Protein Engineering Allows for Mild Affinity-based Elution of Therapeutic Antibodies

Article KDC

YJMBI-65756; No. of pages: 12; 4C:

Protein Engineering Allows for Mild Affinity-based Elution of Therapeutic Antibodies Sara Kanje 1 , Raminta Venskutonytė 2 , Julia Scheffel 1 , Johan Nilvebrant 1 , Karin Lindkvist-Petersson 2 and Sophia Hober 1 1 - CBH, KTH—Royal Institute of Technology, SE-10691 Stockholm, Sweden 2 - Experimental Medical Science, Medical Structural Biology, BMC C13, Lund University, SE-221 84 Lund, Sweden

Correspondence to Sophia Hober: [email protected] https://doi.org/10.1016/j.jmb.2018.06.004 Edited by Sachdev Sidhu

Abstract Presented here is an engineered protein domain, based on Protein A, that displays a calcium-dependent binding to antibodies. This protein, ZCa, is shown to efficiently function as an affinity ligand for mild purification of antibodies through elution with ethylenediaminetetraacetic acid. Antibodies are commonly used tools in the area of biological sciences and as therapeutics, and the most commonly used approach for antibody purification is based on Protein A using acidic elution. Although this affinity-based method is robust and efficient, the requirement for low pH elution can be detrimental to the protein being purified. By introducing a calcium-binding loop in the Protein A-derived Z domain, it has been re-engineered to provide efficient antibody purification under mild conditions. Through comprehensive analyses of the domain as well as the ZCa–Fc complex, the features of this domain are well understood. This novel protein domain provides a very valuable tool for effective and gentle antibody and Fc-fusion protein purification. © 2018 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Introduction Antibodies are one of the most widely used reagents in biological sciences, and the therapeutic antibody field is ever increasing with worldwide annual sales approaching 100 billion dollars [1]. Purification of antibodies is generally performed using Protein Abased affinity chromatography [2], a simple and robust method that yields pure and concentrated product in a single purification step [3, 4]. The main drawback is the need for acidic pH (3–4) for elution. This hampers the development of new antibodies and antibody-based tools since many antibodies suffer from sensitivity to low pH, which can cause aggregation or loss of function [5–8]. Various attempts have been made to improve the elution conditions for antibodies captured on Protein A columns. Examples include binding site mutations of the Protein A-derived Z domain [9] and loop engineering of the same domain [10], which enabled increased elution pH to 4–4.5; addition of urea or arginine to the elution buffer that was shown to suppress protein

aggregation [6, 11]; and incorporation of mutations that decrease the hydrophobicity of Protein A, which resulted in a thermo-responsive ligand that allows elution at 40 °C [12]. One way for nature to control protein activity is by altering the tertiary structure, which is often accomplished by interaction with ions [13–15]. Proteins containing the EF-hand motif are allosterically controlled through binding of calcium ions [12, 13]. Certain positions within the EF-hand motif are evolutionarily conserved while other positions are more diverse [16]. EF-loop motifs have been grafted onto model proteins and shown to retain the calcium binding activity [17, 18]. Metal binding has also been utilized in protein engineering for purification purposes. The O'Connell lab has developed a split calcium protein based on EF-hand subdomains that can be used as a tag and affinity handle in calciumdependent protein purification [19]. Moreover, one of the antibody binding domains from Streptococcal Protein G has been engineered to contain a metal binding site causing the domain to lose its ability to

0022-2836/© 2018 The Author. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). J Mol Biol (2018) xx, xxx–xxx Please cite this article as: S. Kanje, et al., Protein Engineering Allows for Mild Affinity-based Elution of Therapeutic Antibodies, J. Mol. Biol. (2018), https://doi.org/10.1016/j.jmb.2018.06.004

2

Protein Engineering

bind immunoglobulin G (IgG) when a transition metal is present [20]. To circumvent the harsh conditions needed when purifying antibodies by affinity chromatography while keeping the very high selectivity of the Protein A matrix, we have developed a calcium-dependent IgGbinding domain. The Z domain derived from the B domain of Protein A [21], which is a small and stable three-helix bundle protein that binds the Fc-region of IgG with high affinity, was used as a parental scaffold. To attain an ion dependent behavior, a randomized calcium-binding loop was grafted between helix two and three of the Z domain. Phage display selections were performed to isolate protein variants exhibiting a calcium-dependent binding to IgG. The final domain, denoted ZCa, can selectively capture IgG from a complex cell culture supernatant and, as demonstrated by the crystal structure, the new domain binds Fc in a similar manner as Z. After a single purification step with calcium-dependent elution at pH 5.5, the purity and yield achieved is comparable to conventional purification using the parental Z domain. Thus, this novel protein domain provides an important new tool for significantly milder antibody purification.

Results

ed using surface plasmon resonance (SPR) (Supporting Table 2). The affinity of the Z domain to IgG was decreased by the mere introduction of a calmodulin loop, but introduction of a leucine N-terminally of the loop resulted in a recovered affinity for IgG. Introduction of glycine or serine linkers N- and C-terminally of the loop caused the largest loss in affinity for IgG. However, no shift in melting temperature of these variants could be detected between calcium- and EDTA-containing buffers in CD. Z-loop and Z-GGG-loop were chosen for further work since they showed the largest differences in melting temperature between the calcium- and EDTA-containing buffers, indicative of a calciumdependent stability. To generate variants with more pronounced calcium dependency, a combinatorial phagemid library was designed built on the two lead variants. Based on sequences of a large number of calcium-binding loops [16, 29], degenerate codons were chosen to cover a large diversity of canonical EF-hand loops (Supporting Table 3). To enable higher structural dependence on the calcium-binding loop, structurally destabilizing mutations were included in 50% of the library by allowing variations in positions 30, 44, 48 and 51 (Supporting Table 4) [30–32]. The final library had a size of 2 × 10 8 after transformation, corresponding to half the theoretical genetic size.

Library design Selections To investigate the structural and functional dependence of a calcium-binding loop in the Z domain, one of its original loops was exchanged for one of the calciumbinding loops from calmodulin (sequence: DKDGDGTITTKE). A suitable position for introduction of a calcium-binding loop in Z was identified by measuring the distances between the N-terminus and C-terminus of several EF-hand loops both with calcium bound as well as for the Apo-form [22–28]. These distances were compared to the measured distance between the N- and C-termini of the loops between helices one and two, and two and three, respectively, of the Z domain (Supporting Table 1). As the distance between the termini of the loop between helix two and three of the Z domain matched the average distance measured in the calcium bound form of calcium-binding loops, while differing from the Apo form of the calmodulin loop, this site was selected for loop grafting. The effect of adding flexible Gly/Ser linkers before and after the loop was investigated as well as the effect of leucine N-terminally of the EF-hand loop. Six variants were designed and investigated (Supporting Table 2). The variants as well as Z were produced and purified to homogeneity by IgG affinity chromatography. Proteins were analyzed by circular dichroism (CD) spectroscopy for secondary structure content and melting temperature in the presence of calcium or ethylenediaminetetraacetic acid (EDTA) and their interaction with monoclonal human IgG was investigat-

Four rounds of phage display selection were performed to isolate desired variants from the library (Supporting Table 5). Each round started with a negative selection toward IgG in the presence of EDTA to remove variants that bound under such conditions. The supernatant with non-bound phages was buffer exchanged into a calcium containing buffer and used in a positive selection round. Urea (2 M) was present in all steps in order to further structurally destabilize the displayed library members. Washing was gradually increased and elution was done with EDTA. The output was screened using ELISA where domains able bind to IgG in the presence of calcium or EDTA were detected. Out of 282 screened phage supernatants, one clone (A5) exhibited a large preference to bind IgG in the presence of calcium versus EDTA (Fig. 1a). A5, derived from the template with a GGG-linker Nterminally of the selected loop (Supporting Fig. 1), was cloned, produced and purified to homogeneity. The purified domain was evaluated by SPR for its affinity toward IgG in the presence of calcium or EDTA with or without urea present. The dissociation equilibrium constant (KD) of A5 to IgG was estimated to be 20 and 50 nM with calcium or EDTA, respectively, and 170 and 330 nM, when 2 M urea was added to the running buffer. Sensorgrams recorded on the same ligand surface illustrate the observed differences in binding

Please cite this article as: S. Kanje, et al., Protein Engineering Allows for Mild Affinity-based Elution of Therapeutic Antibodies, J. Mol. Biol. (2018), https://doi.org/10.1016/j.jmb.2018.06.004

3

Protein Engineering

Abs 450 nm

(a)

CaCl2

EDTA

(b)

CaCl2 urea EDTA urea

(c) CaCl2 urea

EDTA

EDTA urea

20

Response (RU)

0

0

10

20 10

Response (RU)

30

30

CaCl2

0

200

400

600

800

Time (s)

0

200

400

600

800

Time (s)

Fig. 1. ELISA and SPR data of variant A5 showing partial calcium dependence in its binding to IgG. (a) Phage ELISA signal for A5 run in triplicate from phage supernatant incubated in a plate coated with polyclonal IgG in the presence of calcium (white), EDTA (gray), urea and calcium (spotted) and urea and EDTA (striped). A higher binding capacity in the presence of calcium than EDTA was detected. (b and c) Sensorgrams for 25 nM A5 (b) and 125 nM A5 (c) injected over immobilized polyclonal IgG. In panel b, the running buffer contains calcium (black) or EDTA (gray). In panel c, the running buffer contains 2 M urea and calcium (black) or EDTA (gray). The interaction between A5 and IgG is stronger with calcium present than with EDTA.

(Fig. 1b and c) with faster dissociation and lower binding signal when EDTA is present. Moreover, the secondary structure of A5 was shown to be calcium-dependent (Supporting Fig. 2). However, the affinity for IgG with EDTA present was still too high to enable efficient elution by EDTA-mediated calcium depletion in a protein purification setup (data not shown). Therefore, a maturation library was made using error-prone polymerase chain reaction with A5 as template, which, based on sequencing of N 250 clones, resulted in a library of 2 × 10 7 variants with on average 2–3 random amino acid mutations per gene (Supporting Fig. 3).

Selection from the second library was performed in three parallel tracks with alternating rounds of negative and positive selection with tracks that differed in urea concentration (Supporting Table 6). The output (192 clones per track) was screened for calcium-dependent IgG-binding. The most promising candidates were reanalyzed in triplicate (Fig. 2). Sequences of Zmat1–Zmat9 (Supporting Fig. 4) show substitutions in positions described to participate in the IgG binding (F5, H18, N28, Q32 and K35; numbering according to Supporting Fig. 4) [28, 33–35]. Zmat3 and Zmat8 also contain a mutation at position 55, Q55R, an amino acid shown to form hydrogen

Please cite this article as: S. Kanje, et al., Protein Engineering Allows for Mild Affinity-based Elution of Therapeutic Antibodies, J. Mol. Biol. (2018), https://doi.org/10.1016/j.jmb.2018.06.004

4

Protein Engineering 2.5

Abs 450 nm

2

1.5 CaCl2 1

EDTA

0.5

0 Zmat1

Zmat2

Zmat3

Zmat4

Zmat5

Zmat6

Zmat7

Zmat8

Zmat9

Screen variant

Fig. 2. Phage-ELISA data for the top nine variants from the maturation selection analyzed in triplicate. ELISA plates coated with IgG were subjected to phage supernatant in the presence of calcium (white) or EDTA (gray). All variants display large differences between calcium- and EDTAcontaining buffers, and low signals with EDTA present, showing a calcium-dependent interaction with IgG.

bonds to other amino acids in the original scaffold [36]. Interestingly, some variants (Zmat1-3) have a G/S mutation in the linker before the loop and only one of the variants (Zmat3) has a change in the calcium-binding loop compared to A5. Zmat1–9 were cloned, produced and purified to homogeneity, and analyzed by CD and SPR in the presence of calcium or EDTA (Table 1). Zmat8 showed a striking 24-fold difference in ELISA signal with calcium present compared to EDTA and no binding in the SPR experiment with EDTA present could be detected for Zmat3, Zmat6 and Zmat8. Furthermore, a 10-degree difference in melting temperature for Zmat1 and Zmat2 was observed between two different buffers and, in contrast to the other variants, no cooperative two-state folding behavior could be detected for Zmat8 in the presence of EDTA.

elution was performed using EDTA followed by traditional elution at pH 3.2 to collect possible residual protein from the column. It was concluded that the original Z domain could only be eluted at pH 3.2 (Supporting Fig. 5a), while Zmat8 (from here on denoted ZCa) was shown to be the variant that most effectively could be eluted in a calcium-dependent manner at pH 5 (Supporting Fig. 5b–d). To further characterize the behavior of ZCa, elution buffers with different pH (5.5, 5.7, 6, 6.5 and 7) were investigated. ZCa was shown to be quantitatively eluted with EDTA at pH 5.5, whereas some of the protein was retained on the column at higher pH (data not shown). Furthermore, in SPR experiments, ZCa showed a similar recognition pattern for different species as the Z domain by interacting with human IgG1 and rabbit IgG but not with mouse IgG1, albeit with weaker binding (Supporting Fig. 8). In order to challenge ZCa in a relevant antibody purification setup, ZCa and Z were coupled to prepacked NHS-activated columns. IgG was eluted from the ZCa column in a calcium-dependent manner at pH 5.5 (Fig. 3a), whereas no protein was eluted from the Z column (Fig. 3b). As a control, IgG was eluted from both columns by lowering the pH to 3.2, which showed that similar amounts of IgG were eluted with EDTA and acid from the ZCa column. The elution profiles obtained with low pH from the ZCa and Z column were shown to be very similar, while the EDTA elution gave a somewhat broader peak. A cell culture supernatant spiked with polyclonal human IgG and a mammalian cell culture supernatant from IgG-producing CHO-cells (Supporting Fig. 6) were used for test purifications. IgG from both supernatants was purified to high purity using a ZCa column and EDTA elution at pH 5.5, which demonstrates that the inherent specificity of the Z domain is retained in ZCa (Fig. 4).

Functional assessment in protein purification Based on all differences that were detected with and without calcium, Zmat1, Zmat3, Zmat6 and Zmat8 were further investigated in a chromatography setup. A fixed amount of protein was loaded on an IgG-column and

X-ray structure of the complex between ZCa and Fc of human IgG1 To clarify the role of the grafted calcium-binding EFloop of ZCa for Fc binding, the structure of ZCa in

Table 1. Affinity measurements Variant

ELISA Signal ELISA CaCL2 (A.U.)

Z A5 Zmat1 Zmat2 Zmat3 Zmat4 Zmat5 Zmat6 Zmat7 Zmat8 Zmat9

2.47 2.14 ± 0.09 2.06 ± 0.04 1.15 ± 0.24 2.26 ± 0.08 1.37 ± 0.07 1.51 ± 0.03 1.13 ± 0.04 1.53 ± 0.26 2.41 ± 0.09 2.41 ± 0.07

Signal ELISA EDTA (A.U.) 2.68 1.04 ± 0.5 0.51 ± 0.03 0.26 ± 0.02 0.41 ± 0.01 0.26 ± 0.02 0.35 ± 0.05 0.16 ± 0.02 0.34 ± 0.04 0.1 ± 0.01 0.45 ± 0.03

SPR Signal ELISA CaCl2/EDTA 0.9 2.1 4.0 4.4 5.5 5.3 4.3 7.1 4.5 24 5.4

CD

KD CaCl2 KD EDTA KD EDTA/KD (M) (M) CaCl2 6.1E −9 1.9E −8 3.9E −8 6.6E −8 8.6E −8 2.7E −6 8.1E −8 2.2E −7 8.2E −8 4.3E −7 6.0E −8

3.3E −9 5.2E −8 2.5E −6 1.2E −5 ND 9.3E −6 1.7E −6 ND 3.7E −6 ND 7.8E −7

0.5 2.7 63 180 ND 3.4 21 ND 45 ND 13

Tm CaCl2 (°C)

Tm EDTA (°C)

ΔTm (°C)

73 51 56 62 52 48 55 46 55 50 51

73 47 46 52 43 43 50 39 50 ND 47

0 4 10 10 9 5 5 7 5 ND 4

Please cite this article as: S. Kanje, et al., Protein Engineering Allows for Mild Affinity-based Elution of Therapeutic Antibodies, J. Mol. Biol. (2018), https://doi.org/10.1016/j.jmb.2018.06.004

5

Protein Engineering

(a)

(b)

kDa 250 130 100 70 55

kDa 250 130 100 70 55

35 25

35 25

15

15

10

10 M S FT

Fig. 3. Chromatograms from purification of polyclonal IgG using a ZCa column (a) and a Z column (b). Comparison of different elution conditions shows a calcium-dependent elution of IgG from the ZCa column but not from the Z column. IgG was eluted from both columns by lowering the pH to 3.2. Elution buffers: pH 3.2 (dashed line), EDTA in pH 5.5 (solid line), and calcium in pH 5.5 (dotted line). Integration of the eluted peaks shows that similar amounts were eluted from the ZCa-columns with EDTA and pH 3.2, even if the peak from the EDTA elution shows a different shape.

complex with the Fc domain of human IgG1 (trastuzumab) was determined by X-ray crystallography to 2.5 Å resolution (Fig. 5, Supporting Table 7). The structure was solved in P21212 space group with two Fc dimers and four bound ZCa molecules in the asymmetric unit (Fig. 5a). Fc consists of residues 237–446, of which all are included in the structure model, except for 1–2 Cterminal residues in different chains. ZCa consists of residues 1–69, and here 1–4 residues in N or Cterminus were not modeled, due to poor electron density. As expected, ZCa adopts a three-helix bundle fold, and in three out of four ZCa molecules, the calcium ion was clearly present and displayed expected coordination to the residues in the EF-loop (Fig. 5b). Calcium was modeled into the fourth ZCa molecule as well (chain H in the structure); however, the density was poorly defined for this EF-loop. In general, chain H, as well as its interacting Fc molecule (chain D), showed much higher B-factors (84 Å 2 for chain D and 47 Å 2 for chain A) than the other molecules in the structure, indicating high flexibility. Nevertheless, unambiguous density in other chains confirmed the presence of a

E

M S FT E

Fig. 4. SDS-PAGE (a) and Western blot (b) analysis of a cell culture supernatant purification experiment using a ZCa column. The analysis shows complete capture of IgG and excellent purity of the eluted sample. Cell culture supernatant containing approximately 10 mg/l IgG was purified on a ZCa column. The flow through (FT) and the eluted fractions containing proteins (E) were run on an SDS-PAGE next to supernatant (S) and a molecular weight marker (M). The SDS-PAGE was stained with GelCode® Blue (Thermo Fisher Scientific). The SDS-PAGE was transferred to a PVDF membrane and detected with anti-human-HRP coupled antibody, detecting IgG in the supernatant and the eluate but not in the flow through.

calcium ion in the EF-loop. Calcium forms contacts between 2.3 and 2.6 Å to the side chains of Asp40, Asn42, Asn44, Asp48, Glu51 and to the carbonyl oxygen of Tyr46 (Fig. 5b). Interestingly, the overall complex structure is very similar to the Z complex with Fc (PDB code 5U4Y) (Fig. 5c) [35]. As in the Z–Fc complex, ZCa interacts with Fc using helices 1 and 2. The introduced calciumbinding loop, between helices 2 and 3, faces away from Fc and does not form direct contacts to its partner Fc molecule (Fig. 5d). Although the calcium loop in two out of four molecules of ZCa is within hydrogen bonding distances to other nearby Fc domains, these additional interfaces were ruled out as crystal contacts and did not contribute to complex formation according to substitutional analysis (Supporting Fig. 7). On average, among the four complexes, the buried surface area (BSA) is around 700 Å 2, which is in the same range as for the Z–Fc complex (with BSA of 802 and 689 Å 2, for the two chains, respectively, structure 5U4Y), as calculated by PISA server [37]. Moreover, the main interacting residues are conserved between Z and ZCa and none of the substituted residues in ZCa form hydrogen bonds to Fc. Specifically, residues Gln10, Asn11 and Glu15 of ZCa form hydrogen bonds to the Fc (Fig. 5d). In addition, Glu24 forms a salt bridge to Lys317 in Fc, which is clearly seen in one out of four complexes (chains A and E). The interactions by the corresponding residues in Z and the Fc are also seen in the Z–Fc complex; only the Glu15 adopts a

Please cite this article as: S. Kanje, et al., Protein Engineering Allows for Mild Affinity-based Elution of Therapeutic Antibodies, J. Mol. Biol. (2018), https://doi.org/10.1016/j.jmb.2018.06.004

6

Protein Engineering

(a)

(b)

(c)

(d)

Fig. 5. X-ray structure of the ZCa–Fc complex. (a) Ribbon representation of the overall Fc-dimer, with the Fc chains colored in green and ZCa in blue. (b) The 2Fo–Fc electron density map (cutoff at 2.0σ) of the grafted EF-hand with the calcium ion in yellow and ionic bond formations depicted as dashed lines. In the overall ZCa structure, the 2Fo–Fc electron density map at 5.0σ is shown for calcium ion. (c) Ribbon representation of the overlay between the ZCa–Fc (green and blue) and Z–Fc (gray) superimposed on ZCa. (d) Close-up view of the interaction interface between ZCa and Fc, with hydrogen bonds shown as dashed lines.

different conformation and does not form a contact to Fc. In contrast, the parental Z domain establishes an additional hydrogen bond between Asn126 (5U4Y numbering) and Gln311 in Fc. This interaction is not present in the ZCa–Fc complex, as the corresponding asparagine residue was substituted to a lysine. In summary, the structure of the ZCa–Fc complex confirmed the presence of the calcium ion in the grafted EF-loop and demonstrated that the engineered ZCa interacts with Fc in a similar, albeit somewhat weaker, manner to the wild-type Z domain. The affinity (KD) of ZCa to Fc of human IgG1 in the presence of calcium is 440 nM as estimated by the SPR analysis, which is in the same range as the earlier measured affinity to polyclonal human IgG (Table 1).

Discussion Presented in this paper is a novel Protein A-based domain, ZCa, that enables calcium-dependent elution of bound antibodies. Interestingly, neither the firstgeneration lead candidate A5 nor the interesting domains from the second round of selections contained any destabilizing core mutations. This illustrates the delicate balance of affinity, loop-dependence and structural stability of the domain that was required to achieve the envisioned characteristics.

Although the selections were performed at physiological pH, efficient elution of IgG from the final protein domain by merely removing calcium by addition of EDTA was not possible. Interestingly, upon lowering of the pH to 5.5 in combination with EDTA, IgG was quantitatively eluted. This behavior could be explained either by pH-dependent calcium affinity [38] or a pH-dependence of residues stabilizing the protein complex. From the crystal structure of the Fc–ZCa complex (at pH 6), the residues in ZCa that form bonds to Fc (Glu, Asp, Gln and Asp) do not change their protonation state upon lowering the pH to 5.5. Thus, an altered calcium affinity is the more likely explanation for the loss of binding as addition of EDTA at pH 5.5 destabilizes the complex further. This is supported by the structure, as the electron density for the calcium is poor in one of the complexes in the asymmetric unit, indicating flexibility and destabilization. In addition, as functional data show, in the absence of calcium, there is no binding between ZCa and Fc, which also indicates a destabilizing effect on ZCa. Finally, the introduced substitutions resulted in loss of one contact between ZCa and Fc compared to the Z and Fc interface, which may explain the lowered overall affinity (Table 1). During selections, binding and washing were performed in the presence of calcium and elution was performed by incubation with EDTA for 5 min. The Z domain is structurally a very stable

Please cite this article as: S. Kanje, et al., Protein Engineering Allows for Mild Affinity-based Elution of Therapeutic Antibodies, J. Mol. Biol. (2018), https://doi.org/10.1016/j.jmb.2018.06.004

7

Protein Engineering

protein, also with the grafted loop, and the domain is further stabilized when bound to IgG. This may explain that, in addition to adding a chelating agent, a slight decrease of the pH is necessary to release IgG from the ZCa-column. In order to distinguish the interesting candidates, screening of hundreds of clones using phage ELISA was performed. Many of the screened candidates exhibited a large difference in binding between calcium and EDTA containing buffers, proving that the selection strategy was successful in isolating fully or partly calcium-dependent variants. Still, many candidates had too high affinity for IgG in the presence of EDTA to be useful in the intended purification setup. Thus, the key challenge was to find a protein domain with high enough affinity with calcium present to work in a purification setup, still with sufficiently low affinity with EDTA present to efficiently release IgG in the elution step. In the output from the second round of selections, all interesting variants had substitutions in positions known to be important for IgG binding, which indicates that decreased affinity between the proteins is part of the successful calcium-dependent elution. A concern with the post-selection analysis of the protein affinity was that the interaction with IgG was measured with either calcium or EDTA present in all steps. This differs from the intended purification setup where IgG is first allowed to bind in the presence of calcium and then expected to elute when calcium is depleted upon the exposure to excess EDTA. The selection method mimicked the intended purification setup, whereas the analysis methods did not. The top performing clone, ZCa, was the variant with the largest difference in ELISA signal when comparing binding in the presence of calcium or EDTA, as well as having the signal closest to background for the EDTA-ELISA (Fig. 2). This variant did not show any binding to IgG in the SPR analysis in the presence of EDTA, whereas the other variants showed at least some interaction with IgG, albeit lower than with calcium present. Furthermore, this domain did not show a cooperative melting behavior in the CD spectrum analysis when EDTA was present. Taken together, this shows that the methods for analysis were still sufficiently sensitive for identifying the most promising candidates. When comparing the elution profiles from the ZCa-column and a Z-column, the IgG eluted from the ZCa-column with EDTA was released at the same elution time as IgG from the Z column when using HAc (Fig. 3). However, elution from the ZCa-column with EDTA gives a slightly lower and broader peak than acidic elution, but still a sharp and quantitative elution peak with a concentrated product was achieved. This difference might be due to slightly slower kinetics when breaking the interaction with EDTA. Furthermore, in Fig. 4, it is clearly shown that the eluted product is highly pure and that the inherent specificity for IgG of the Z domain is retained.

Presented here is a novel engineered Protein Aderived Z domain that possesses a calciumdependent interaction with IgG. This feature enables selective capture of IgG from a complex sample in the presence of calcium and elution of the captured IgG by EDTA at pH 5.5. The mild elution is a major advantage in the purification of acid-labile antibodies as well as other sensitive proteins fused to Fc. Moreover, the risk of aggregation of the eluted product decreases if low pH can be avoided. Within this work, Tris-based buffers have been used since calcium is prone to precipitate in phosphate buffers. However, for applications that are incompatible with Tris, Hepes-based buffers are good alternatives. For the loading of the sample and elution of the captured antibodies, phosphate-based buffers are useable since the presence of calcium in these steps is not desired. Furthermore, to utilize the full potential of this novel IgG-binding domain in industrial application, the commonly used virus inactivation step of low pH needs to be substituted to another, for example, detergent-based unit operation [39–41]. Hence, this domain offers an improved purification method that in a single purification step yields a highly pure and concentrated product while all purification steps are performed under mild conditions. This novel protein domain provides a very valuable tool for effective and gentle antibody and Fc-fusion protein purification.

Material and Methods All enzymes (New England Biolabs) and purification kits (Qiagen) were used according to instructions. PCR reactions were performed with Phusion polymerase and PCR screens with Dynazyme II DNA polymerase (ThermoFisher). Cloning Pre-study variants Single-stranded DNA corresponding to helix one and two of Z was mixed with a reverse primer encoding part of helix three, the calmodulin loop and linkers. PCR was run for six cycles, after which primers were added and the PCR was run for an additional 50 cycles. The purified Z-loop constructs as well as the pET-26b(+) vector (Novagen) were cleaved, purified and ligated. A5 and Zmat1–9 and ZCa mutants The genes were amplified by PCR, cleaved, purified and ligated to the vector pHis [42]. Genes encoding ZCa[Q10A, N11A, E15A] and ZCa[Q10A, N11Y, E15A] were amplified by PCR using a forward primer containing a His6-tag, cleaved, purified and ligated into the vector pET-45b(+).

Please cite this article as: S. Kanje, et al., Protein Engineering Allows for Mild Affinity-based Elution of Therapeutic Antibodies, J. Mol. Biol. (2018), https://doi.org/10.1016/j.jmb.2018.06.004

8

Protein Engineering

Library construction

Selection

Original library

Negative selections were performed in EIA/RIA tubes (Greiner bio-one) coated with polyclonal human IgG (20 μg/ml) in coating buffer (50 mM carbonate, pH 9.6). The tubes were blocked with 1 × TBSTB-E (1× TBSTB, 100 mM EDTA). Phage stock was diluted in 1 × TBSTB-E and added to a blocked tube. After incubation for 20 min at RT with rotation, the phage mix was transferred to a new tube and incubated for 20 min. The remaining phages were concentrated and washed with 1 × TBST (1 × TBS, 0.1% Tween20) four times and diluted with 1 × TBSTB with 2 M urea and 5 mM CaCl2 using a 30-kDa cutoff concentrator (Amicon, Merck) (input positive selection). Target polyclonal IgG was biotinylated with EZlink TM Sulfo-NHS-Biotin (ThermoFisher). Streptavidin-coated beads [Dynabeads M-280 (Invitrogen)] were washed, blocked and used in pre- and positive selections. The positive selection input was pre-selected against streptavidin beads and blocked microtubes. The pre-selected library was incubated with 50 nM biotinylated IgG. The phages and target were incubated with streptavidin-coated beads. The beads were washed in 1 × TBST with 2 M urea and 1 mM CaCl2 and phages were eluted with 1 × TBSTB with 2 M urea and 100 mM EDTA. The supernatant was diluted with 1 × TBST and 1 mM CaCl2. After selection, the eluate was amplified by infecting ER2738 cells in early log-phase 30 min at 37 °C. Amp (100 μg/ml) was added followed by incubation at 37 °C. An excess of M13K07 helper phage was added and incubated 30 min, 37 °C. The cells were pelleted, re-suspended in TSB + Y with Amp (100 μg/ml), Km (25 μg/ml), CaCl2 (1 mM) and IPTG (0.1 mM) 150 rpm, and amplified at 30 °C ON followed by precipitation.

The pAY02592 phagemid (similar to pAffi1 [43]) was modified to contain the Z-loop gene followed by geneIII (pAYZ-loop). The two libraries, with and without GGGlinker, were prepared separately. The forward stock contained primers encoding helix one and two with 50% encoding the F30A mutation. The reverse stock contained primers encoding an overlap with the 3′ end of helix two, linker (GGG-loop variant only), the loop library and helix three. Fifty percent had mutations in position L44, A48, L51 or any combination of the three. Five picomoles of each library stock was annealed and extended for six cycles. One hundred picomoles of forward and reverse primer was added and the library insert was amplified for 15 cycles and purified. The fragments and pAYZ-loop were cleaved and purified. pAYZ-loop was ligated with library fragments at 16 °C, overnight (ON) and purified. The libraries were pooled and transformed to ER2738 cells (Lucigen). After phenotyping, the cells were grown at 37 °C, ON in Tryptic Soy Broth (TSB, 30 g/l Merck) and 100 μg/ml ampicillin (Amp). The cells were harvested by centrifugation after 16 h and dissolved in glycerol (15%), aliquoted and stored at − 80 °C. Maturation library Five consecutive rounds of error prone-PCR using 0.1-pg template were run [GeneMorph II kit (Agilent)]. Fragment was purified and introduced in the phagemid by a restriction-free cloning reaction. pAYZ-loop containing the A5 gene was mixed with library fragments and thermo cycled [95 °C 1 min (95 °C 50 s, 60 °C 50 s, 68 °C 9 min) × 25]. DpnI enzyme (1μl) was added and incubated at 37 °C, 2 h. The plasmid was purified and electroporated to ER2738 cells. Library amplification Library variants were inoculated in TSB with 2% glucose, 100 μg/ml Amp, 10 μg/ml tetracycline (Tet) and 1 mM CaCl2 and incubated at 37 °C. An excess of M13K07 helper phages was added and incubated. The culture was harvested and dissolved in TSB with yeast extract (5 g/l, Merck, TSB + Y) 100 μg/ml Amp, 50 μg/ml Km, 1 mM IPTG and 1 mM CaCl2. The cultures were incubated at 30 °C, 250 rpm, ON. Phage precipitation Cells were harvested, the supernatant was precipitated with 5× PEG/NaCl and the pellet was dissolved in 1 × TBSTB [1 × TBS (50 mM Tris, 150 mM NaCl, pH 7.5), 0.1% Tween20, 3% BSA].

Phage ELISA Colonies from the output were inoculated in deep-well plates in TSB+Y with Amp (100 μg/ml), Tet (10 μg/ml) and CaCl2 (1 mM) and incubated at 37 °C, 250 rpm ON. The cultures were diluted and incubated at 37 °C, 250 rpm, 2 h. M13K07 helper phages were added at 37 °C for 30 min. Km (250 μg/ml) and IPTG (0.7 mM) were added and incubated at 30 °C, 250 rpm ON. The phage cultures were harvested and the supernatants saved. Ninety-six-well plates (Corning) were coated with polyclonal human IgG. Each phage supernatant was subjected to two ELISAs, one with buffers containing 100 mM EDTA and one with buffers containing 1 mM CaCl2. Positive control: original Z scaffold, negative control: Her2-binder. Phage supernatants were diluted 1:20 in 0.5% casein and added to two casein blocked plates and incubated for 1 h at RT, washed with 1 × TBST, and detected by anti-M13-HRP antibody (GE Healthcare). The plates were washed and developed

Please cite this article as: S. Kanje, et al., Protein Engineering Allows for Mild Affinity-based Elution of Therapeutic Antibodies, J. Mol. Biol. (2018), https://doi.org/10.1016/j.jmb.2018.06.004

9

Protein Engineering

with TMB substrate (Pierce) for 15–30 min before adding 2 M H2SO4. The absorbance was read in a Sunrise TM microplate reader (Tecan) at 450 nm. Protein production, purification and analysis Expression Selected variants were sub-cloned and produced as described in Tegel et al. [44] in the presence of 1 mM CaCl2. Cultures were harvested and pellets were dissolved in 1 × TBST-C (1 × TBS, 0.05% Tween20, 1 mM CaCl2) or (8 M Urea, 50 mM NaH2PO4•H2O, 300 mM NaCl, pH 7.5) and sonicated at 40% amplitude 1.0/1.0 s pulsing for 1 min and 30 s using a Vibra-cell (Sonics). The lysed cells were pelleted and the supernatants were saved.

First selection and maturation selection The A5 protein was run on a Biacore 3000 instrument (GE Healthcare). The maturation variants (Zmat1–9), A5 and the original Z domain were run on a Biacore T200 (GE Healthcare). Human IgG was immobilized on a CM5 chip. A dilution series of the proteins in 1 × TBST (0.05% Tween20) with 1 mM CaCl2 or 100 mM EDTA was analyzed. A5 was also analyzed in the same running buffers but with the addition of 2 M urea. Substitutional analysis The ZCa mutants were analyzed on Biacore 3000 with Fc domain of human IgG1 immobilized and 1 × HBST [20 mM Hepes, 150 mM NaCl (pH 7), 0.05% Tween20] with 1 mM CaCl2 as running buffer.

Purification Purification test IgG Sepharose

Columns packed with IgG-Sepharose 6 FF (GE Healthcare) were washed with dH2O, 0.3 M HAc (pH 3.2) and 1 × TBST-C. Protein lysates were added and washed with 1 × TBST-C and 5 mM NH4Ac with 1 mM CaCl2 (pH 5.5) prior to elution with 0.3 M HAc. The eluted protein was freeze-dried, dissolved in 1 × TBS with 1 mM CaCl2, and concentrations were determined using BCA (Pierce). IMAC (TALON® Metal Affinity Resin (Clontech Laboratories)) was used to purify the His-tagged ZCa mutant proteins.

IgG Sepharose 6 FF (GE Healthcare) was packed in a column and pulsed with 0.3 M HAc (pH 3.2) and 1 × TBST-C. One hundred micrograms of protein was added followed by a wash with 1 × TBST-C. Elution was tried with 100 mM NH4Ac with 100 mM EDTA (pH 5, 5.7, 6, 6.5 or 7) or 1 mM CaCl2 at pH 5. After elution, the column was washed with 5 mM NH4Ac with 1 mM CaCl2, pH 5.5, followed by elution with 0.3 M HAc, pH 3.2. All eluted fractions were measured at 280 nm.

Mass spectrometry and SDS-PAGE

Purification test ZCa and Z column

Molecular weight was determined by MS and protein purity by SDS-PAGE as described in Kanje and Hober [45]. Western blot Western blot analysis was performed as earlier described [46]. Human antibody was detected with HRP coupled goat-anti-human antibody (Novex). CD All CD measurements were performed on a Jasco J810. Protein melting curves were measured between 20 and 90 °C at 221 nm with a 5 °C/min temperature ramp. SPR Pre-study The test variants were run on a ProteOn™ XPR36 instrument (Bio-Rad). Monoclonal human IgG was immobilized to 5000 RU on a GLM chip. A dilution series of the variants were analyzed. All proteins were run with 1× PBST (0.05% Tween20) with 30 μM CaCl2 and 1× PBST with 30 μM EDTA.

Ten milligrams of ZCa and Z was coupled to 1 ml HiTrap™ NHS-Activated HP columns (GE Healthcare). Purifications of polyclonal human IgG, spiked CHO cell supernatant (100 mg/l polyclonal human IgG) and CHO cell supernatant from cells producing monoclonal human IgG (10 mg/l) were performed with a flow rate of 1 ml/min on Äkta Explorer system (Amersham Pharmacia Biotech). Equilibration was done with 1× TBST-C prior to sample injection using a 2-ml sample loop. Washing was performed with 1 × TBST-C followed by 5mM NH4Ac and 1 mM CaCl2 (pH 5.5). Protein was eluted with 0.3 M HAc (pH 3.2) or 100 mM NH4Ac and 100 mM EDTA (pH 5.5) or 100 mM NH4Ac and 1 mM CaCl2 (pH 5.5). Crystallization of the Fc–ZCa complex Fc domain of human IgG1 (trastuzumab) was provided by Genovis. Prior to crystallization trials, the proteins were run on a Superdex 75 column (GE Healthcare) in 20 mM Hepes (pH 7.0), 150 mM NaCl and 1 mM CaCl2. Peak fractions were pooled and concentrated. A high-throughput crystallization trial was carried out using a Mosquito robot (TTP Labtech, UK) crystallization screens at Lund Protein Production

Please cite this article as: S. Kanje, et al., Protein Engineering Allows for Mild Affinity-based Elution of Therapeutic Antibodies, J. Mol. Biol. (2018), https://doi.org/10.1016/j.jmb.2018.06.004

10

Protein Engineering

Platform. Several conditions yielded crystals, which were further reproduced manually. Data were collected on a single crystal obtained by vapor diffusion hanging drop method. The drop contained 5 mg/ml ZCa, 15 mg/ml Fc and ~3.5 mM CaCl2 in 20 mM Hepes (pH 7.0) and 150 mM NaCl mixed with reservoir solution consisting of 24% PEG3350, 0.1 M LiCl2 and 0.1 M Mes (pH 6.0). Crystals were immersed in a cryosolution consisting of the reservoir solution with an addition of 20% glycerol and flash cooled in liquid nitrogen. Data collection and structure determination X-ray diffraction data were collected at the ID23-1 beamline at ESRF, Grenoble [47], at the wavelength of 1.00000 Å and processed using XDS [48] indexed and scaled in POINTLESS and SCALA within the CCP4 program suite [49]. The structure was solved by molecular replacement using PHASER [50] within PHENIX [51]. The search model used for molecular replacement consisted of chain A of the Fc fragment (PDB code 5U4Y [35]) and a model of ZCa, which was generated in SWISS-MODEL server [52] using chain C of the Z domain from the same structure (PDB code 5U4Y). The calcium binding loop was deleted from the model for the molecular replacement. The resulting model was then rebuilt in AutoBuild [53] within PHENIX and refined using PHENIX [54] and manually inspected and edited in COOT [55]. Glycosylation sites were observed at Asn297 at each molecule of the Fc, and sugar molecules were introduced to these sites. The structure model was refined using NCS and TLS restraints and riding hydrogens, which were removed from the final model before deposition to the PDB (PDB ID 6FGO). Data and refinement statistics are reported in Supporting Table 7. Structure figures were prepared in PyMol (Schrödinger LLC).

Acknowledgments This work was funded by Vinnova. We thank our partner companies GE Healthcare and Genovis for valuable input and Prof. Nygren and Dr. Löfblom for fruitful discussions. We thank ESRF for providing beamtime and local scientists for assistance during data collection. Lund Protein Production Platform is acknowledged for assistance with crystallization.

Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.jmb.2018.06.004.

Received 3 April 2018; Received in revised form 25 May 2018; Accepted 4 June 2018 Available online xxxx Keywords: protein engineering; Protein A; antibody purification; Z domain; calcium-dependent binding Abbreviations used: IgG, immunoglobulin G; CD, circular dichroism; EDTA, ethylenediaminetetraacetic acid; SPR, surface plasmon resonance; BSA, buried surface area.

References [1] D.M. Ecker, S.D. Jones, H.L. Levine, The therapeutic monoclonal antibody market, MAbs 7 (2015) 9–14. [2] P. Gagnon, Technology trends in antibody purification, J. Chromatogr. A 1221 (2012) 57–70. [3] S. Hober, K. Nord, M. Linhult, Protein A chromatography for antibody purification, J. Chromatogr. B Anal. Technol. Biomed. Life Sci. 848 (2007) 40–47. [4] T. Boström, J. Nilvebrant, S. Hober, Purification Systems Based on Bacterial Surface Proteins, INTECH Open Access Publisher, 2012. [5] M. Vazquez-Rey, D.A. Lang, Aggregates in monoclonal antibody manufacturing processes, Biotechnol. Bioeng. 108 (2011) 1494–1508. [6] A.A. Shukla, P. Gupta, X. Han, Protein aggregation kinetics during Protein A chromatography. Case study for an Fc fusion protein, J. Chromatogr. A 1171 (2007) 22–28. [7] B. Liu, H. Guo, J. Xu, T. Qin, L. Xu, J. Zhang, et al., Acidinduced aggregation propensity of nivolumab is dependent on the Fc, MAbs 8 (2016) 1107–1117. [8] A.R. Mazzer, X. Perraud, J. Halley, J. O'Hara, D.G. Bracewell, Protein A chromatography increases monoclonal antibody aggregation rate during subsequent low pH virus inactivation hold, J. Chromatogr. A 1415 (2015) 83–90. [9] T.M. Pabst, R. Palmgren, A. Forss, J. Vasic, M. Fonseca, C. Thompson, et al., Engineering of novel Staphylococcal Protein A ligands to enable milder elution pH and high dynamic binding capacity, J. Chromatogr. A 1362 (2014) 180–185. [10] S. Gulich, M. Uhlen, S. Hober, Protein engineering of an IgGbinding domain allows milder elution conditions during affinity chromatography, J. Biotechnol. 76 (2000) 233–244. [11] T. Arakawa, J.S. Philo, K. Tsumoto, R. Yumioka, D. Ejima, Elution of antibodies from a Protein-A column by aqueous arginine solutions, Protein Expr. Purif. 36 (2004) 244–248. [12] I. Koguma, S. Yamashita, S. Sato, K. Okuyama, Y. Katakura, Novel purification method of human immunoglobulin by using a thermo-responsive protein A, J. Chromatogr. A 1305 (2013) 149–153. [13] L. Banci, I. Bertini, Metallomics and the cell: some definitions and general comments, Met. Ions Life Sci. 12 (2013) 1–13. [14] Z. Grabarek, Structural basis for diversity of the EF-hand calcium-binding proteins, J. Mol. Biol. 359 (2006) 509–525. [15] M. Yanez, J. Gil-Longo, M. Campos-Toimil, Calcium binding proteins, Adv. Exp. Med. Biol. 740 (2012) 461–482.

Please cite this article as: S. Kanje, et al., Protein Engineering Allows for Mild Affinity-based Elution of Therapeutic Antibodies, J. Mol. Biol. (2018), https://doi.org/10.1016/j.jmb.2018.06.004

Protein Engineering

[16] J.L. Gifford, M.P. Walsh, H.J. Vogel, Structures and metalion-binding properties of the Ca2+-binding helix-loop-helix EF-hand motifs, Biochem. J. 405 (2007) 199–221. [17] Y. Ye, H.W. Lee, W. Yang, S.J. Shealy, A.L. Wilkins, Z.R. Liu, et al., Metal binding affinity and structural properties of an isolated EF-loop in a scaffold protein, Protein Eng. 14 (2001) 1001–1013. [18] S.J. Kellmann, S. Dubel, H. Thie, A strategy to identify linkerbased modules for the allosteric regulation of antibodyantigen binding affinities of different scFvs, MAbs 9 (2017) 404–418. [19] D. O'Connell, S.S. Linse, E. Thulin, A. Merino, Affinity Tag System, (PCT/EP2013/050169) 2013. [20] S.F. Marino, D. Shechner, L. Regan, ‘Morphs’ (MRFs): metalreversible folding domains for differential IgG binding, Chem. Biol. 8 (2001) 1221–1229. [21] B. Nilsson, T. Moks, B. Jansson, L. Abrahmsen, A. Elmblad, E. Holmgren, et al., A synthetic IgG-binding domain based on staphylococcal protein A, Protein Eng. 1 (1987) 107–113. [22] M.A. Wilson, A.T. Brunger, The 1.0 A crystal structure of Ca (2 +)-bound calmodulin: an analysis of disorder and implications for functionally relevant plasticity, J. Mol. Biol. 301 (2000) 1237–1256. [23] H. Blanchard, P. Grochulski, Y. Li, J.S. Arthur, P.L. Davies, J.S. Elce, et al., Structure of a calpain Ca(2+)-binding domain reveals a novel EF-hand and Ca(2+)-induced conformational changes, Nat. Struct. Biol. 4 (1997) 532–538. [24] M. Nagae, A. Nozawa, N. Koizumi, H. Sano, H. Hashimoto, M. Sato, et al., The crystal structure of the novel calciumbinding protein AtCBL2 from Arabidopsis thaliana, J. Biol. Chem. 278 (2003) 42240–42246. [25] H. Kuboniwa, N. Tjandra, S. Grzesiek, H. Ren, C.B. Klee, A. Bax, Solution structure of calcium-free calmodulin, Nat. Struct. Biol. 2 (1995) 768–776. [26] H. Ishida, K. Takahashi, K. Nakashima, Y. Kumaki, M. Nakata, K. Hikichi, et al., Solution structures of the N-terminal domain of yeast calmodulin: Ca2+-dependent conformational change and its functional implication, Biochemistry 39 (2000) 13660–13668. [27] V.P. Reddy Chichili, Y. Xiao, J. Seetharaman, T.R. Cummins, J. Sivaraman, Structural basis for the modulation of the neuronal voltage-gated sodium channel NaV1.6 by calmodulin, Sci. Rep. 3 (2013) 2435. [28] M. Tashiro, R. Tejero, D.E. Zimmerman, B. Celda, B. Nilsson, G.T. Montelione, High-resolution solution NMR structure of the Z domain of staphylococcal protein A, J. Mol. Biol. 272 (1997) 573–590. [29] B.J. Marsden, G.S. Shaw, B.D. Sykes, Calcium binding proteins. Elucidating the contributions to calcium affinity from an analysis of species variants and peptide fragments, Biochem. Cell Biol. 68 (1990) 587–601. [30] S.P. Bottomley, A.G. Popplewell, M. Scawen, T. Wan, B.J. Sutton, M.G. Gore, The stability and unfolding of an IgG binding protein based upon the B domain of protein A from Staphylococcus aureus probed by tryptophan substitution and fluorescence spectroscopy, Protein Eng. 7 (1994) 1463–1470. [31] S. Chowdhury, H. Lei, Y. Duan, Denatured-state ensemble and the early-stage folding of the G29A mutant of the B-domain of protein A, J. Phys. Chem. B 109 (2005) 9073–9081. [32] L. Cedergren, R. Andersson, B. Jansson, M. Uhlen, B. Nilsson, Mutational analysis of the interaction between staphylococcal protein A and human IgG1, Protein Eng. 6 (1993) 441–448.

11 [33] L. Jendeberg, B. Persson, R. Andersson, R. Karlsson, M. Uhlen, B. Nilsson, Kinetic analysis of the interaction between protein A domain variants and human Fc using plasmon resonance detection, J. Mol. Recognit. 8 (1995) 270–278. [34] J. Deisenhofer, Crystallographic refinement and atomic models of a human Fc fragment and its complex with fragment B of protein A from Staphylococcus aureus at 2.9and 2.8-Å resolution, Biochemistry 20 (1981) 2361–2370. [35] M. Ultsch, A. Braisted, H.R. Maun, C. Eigenbrot, 3–2–1: structural insights from stepwise shrinkage of a three-helix Fc-binding domain to a single helix, Protein Eng. Des. Sel. 30 (2017) 619–625. [36] J. Feldwisch, V. Tolmachev, C. Lendel, N. Herne, A. Sjoberg, B. Larsson, et al., Design of an optimized scaffold for affibody molecules, J. Mol. Biol. 398 (2010) 232–247. [37] E. Krissinel, K. Henrick, Inference of macromolecular assemblies from crystalline state, J. Mol. Biol. 372 (2007) 774–797. [38] M. Simonovic, K. Dolmer, W. Huang, D.K. Strickland, K. Volz, P.G. Gettins, Calcium coordination and pH dependence of the calcium affinity of ligand-binding repeat CR7 from the LRP. Comparison with related domains from the LRP and the LDL receptor, Biochemistry 40 (2001) 15127–15134. [39] I. Nur, L. Bar, Method of the inactivation of viruses by a solventdetergent combination and nanofiltration, (US6468733B2) 2002. [40] P.L. Roberts, Virus inactivation by solvent/detergent treatment using Triton X-100 in a high purity factor VIII, Biologicals 36 (2008) 330–335. [41] P.L. Roberts, D. Lloyd, P.J. Marshall, Virus inactivation in a factor VIII/VWF concentrate treated using a solvent/detergent procedure based on polysorbate 20, Biologicals 37 (2009) 26–31. [42] T. Alm, L. Yderland, J. Nilvebrant, A. Halldin, S. Hober, A small bispecific protein selected for orthogonal affinity purification, Biotechnol. J. 5 (2010) 605–617. [43] C. Gronwall, A. Jonsson, S. Lindstrom, E. Gunneriusson, S. Stahl, N. Herne, Selection and characterization of Affibody ligands binding to Alzheimer amyloid beta peptides, J. Biotechnol. 128 (2007) 162–183. [44] H. Tegel, S. Tourle, J. Ottosson, A. Persson, Increased levels of recombinant human proteins with the Escherichia coli strain Rosetta(DE3), Protein Expr. Purif. 69 (2010) 159–167. [45] S. Kanje, S. Hober, In vivo biotinylation and incorporation of a photo-inducible unnatural amino acid to an antibody-binding domain improve site-specific labeling of antibodies, Biotechnol. J. 10 (2015) 564–574. [46] S. Kanje, E. von Witting, S.C. Chiang, Y.T. Bryceson, S. Hober, Site-specific photolabeling of the IgG Fab fragment using a small protein G derived domain, Bioconjug. Chem. 27 (2016) 2095–2102. [47] D. Nurizzo, T. Mairs, M. Guijarro, V. Rey, J. Meyer, P. Fajardo, et al., The ID23-1 structural biology beamline at the ESRF, J. Synchrotron Radiat. 13 (2006) 227–238. [48] W. Kabsch, Xds, Acta Crystallogr. D Biol. Crystallogr. 66 (2010) 125–132. [49] M.D. Winn, C.C. Ballard, K.D. Cowtan, E.J. Dodson, P. Emsley, P.R. Evans, et al., Overview of the CCP4 suite and current developments, Acta Crystallogr. D Biol. Crystallogr. 67 (2011) 235–242. [50] A.J. McCoy, R.W. Grosse-Kunstleve, P.D. Adams, M.D. Winn, L.C. Storoni, R.J. Read, Phaser crystallographic software, J. Appl. Crystallogr. 40 (2007) 658–674.

Please cite this article as: S. Kanje, et al., Protein Engineering Allows for Mild Affinity-based Elution of Therapeutic Antibodies, J. Mol. Biol. (2018), https://doi.org/10.1016/j.jmb.2018.06.004

12 [51] P.D. Adams, P.V. Afonine, G. Bunkoczi, V.B. Chen, I.W. Davis, N. Echols, et al., PHENIX: a comprehensive Pythonbased system for macromolecular structure solution, Acta Crystallogr. D Biol. Crystallogr. 66 (2010) 213–221. [52] M. Biasini, S. Bienert, A. Waterhouse, K. Arnold, G. Studer, T. Schmidt, et al., SWISS-MODEL: modelling protein tertiary and quaternary structure using evolutionary information, Nucleic Acids Res. 42 (2014) W252–W258. [53] T.C. Terwilliger, R.W. Grosse-Kunstleve, P.V. Afonine, N.W. Moriarty, P.H. Zwart, L.W. Hung, et al., Iterative model building, structure refinement and density modification with

Protein Engineering

the PHENIX AutoBuild wizard, Acta Crystallogr. D Biol. Crystallogr. 64 (2008) 61–69. [54] P.V. Afonine, R.W. Grosse-Kunstleve, N. Echols, J.J. Headd, N.W. Moriarty, M. Mustyakimov, et al., Towards automated crystallographic structure refinement with phenix.refine, Acta Crystallogr. D Biol. Crystallogr. 68 (2012) 352–367. [55] P. Emsley, B. Lohkamp, W.G. Scott, K. Cowtan, Features and development of Coot, Acta Crystallogr. D Biol. Crystallogr. 66 (2010) 486–501.

Please cite this article as: S. Kanje, et al., Protein Engineering Allows for Mild Affinity-based Elution of Therapeutic Antibodies, J. Mol. Biol. (2018), https://doi.org/10.1016/j.jmb.2018.06.004