes
`(in number of cycles). But in a
`multiproduct facility, 200 cycles
`correspond to quite a large number of
`batches, which isn't always achievable."
`
`"There's been more interest in protein A
`replacement technologies than anything
`else for over 15 years, and it is still the #1
`matrix for MAbs. Material cost is not the
`
`only consideration; there are also time
`of development and GMP facility use.
`Now that we have two—step processes
`based on new protein A matrices, I’m
`more a believer than I was 20 years ago."
`
`In Support of Alternatives
`"A number of companies are actively
`developing fully synthetic mixed and
`multimodal-ligand designs and resins
`that I believe can challenge protein A
`performance. It remains a constant
`technical challenge to achieve the
`conformational specificity that a
`proteinaceous ligand can offer. But with
`low-cost, near-physiological pH elution;
`chemical resistance for cleaning in place;
`and improved solid—phase architectures,
`
`these products will continue to develop
`interest for whole and fragmented
`antibody-derived biologics. Such low-
`cost, high-performance products will
`help us apply the increasingly important
`single-use strategy to biomanufacturing.
`And as biotherapeutic discovery and
`development moves toward more
`complex, engineered antibody-related
`products, the highly specific binding
`characteristics of protein A may
`eventually hinder its application. We are
`only one iterative design away from the
`next big antibody scaffold that it can’t
`capture."
`"I think the monolith and membrane
`
`formats might be taking over from
`conventional resins. If these work well,
`protein A will be around for 20 years or
`more."
`
`"Capacity of current protein A gels needs
`to be increased to >100 g/L for it to
`remain a competitive technology."
`
`"Protein A is already antiquated in
`laboratory applications. Industry, as
`always, will take a decade to catch up."
`
`capacity, cation—exchange capture step for MAbs using a C
`membrane from Natrix. They reported a capacity of 55 mg
`of antibody per milliliter of membrane, which is about five
`times that of other membrane adsorbers (35). Because of low
`
`capacities and dilutions created by the void volume and
`variations in membrane thickness, most of these adsorbers
`
`are preferentially used in flow-through mode.
`Alternative Resins and Ligands: With development of
`high—capacity cation exchange (CEX) resins capturing
`2100 mg IgG per milliliter of resin, it has become more
`attractive to implement CEX as a capture alternative to
`protein A. I led a group demonstrating how a high-capacity
`CEX capture step could deliver MAb of the same purity
`and quality of protein A produced material (42). Although
`CEX could require more development work than protein A
`for each antibody, the benefits of high capacity and
`significant cost savings make a strong case for its
`implementation in some manufacturing processes.
`Finally, what are the alternative ligands to protein A?
`Among bioaffinity ligands are some other naturally
`occurring immunoglobulin—binding proteins such as protein
`G and protein L. But both suffer the same shortcomings as
`protein A in terms of low capacity and harsh elution
`conditions. Camelid mammals produce IgG analogs made up
`of only heavy chains: VHH single-variable heavy-chain
`fragments from camels and llamas. The molecules are
`relatively small in size and very stable. Dutch company BAC
`BV (now part of Life Technologies) has cloned and expressed
`them in yeast. The ligands offer high selectivity and show
`good stability in 0.1N NaOH, both free in solution and
`
`36 BioProcesslnternational
`
`11(8)
`
`SEPTEMBER 2013
`
`immobilized on agarose (43). Because of their small size, they
`can penetrate deeply into particles. But their capacity may be
`limited because they are monomeric and thus cannot bind
`multiple IgG molecules on a single VHH fragment.
`Synthetic ligands target the Fc region of IgG antibodies
`by mimicking an epitope (mimetic ligands) or by
`nonmimetic targeting of the paratope. But these ligands
`generally lack the selectivity of protein A and show some
`degree of nonspecific adsorption. Examples include amines,
`thiophilic compounds, mixed-mode adsorbents, and
`peptide and chemical libraries.
`Instead of trying to find a highly specific ligand to an
`antibody, users could modify it and add a tag. But such tags
`ultimately need to be removed because the FDA considers
`them to be impurities. But if you insert a protease cleavage
`site to remove a tag, for example, that raises concerns
`regarding nonspecific proteolysis. Some novel systems
`simplify the cleavage step with tags derived from protein-
`splicing elements (inteins). But all of these strategies are
`more applicable to a research and development (R&D)
`setting than to a manufacturing environment.
`
`COMING ATTRACTIONS
`
`Reviews in 2007 (5, 6) predicted that protein A was here to
`stay at least for five more years. Some people responding to
`our 2013 survey (see the “Survey Says” boxes for more detail)
`still say that it will not be phased out of manufacturing for
`another five or 10 years. But a number of recent examples
`have shown that it could be successfully replaced (35, 42).
`Others apply SMB for continuous manufacturing of
`
`

`

`proteins, including at least one report involving a monoclonal
`antibody (40). Those could be early indicators of changes to
`come — probably sooner than expected — that could very
`well transform the field of antibody purification.
`
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`2 Therapeutic MonoclonalAntibodies: WorldMarket 2011—2021.
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`3 Next— Generation Antibody Therapies: Pipeline and Market 2011—
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`4 Carter—Franklin JN, et al. Fragments of Protein A Eluted During
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`5 Low D, O’Leary R, Pujar NS. Future of Antibody Purification. J.
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`7 Gagnon P. Technology Trends in Antibody Purification. J.
`Chromatogr. A 1221, 2012: 57—70.
`8 Sjoquist J, et al. Localization of Protein A in the Bacteria. Eur. J.
`Biochem. 30(1) 1972: 190—194.
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`9 Langone JJ. Protein A of Staphylococcus aureus and Related
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`Pneumonococci. Adv. Immunol. 32, 1982: 157—252.
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`10 Lofdahl S, et al. Gene for Staphylococcal Protein A. Proc. Natl.
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`11 Guss B, et al. Region X, the Cell—Wall—Attachment Part of
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