50 BioProcess International February 2010

Downstream processing of biopharmaceuticals is costly and time-consuming, often involving multiple steps with

significant time and energy expended on maximizing product quality and yield. Affinity chromatography is one of the simplest and most effective methods for purifying protein and peptide therapeutics, offering reduced process steps and therefore higher yields than nonaffinity methods can provide. Protein A is widely used for affinity purification of monoclonal antibodies (MAbs), Fc fragments, and Fc fusion proteins. But it is a challenge to find a protein A equivalent for nonantibody targets.

To be useful in bioprocess applications, an affinity medium must provide the specificity required to purify a product effectively while allowing elution conditions that preserve product structure and activity. It is also important that an affinity medium can withstand cleaning processes required for effective adsorbent cleaning to meet regulatory standards in therapeutics production.

To address these challenges, BAC derived a class of affinity ligands from unique heavy-chain antibodies found in genus Camelidae (trade named CaptureSelect). Heavy-chain antibodies

are devoid of the entire light chain and CH1 domain found in conventional antibodies (Figure 1). Because of that absence, antigens are bound only by the variable domain of the heavy chain (VHH) without any less binding affinity than conventional antibodies. Derivative VHH ligands are ~12-kD single-domain fragments that make up the antigen-binding domain. They are efficiently produced by the yeast Saccharomyces cerevisiae in a system that is completely free of animal components. This new class of ligand has been shown to provide not only a suitable alternative to protein A (1), but also an equivalent for a wide range of nonantibody targets, a number of which are being used in manufacturing clinical trial materials (CTMs).

The unique suitability of the VHH domain for use as an affinity ligand lies in the stability of its structure as a fragment. It is the smallest known functional antigen-binding antibody fragment that shows high affinity and stability. The Fv domain is its equivalent fragment in conventional antibodies that consists of a heavy-chain variable domain (VH) and a light-chain variable domain (VL) held together by a noncovalent bond. If that bond is broken, all antigen-binding functionality is lost. Although the method of synthetic production — linking the two variable domains together into a single chain — retains the antigen binding functionality of scFv fragments, they often show less affinity than their parent antibody. Because the VHH domain exists as a single polypeptide chain, it is an extremely stable and easily produced fragment that retains the full binding activity of its parent heavy-chain antibody.

VHH fragments have been used successfully as affinity ligands in chromatography processes for purifying many types of protein, including

antibodies (2–5), blood factors (6), and adenoassociated viruses (7, 8). Ligands for a specific target molecule are identified from expression libraries that represent the heavy-chain antibody repertoire of an immunized llama. Coming from a highly variable mammalian immune system, these expression libraries provide an opportunity to select VHH molecules that meet very specific requirements for specificity, affinity, elution profile, and cleaning conditions. Thus, VHH affinity ligands can be created to match exactly the processes for which they are intended. Immobilization onto resins suitable for large-scale bioprocess applications make these ligands good tools for downstream processing.

Platform technology for multiProduct facilitiesVHH affinity media enable a “plug-and-play” platform approach providing a highly specific primary capture step that can be slotted into existing downstream processes. Figure 2 illustrates how VHH media can be incorporated into any “generic” biopharmaceutical purification process for comparable results with current MAb purification platforms using protein A as a primary capture step.

V E N D O RVoiceNovel Affinity Ligands Provide for Highly Selective Primary Capture Frank Detmers, Pim Hermans, Jin-An Jiao, and Justin T. McCue

Product focus: Non-MAb biologics

Process focus: Downstream processing

Who should read: Process development and manufacturing

KeyWords: Chromatography media, platform technologies, purification, specificity

level: Intermediate

Figure 1: Comparing conventional IgG structure and the Camelid heavy-chain antibody

VH domain VHH

VL domain

Camelid IgGIgG

CH2

CH3

CH2

CH3

CH1

CL

February 2010 BioProcess International 51

Many biotherapeutics are produced in multiproduct contract manufacturing facilities. It would be significantly convenient if purification processes for most/all products could be made similar — for example, if nonantibody products could be purified by a similar process to the protein A purification of MAbs. To that aim, the use of a VHH ligand for primary capture of recombinant Factor VIII (FVIII) was investigated as an alternative to a traditional multistep process involving chromatographic separation and precipitation techniques.

The VHH adsorbent was found to be highly selective for recombinant FVIII. It provided good viral clearance and significantly improved host-cell DNA and protein clearance (6). Of importance, FVIII elution was achieved at a neutral pH because the protein is relatively unstable, and yields exceeded 70%. In addition, the adsorbent retained capacity following 25 cycles of cleaning and reuse, and it produced consistent results across the range from laboratory to manufacturing scale. That demonstrated the suitability of the VHH ligand for use in FVIII commercial manufacturing. Table 1 summarizes the key features of the anti-FVIII adsorbent.

customized sPecificityVHH ligands are selected and developed from expression libraries obtained from a llama immunized with a target molecule. Each library contains thousands of potential ligands that can be screened and tested for specificity/affinity for the target molecule. Specificity can be as narrow or as broad as required, and binding characteristics can be tuned to produce a desired elution profile. Table 2 summarizes the VHH ligands currently commercially available.

To assess the specificity of the VHH ligand against human IgG (IgSelect, GE Healthcare), an ELISA was used to compare the distribution (by percentage) of IgG subclasses in serum between the load and elution fractions (Table 3). Subclass distribution in the loading material was retained during purification of human IgG from serum on the VHH affinity resin (anti human IgG-Fc). By comparison, because protein A–based affinity resins lack a sufficient binding affinity toward human IgG3, the ratio of subclasses in a protein A–purified pool becomes unbalanced.

Table 3 shows how it is possible to develop a VHH ligand with a relatively broad specificity for all human IgG subclasses. However, VHH ligands have also been developed showing excellent human IgG subclass selectivity (e.g., for IgG1, IgG3, or IgG4). To assess the efficacy of a VHH ligand with that much narrower a specificity (IgG4) an ELISA was used to compare the IgG subclass distribution (by percentage) in IVIG between load and elution fractions (Table 4). Human IgG4 subclass selectivity was demonstrated by a >20-fold enrichment of IgG4 in a one-step purification.

Human IgG–specific VHH ligands have obvious application in MAb

production. The ability to select for species specificity during development of a VHH ligand has provided a powerful tool for one-step purification of therapeutic human IgG from feedstock materials that come from transgenic animals (e.g., cattle). These media enable removal of low levels of endogenous IgG antibodies that may be coexpressed with the human IgG. Table 5 demonstrates a significant clearance of bovine IgG contaminants (≥97%) from a human IgG product using IgSelect human IgG VHH resin from GE Healthcare (www.gelifesciences.com). In a separate study, the human IgG ligand was used in conjunction with a selective

Table 2: Commercially available VHH ligands

Target Specificity Development StageIgG (Fc) Human IgG (all subclasses), Fc-fusions Large-scale processes

AAV Adenoassociated viruses from subclasses 1, 2, 3 and 5 Large-scale processes

FVIII Recombinant full-length and B-domain–deleted Factor VIII

Large-scale processes

Fab kappa All human antibodies and Fab fragments with kappa light chain (IgG, IgA, IgM, IgD and IgE), all subclasses

Large-scale processes

AAT Human alpha-1-antitrypsin Large-scale processes

IgG (Fc) Multispecies IgG (human, mouse, cow, rabbit, rat, goat, horse, sheep), all subclasses

Laboratory research and pilot projects

Fab lambda All human antibodies and Fab fragments with lambda light chain (IgG , IgA, IgM, IgD and IgE), all subclasses

Laboratory research and pilot projects

IgA Human IgA (monomer, dimer and secretory IgA), all subclasses

Laboratory research and pilot projects

IgM Human and mouse IgM Laboratory research and pilot projects

IgG4 Human IgG4, no cross-reactivity with other subclasses Laboratory research and pilot projects

Table 1: Summary of FVIII adsorbent analysis (6)

Feature VIIISelect Performance Additional NotesStatic binding capacity 55,000–67,000 IU FVIII/mL Static capacity independent

of salt concentration over 0.15–1.5M NaCl, pH 7

Dynamic binding capacity

23,000 IU FVIII/mL at 1.2 minutes residence time; 37,000 IU FVIII/mL at 2.4 minutes residence time

500 mM NaCl, pH 7, 10% breakthrough

Host-cell DNA clearance

4.9 logs 1.0M CaCl2 wash

Host-cell protein clearance

3.1–3.5 logs Clearance comparable over different salt washes

Viral clearance X-MLV: 3.8–4.2 logs

MMV: >5.0 logs*

* Virus titers in the column eluate were below the limit of the assay detection

FVIII recovery >70% Elution buffer containing 40–50% (v/v) propylene glycol and 0.8–1.0 M l-arginine

Adsorbent cleaning and reuse

No loss of dynamic binding capacity over 25 cycles

Cell culture feed loaded every fifth cycle; equilibration buffer loaded for every other cycle

Scale-up Performance of adsorbent consistent across laboratory, pilot, and manufacturing scales (maximum 900-fold scale-up)

52 BioProcess International February 2010

bovine IgG scavenging ligand to produce a human polyclonal IgG product at a purity suitable for therapeutic use (5).

matching elution Profile to target requirementsUnstably structured proteins may be difficult to effectively purify using affinity chromatography because of their instability under conditions required for elution, such as low pH or the presence of high solvent concentrations. The inherent flexibility of the VHH ligand development process means that ligands can be selected for development based not only on their specificity, but also on their elution profiles. Elution under mild conditions is assessed by testing ligands with a range of mild elution buffers. Table 6 summarizes those profiles achieved for a range of target molecules purified with highly specific VHH affinity resins.

As described above, FVIII is a relatively unstable molecule. It consists of a 200-kDa heavy chain and an 80-kDa light chain held together with a highly labile noncovalent metal bond (Me2+). The protein is stable in neutral conditions for only a limited time and is not stable at all at low pH. The challenge was therefore to identify a ligand that would enable swift (one-step) purification of the FVIII molecule at neutral pH. The ligand identified was highly specific and selective for FVIII products. An elution buffer optimization step provided the balance of buffer components required to achieve high product recovery at neutral pH (6).

Alpha-1 antitrypsin (AAT) is a 52-kDa serum protease inhibitor that is traditionally hard to purify. Conventional methods of manufacturing it follow ethanol precipitation procedures that produce only a 10% yield because the protein is inactivated by high ethanol concentrations (9). Because AAT is obtained from human blood plasma, the efficiency of extraction needed improvement. To that end, a VHH ligand was developed.

To assess the selected VHH ligand for functionality under the mild conditions required for this protein, AAT was purified from human plasma using a VHH affinity resin specific for it. AAT was successfully eluted from the resulting affinity resin with a neutral buffer (2 M MgCl2, pH 7.4). To check the comprehensiveness of that neutral elution, the mild elution was then followed with an acidic elution (PBS,

pH 2.0). No significant elution of product was observed in the acidic fraction, indicating that elution had been complete with 2 M MgCl2. Fractions were analyzed using Coomassie-stained sodium-dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The results in Figure 3 confirm that the affinity resin achieved a high-yield, high-purity purification of AAT from human plasma in a single step at neutral pH.

loWering cost and imProving qualityA key benefit to using affinity chromatography for purifying proteins is the simplicity of the process, which can often reduce a complex multistep procedure to a single step that results in a high-purity, high-quality product. Step reduction not only reduces production time and associated costs, but decreasing the length of time a protein product is “in process” limits potential degradation or damage, thereby ensuring better final product. Most important, each step of a downstream process reduces final product yield, so it’s highly desirable to keep steps to a minimum.

Adenoassociated virus (AAV) is traditionally hard to purify. Over the past decade, it has received increased interest as a potential gene therapy vector. Current, conventional purification processes require multiple purification steps including density-gradient purification, several chromatography steps (ion-exchange, hydrophobic-interaction, heparin, and A-20 affinity), and an associated lengthy cleanroom residence time. With such a multistep process, the typical AAV yield is low. The process also suffers from poor scalability, limiting the commercial feasibility of this promising vector.

To address that challenge, a VHH ligand against AAV was identified and developed. Screening delivered a 14-kDa VHH fragment that was shown to bind AAV subclasses 1, 2, 3, and 5 and provide an eluate with high purity (7). Process steps were reduced from the typical four or five to just one or two steps, with a related reduction in cleanroom time for the recovery process from weeks to days. In this case, step reduction improved product recovery from 30% to >60%. Figure 4 shows the one-step purification of AAV serotype 1 using AVB Sepharose High Performance anti-AAV VHH ligands from GE Healthcare. The VHH ligand produced similar product purity to that of the reference standard.

Table 3: IgG subclass distribution (%) in load and elution fractions determined by ELISA

Antihuman IgG-Fc hu IgG1 hu IgG2 hu IgG3 hu IgG4Load material

(human serum)62.6 28.4 5.9 3.2

Elution fraction VHH resin

62.3 28.2 5.9 3.7

Elution fraction Protein A resin

62.9 34.2 0.5 2.5

Table 4: IgG subclass distribution (%) in load and elution fractions determined by ELISA

Antihuman IgG4 hu IgG1 hu IgG2 hu IgG3 IgG4Load material

(IVIG)57.1 34.2 4.0 4.7

Elution fraction anti–hu-IgG4

1.7 0 0.6 97.7

Figure 2: CaptureSelect VHH affinity media enable a “plug-and-play” platform approach, shown here replacing the protein A primary capture step in a standard MAb purification process.

For Monoclonal Antibodies

Cell culture harvest

Protein Achromatography

Viral inactivation

Polishing

Viral �ltration

UF/DF

For All Biopharmaceuticals

Cell culture harvest

CaptureSelect chromatography

Viral inactivation

Polishing

Viral �ltration

UF/DF

54 BioProcess International February 2010

contaminant removalA highly desirable feature for all purification steps is the ability to remove contaminants such as host-cell proteins (HCPs) and DNA or virus particles. HCP and DNA clearance are good indications of the selectivity of purification media. In an analysis of protein A alternative chromatographic media for antibody purification, VHH ligands were found to be the only alternative that provided comparable HCP removal (10).

Viral clearance studies of the FVIII VHH ligand were performed by McCue, et al. (6). In their studies, clarified cell culture samples were spiked with one of two model viruses: xenotropic murine leukemia virus (X-MLV) to evaluate the adsorbent’s ability to remove retroviruses or retrovirus-like particles; and mouse minute virus (MMV) to evaluate the ligand’s ability to remove a small nonenveloped virus. Column elution pools were evaluated for virus presence, and polymerase chain reaction (PCR) was used to quantify the amount of virus in both the load and elution fractions. As Table 1 shows, the FVIII adsorbent provided significant clearance of both model viruses (6).

The ability of VHH ligands to remove HCP, DNA, and adventitious or endogenous viruses is expected to be good because of the ligand’s complexity. The more complex a ligand is, generally the more selective it is.

a Platform technologyVHH ligands provide a highly flexible platform technology for purification of almost any target protein with high specificity and selectivity and good contaminant clearance under mild elution conditions. A number of VHH affinity adsorbents are commercially available already and being used for production of CTMs. Many more are being developed for future research and bioprocess use in the future (11).

Use of VHH media as a platform could shorten development times for purification of a given molecule by providing a plug-and-play solution for downstream processing. The simplicity and high specificity of the process reduces overall complexity in multiproduct facilities and ensures a minimum number of process steps. That increases yield while retaining excellent purity and quality of the end product. VHH ligands offer a clear advantage for multiproduct facilities and groups working with typically hard-to-purify molecules.

references1 Liu J, et al. Comparison of Camelid

Antibody Ligand to Protein A for Monoclonal Antibody Purification. BioPharm Int. September 2009: 35–43.

2 Detmers F, Hermans P, ten Haaft M. Affinity Chromatography Based Upon Unique Single Chain Antibodies. The Peak September 2007: 7–14.

3 Beyer T. Serum-Free Production and Purification of Chimeric IgA Antibodies. J. Immunol. Meth. 346, 2009: 26–27.

4 Klooster R, et al. Improved Anti-IgG and HSA Affinity Ligands: Clinical Application of VHH Antibody Technology. J. Immunol. Meth. 324, 2007: 1–12.

5 Kuroiwa Y, et al. Antigen-Specific Human Polyclonal Antibodies from Hyperimmunized Cattle. Nature Biotechnol. 27(2) 2009: 173–181.

6 McCue JT, Selvitelli K, Walker J. Application of a Novel Affinity Adsorbent for the Capture and Purification of Factor VIII Compound. J. Chromatog. A 1216 2009: 7824–7830.

7 Hellström M, et al. Cellular Tropism and Transduction Properties of Seven Adeno-Associated Viral Vector Serotypes in Adult Retina After Intravitreal Injection. Gene Ther. 16, 2008: 521–532.

8 Smith RH, Levy JR, Kotin RM. A Simplified Baculovirus-AAV Expression Vector System Coupled with One-Step Affinity Purification Yields High-Titer rAAV Stocks from Insect Cells. Molecular Therapy 17(11) 2009: 1888–1896.

9 Kumpalume P, et al. New Process for the Manufacture of α-1 Antitrypsin. J. Chromatog. A 1148, 2007: 31–37.

10 Low D, O’Leary R, Pujar NS. Future of Antibody Purification. J. Chromatog. B 848, 2007: 48–63.

11 BAC’s Pipeline. http://www.captureselect.com/downloads/pipeline.pdf. •

Frank Detmers, PhD, is director of ligand applications, and Pim Hermans is director of ligand discovery at BAC BV, Huizerstraatweg 28, 1411 GP Naarden, The Netherlands; info@bac.nl; www.bac.nl. Dr. Jin-An Jiao is executive vice president at Hematech, Inc. in Sioux Falls, SD. Dr. Justin T. McCue is a senior engineer in bioprocess development at Biogen Idec in Cambridge, MA.

Figure 3: Coomassie-blue–stained SDS-PAGE shows purified AAT eluted at pH 7.4.

1 2 3 4 5 6

Figure 4: Adenoassociated virus purification with anti-AAV VHH ligand

VP1VP2VP3

rAAV1: C

ontrol

Marker

LoadFlow-th

rough

Elution

Elution

Table 5: Removal of bovine IgG from human IgG samples using 5 mL IgSelect human IgG VHH resin

Sample Loaded Human IgG

RecoveryBovine IgG Clearance

25 mg human, 5 mg bovine

91.0% 97.3%

50 mg human, 10 mg bovine

86.3% 97.4%

75 mg human, 15 mg bovine

85.6% 97.0%

Table 6: Elution of a range of target molecules under mild conditions.

Target MoleculeMgCl2 pH 7

50% (v/v) Propylene Glycol pH 7

Arginine pH 6-8

Glycine pH 4.5

Glycine pH 3

Human IgG – ND ND ++ ++Factor VIII – ++ + +/– ++Human albumin, albumin fusions

++ ++ + +/– ++

α-1 antitrypsin ++ – + +/– ++Transferrin, transferrin fusions

ND ++ ND +/– ++

Fibrinogen + ++ ND – ++++ elution 85–100% + Elution 75–85% +/– Elution 60–75% – No efficient elution ND Not determined