1、Advancing Monoclonal Antibody Production with Improved Purification Technology,4th International Conference and Expo on Bioprocess & Biosystems Houston, USA, October 20-21, 2016,Nian Rui Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences,Outline,Chromatin-directed
2、cell culture clarification Improved protein A chromatography,Jun 2012-Sep 2015,2,Introduction The standard model of protein A is simple. IgG binds. Contaminants do not. Load, wash, elute pure IgG. But in the real world, protein A does not elute pure IgG.,Pg 3,Introduction Hypotheses to explain exces
3、s contamination include non-specific interactions between contaminants and:the protein A ligand,the base matrix of the chromatography media, and with IgG. Other hypotheses have been suggested to explain how protein A chromatography promotes aggregation or doesnt. But all of them overlook two rogue e
4、lements in the equation. The first is Chromatin. The second is protein A itself.,Pg 4,Starting from a clean slate Chromatin from dead cells persists in cell culture harvests as hetero-aggregates. Nucleosomal arrays provide their structural core.,Individual nucleosomes contain 147 bp of DNA wrapped 1
5、.65 times around a core octamer of histones (H2A,H2B,H3,H4)2). DNA and histones each contribute about 100 kDa. Nucleosomes are linked in linear arrays by 20-80 bp linker DNA segments associated with histone H1. Histones are strongly hydrophobic with pI ranges from 11-12; DNA about 2.6. Image modifie
6、d from scbt.sastra.edu,Pg 5,Composition of chromatin heteroaggregates The nucleosomal array cores of heteroaggregates are revealed by electrophoresis. Band size shows number of nucleosomes per array. Agarose electrophoresis of SEC fractions from cell culture harvest.,Pg 6,Composition of chromatin he
7、teroaggregates Diversity of host cell proteins in chromatin heteroaggregates Reduced SDS-PAGE, fractions from SEC cell culture harvest,Chromatin heteroaggregates contain more species than all other fractions combined. Histone identities confirmed by Mass Spec and Western blots.,Pg 7,Composition of c
8、hromatin heteroaggregates Native size distribution of major host contaminant classes SEC, harvest clarified by centrifugation-microfiltration,15-20% of the host contaminant mass is in the form of HMW chromatin heteroaggregates. Non-histone proteins contribute 80-90% of that mass. Their native IgG co
9、ntent is low and they mostly contain aberrant forms. Histones are typically less than 3% of total HCP. DNA:histone is typically 1:6, indicating advanced degradation of DNA. DNA:RNA 1:4. Chromatin heteroaggregates include every contaminant class!,Pg 8,What happens during protein A loading? Chromatin
10、heteroaggregate size in cell culture harvest ranges from 50-400 nm (DLS).,Unrestricted diffusive mass transport requires that pore size be at least 10 times larger than solute size. Jungbauer, J. Chromatogr. A 1065 (2005) 3-12. Pores and solutes drawn to scale. Solutes represented as hydrodynamic sp
11、heres without respect to detailed conformation.,J. Chromatography A 1374 (2014) 145-155.,Pg 9,What happens during protein A loading? Chromatin heteroaggregates bind to protein A.,Chromatin heteroaggregates obstruct access of IgG to particle pores and depress dynamic binding capacity by up to 20%. Ex
12、perimental data indicate that protein A binding of chromatin heteroaggregates is mediated through multipoint attachments to surface-accessible histone elements. Alkaline hydrophobic histones are a good multimodal match for the acidic hydrophobic surface of protein A.,Pg 10,What happens during protei
13、n A loading? Chromatin heteroaggregates bind protein A more strongly than IgG.,In addition to limiting dynamic capacity, strong chromatin binding has importance at later process stages: In particular, it makes a broad diversity of host contaminants accessible to IgG during elution.,J. Chromatography
14、 A 1340 (2014) 68-78.,Pg 11,X-ray crystallography results redrawn from Deisenhofer, Biochemistry 20 (1981) 2361.,Destabilization occurs at first contact but increases during elution.,Shown positions in the destabilized region are inferred from native IgG. Their mobility in the destabilized form rend
15、ers electron densities too low to assign coordinates.,Destabilization of the upper third of Cg2 results from re-positioning of one of the beta sheets due to re-positioning of one of the residues that contacts protein A in the cleft between Cg2 and Cg3.,What happens during protein A loading? IgG is c
16、onformationally modified by contact with protein A.,Pg 12,What happens during protein A elution? The low pH of elution compounds the denaturing effect of initial contact with protein A. IgG elutes as an unstable intermediate about half the size of native IgG. DLS post-elution in 100 mM acetate, pH 3
17、.5, vs native at physiological,The antibodies used to generate these results were all IgG1s. HE, HU, and AV are prospective biosimilars for Herceptin, Humira, and Avastin.,J. Chromatography A 1395 (2015) 136-142.,Pg 13,What happens during protein A elution? The compound denaturation mediated by prot
18、ein A and subsequent exposure to low pH is accompanied by a dramatic loss of structure in the Cg2 domain. Circular dichroism spectroscopy,CD spec shows a 45% loss of beta-sheet and corresponding increase in alpha-helix. Loss of structure contributes to aggregate formation. Note that native IgG expos
19、ed to pH 3.5 tracks closely with the native protein at pH 7.0, but there are differences. Those differences correlate with a tendency for IgG to associate nonspecifically with surfaces.,Pg 14,What happens during protein A elution? Conformational options for the 5.5 nm species suggest a folded-over c
20、onfiguration.,Creation of the folded-over form is facilitated by the structural collapse of the Cg2 domain, compounding the inherent flexibility of the hinge region.,Pg 15,What happens during protein A elution? The reduced size conformation is more vulnerable to aggregation by secondary stress than
21、native IgG. Analytical SEC,The trailing contaminant is H2L. It is also present in the third profile but obscured by broadening of the IgG peak. Peak broadening is itself a concern because it suggests the antibody is more prone to non-specific interactions, potentially leading to future aggregation.,
22、Pg 16,What happens during protein A elution? Elution conditions dissociate IgG from its direct associations with protein A, but also cause IgG to form stable associations with chromatin elements.,These data are from experiments with cell culture harvest, but the same phenomena occur during elution o
23、f protein A. Note the weak insolubility of pure IgG at acidic pH. Note that non-histone host protein reduction is about 20% at pH 4.5 where most of the chromatin has precipitated. This mostly corresponds to the non-histone HCP resident in chromatin heteroaggregates.,Pg 17,What happens during protein
24、 A elution? Interactions between chromatin and IgG at low pH promote formation of aggregates that persist after neutralization.,Dominance of UV absorbance at 254 nm highlights DNA. 280 dominance of the induced aggregate peak shows that it consists mainly of IgG. This suggests that DNA acts as an agg
25、regate-forming template. Histone-IgG interactions also create aggregates at pH 3.2.,J. Chromatography A in press, doi:10.1016/j.chroma.2015.07.017.,Pg 18,What happens during protein A elution? Elution conditions also cause subsets of contaminants to dissociate from chromatin elements that remain bou
26、nd to protein A.,Eluted IgG can bind to either leached chromatin elements, or to chromatin elements that remain bound to protein A. IgG bound to leached chromatin forms aggregates. IgG bound to protein A-bound chromatin is lost from elution. Typically 5%.Leached chromatin in the elutionfraction is t
27、he main cause of turbidity.,Pg 19,What happens during eluate neutralization? Physiological conditions restore native IgG size. Data from DLS,These data indicate that IgG is in a non-native configuration throughout the duration of virus inactivation by low pH, and potentially when being loaded to cat
28、ion exchangers. They also suggest, in both cases, that if the IgG is restored to native size before subsequent treatment, it may be less prone to aggregation.,Pg 20,What happens during eluate neutralization? Neutralization has two important additional effects:,Native IgG becomes dissociated from chr
29、omatin heteroaggregates. Chromatin elements meanwhile re-associate into very large particles that cause the high turbidity commonly observed among neutralized eluates. This is what enables contaminant and turbidity reduction by microfiltration, and why it does not reduce IgG recovery.,Pg 21,What hap
30、pens during eluate neutralization? Neutralization pH controls contaminant content of filtered eluate.,Chromatin elements leached from protein A during elution re-associate during neutralization, forming large particles that are easily eliminated by microfiltration. The higher their stability, the mo
31、re effectively they accumulate chromatin elements from the local environment, leaving fewer in the filtrate with IgG. Since the mechanism is driven by the behavior of chromatin, pH 5.5 should give similar results with all antibodies.,Pg 22,50 mM NaOH is more effective than 4 M guanidine, but not eno
32、ugh to prevent cumulative fouling. This seems likely to emerge as a performance issue in small-column continuous capture systems.,What happens during cleaning-sanitization? If not removed by aggressive treatment, chromatin accumulates, depresses column performance, and increases bioburden. Elution p
33、rofile, protein A, sanitization step,Pg 23,What if you apply aggressive pre-elution washes? Protein A chromatograms, with and without a 2.0 M NaCl wash prior to elution.,Pre-elution washes pre-leach contaminant subsets from strongly retained chromatin hetero-aggregate elements. They reduce the mass
34、of chromatin-associated contaminants that can leach during elution of IgG, but leachable contaminants remain. Washes do not help with capacity reduction by pore blockage during binding, nor do they prevent accumulation of bioburden.,Pg 24,What if you extract chromatin in advance? Protein A chromatog
35、rams, with and without advance removal of chromatin heteroaggregates.,Advance chromatin extraction enables unrestricted access of IgG to the pores of the protein A media and increases capacity, it virtually suspends contamination from leaching, it removes the opportunity for still-bound chromatin to
36、 trap eluted IgG, and it removes the opportunity for chromatin-IgG interactions to create aggregates during elution.,Pg 25,What if you extract chromatin in advance? Protein A, performance summary, with and without advance removal of chromatin heteroaggregates PA perform/post: Cen/mcF ChromX Improvem
37、ent DBC 25 g/L 31 g/L 20% Recovery 93% 99% 6% Productivity/cycle 23 g/L 30 g/L 24% Non-histone HCP 2006 ppm 10 ppm 200x Histone HCP 227 ppm LOD 200x DNA 13 ppm 1 ppb 13,000x Turbidity 108 NTU 6 NTU 18x Aggregates 1.7% 0.8% 2x All data from a Herceptin analog on TOYOPEARL AF-rProtein A-650F. More inf
38、ormation: J. Chromatogr. A 1340 (2014) 68-78.,Pg 26,Conclusions IgG purification by affinity chromatography with protein A is more complicated than the traditional model suggests. Protein A-induces a major conformational change in IgG that persists as long as elution conditions are maintained. This
39、appears to be universal among IgG1 antibodies, likely occurs among other subclasses, other species, and probably among Fc-fusion proteins. How it affects subsequent downstream operations remains to be investigated.,Pg 27,Conclusions Chromatin heteroaggregates interfere with protein A purification in
40、 many important ways:They block IgG access to particle pores, reduce capacity.They induce aggregation under elution conditions.They inflate host contamination of the eluted IgG.They mediate on-column loss of IgG.They increase bioburden.Their leachates impair the ability of subsequent purification st
41、eps to remove host contaminants. Since chromatin is a universal element of cell culture harvests, these effects should be understood to be universal.,Pg 28,Conclusions If you extract chromatin in advance, the protein A model becomes nearly as simple as everyone assumes it is. Protein A-mediated conformational change remains a potential concern, but chromatin-mediated effects are suspended. This enables better process economics, reproducibility, and product quality with a single polishing step than is otherwise possible with two polishing steps.,Pg 29,Thank you！,Pg 30,
