Redefining the Interface Between Upstream and Downstream Processing Pete Gagnon, Hui Theng Gan, Lihan Tan, Wei Zhang, Rui Nian Bioprocessing Technology Institute, Singapore BioProcessing Asia, Langkawi, Malaysia, November 3-6, 2014 Introduction One of the most basic principles of process development is that any given purification method should be preceded by a step to remove agents that would interfere with the ability of the method to fulfill its intended role. The need to remove solids is obvious, but stopping there represents an assumption that interference is mediated exclusively through physical clogging. Recent studies show that chemical interference also depresses fractionation performance, severely at capture, and significantly during later polishing steps. Luhrs et al, J. Chromatogr. B 877 (2009) 1543. • Mechetner et al, J. Chromatogr. B 879 (2011) 2583. Gagnon et al, J. Chromatogr. A 1218 (2011) 2405. • Gan et al, J. Chromatogr. A 1291 (2013) 33. Gagnon et al, J. Chromatogr. A 1324 (2014) 171. • Gagnon et al, J. Chromatogr. A 1340 (2014) 68. © 2014, Bioprocessing Technology Institute, all rights reserved. 2 Introduction All data in this presentation were obtained with an IgG1 monoclonal antibody immunospecific for human epidermal growth factor receptor 2, produced at up to 2.8 g/L by ~1011 CHO cells/mL in protein-free media, from a tri-cistronic vector developed at BTI. Ho et al, J. Biotechnol. 157 (2012) 130. © 2014, Bioprocessing Technology Institute, all rights reserved. 3 The chemical interface Chromatin expelled from dead host cells is a major contributor to aggregate formation and source of host cell contamination. Individual nucleosomes contain 147 bp of DNA wrapped 1.65 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. Image modified from scbt.sastra.edu © 2014, Bioprocessing Technology Institute, all rights reserved. 4 The chemical interface Intact nucleosomal arrays persist in cell culture harvest Fractions from SEC in 2 M NaCl, agarose electrophoresis © 2014, Bioprocessing Technology Institute, all rights reserved. 5 The chemical interface Native size distribution of major contaminant classes SEC, harvest clarified by centrifugation-microfiltration Nucleosome arrays provide the structural core of chromatin heteroaggregates that range in size from about 50-700 nm. DNA and histones dominate their chemical behavior, but 80% of their mass is from non-histone proteins. © 2014, Bioprocessing Technology Institute, all rights reserved. 6 The chemical interface Size distribution of host proteins, rSDS-PAGE of SEC fxns HMW fxns contain more species than all others, most <100 kDa. Histone identities confirmed by Mass Spec and Western blots. © 2014, Bioprocessing Technology Institute, all rights reserved. 7 The chemical interface Why has the role of chromatin not been recognized? Broad spectrum HCP immunoassays do not detect histones. Accurate estimation of histone content requires dedicated histone assays. Even dedicated histone assays require up to 16 hour sample extraction at pH values below 1, and some require antibody addition to the sample under those conditions. Accurate determination of total HCP requires that broad spectrum and dedicated histone assays be run, and the results added together. © 2014, Bioprocessing Technology Institute, all rights reserved. 8 The chemical interface Why has the role of chromatin not been recognized? DNA also requires severe sample preparation: up to 16 hours exposure to proteinase K in 0.5% SDS at 50°C. Lacking such preparation, DNA quantitation and determination of size distribution are both confounded. DNA association with chromatin also points to another analytical challenge: All of the DNA in cell culture harvests is histone-associated. It behaves differently than purified DNA. Are clearance results from spike-recovery models with purified DNA valid? © 2014, Bioprocessing Technology Institute, all rights reserved. 9 The chemical interface How does chromatin respond to different conditions? Under physiological conditions: Strong binding of DNA to histones retards its lysis by endogenous nucleases released at cell death. Inter-nucleosomal linker DNA becomes apparent first, then lysis of core DNA releases histone octamers. Liberated core octamers dissociate stepwise to hexamers, tetramers, dimers, then individual histones. Liberated histones bind secondarily to exposed DNA on the perimeter of still-intact nucleosomes. Secondary associations of non-histone HCP with chromatin remain fairly stable. © 2014, Bioprocessing Technology Institute, all rights reserved. 10 The chemical interface How does chromatin respond to different conditions? In 2 M NaCl: DNA dissociates from histone core octamers. Histone core octamers are stabilized and form aggregates. Liberated core octamers co-aggregate with histones released from no-longer-existing nucleosomes. Secondary associations between chromatin and nonhistone proteins are destabilized. © 2014, Bioprocessing Technology Institute, all rights reserved. 11 The chemical interface How does chromatin respond to different conditions? © 2014, Bioprocessing Technology Institute, all rights reserved. 12 The chemical interface Take-home: Composition of chromatin heteroaggregates varies substantially as a function of buffer conditions. That means their composition varies across different fractionation environments – different kinds of chromatography. It also means their composition varies across the stages of a chromatography method, and across processes. © 2014, Bioprocessing Technology Institute, all rights reserved. 13 Interactions with chromatography media Protein A as a model Under loading conditions, the nucleosomal cores of most heteroaggregates are insulated by surrounding non-histone proteins. Their affinity for protein A is nil, and these heteroaggregates are eliminated in the flow-through. Heteroaggregates with exposed histone elements bind strongly to protein A carboxyl groups. At 50-700 nm, they are mostly too large to enter particle pores, so they accumulate on the surface where they interfere with the entrance of IgG into the pores. This depresses binding capacity. © 2014, Bioprocessing Technology Institute, all rights reserved. 14 Interactions with chromatography media Protein A as a model Under elution conditions, histone-protein A contacts and some intra-heteroaggregate associations persist, but others are destabilized and become dissociated. Dissociated contaminant subsets include individual species and heteroaggregates. These leachates are mostly soluble under elution conditions, but become turbid upon neutralization. Note: that means turbidity of protein A eluates is not an indicator of low pH-mediated damage to the antibody. Heteroaggregate components that remain bound after elution are mostly removed by 4 M guanidine, and more effectively by 50 mM NaOH, but complete removal requires 500 mM NaOH. © 2014, Bioprocessing Technology Institute, all rights reserved. 15 Interactions with chromatography media The overall pattern is the same with cation exchange and electronegative multimodals, but the contaminant subsets that flow-through, that are bound, or leach during elution, or remain bound, vary with the chemistry of the solid phase and with the elution conditions. The pattern is conceptually similar with electropositive multimodals applied in bind-elute mode, except that binding is mediated through DNA. The leaching factor is eliminated with polishing methods applied in flow-through mode because there is no change of conditions to dissociate contaminant subsets from strongly bound heteroaggregates. © 2014, Bioprocessing Technology Institute, all rights reserved. 16 Interactions with chromatography media CHA: chromatin heteroaggregate. 1-3) Bound CHA restricts pore access. 4) compound accumulation of CHA. 5) CHA without external histone contact flow through the column. 6,8) histone-dominant CHA remnants restrict pore egress. 7) individual non-histone dissociated from CHA by elution conditions contaminants eluted IgG. 9) CHA dissociated from compound association with still-retained CHA also contaminates eluted IgG. © 2014, Bioprocessing Technology Institute, all rights reserved. 17 Managing chromatin The most effective approach is to remove chromatin in advance. Removal can target the DNA component or the histone component. Management tools: Ethacridine: intercalates and destabilizes DNA, weakens interactions between DNA and histones, co-precipitates proteins. Caprylic acid: co-precipitates proteins by accumulating on their electropositive domains. Very high affinity for histones. Allantoin: co-precipitates aggregates though hydrogen bonding. Solid phases: particles, membranes, monoliths, depth filters, etc., with chromatin-complementary surface chemistry. © 2014, Bioprocessing Technology Institute, all rights reserved. 18 Managing chromatin Comparison of harvest clarified by different methods. Redux of/clar.met. Cent.MicF Ethacridine Caprylate Host DNA nil 5-6 logs 5-6 logs Histone host proteins nil > 95% > 99% Non-histone host proteins nil 60-90% 90-99% Aggregates > 300 kDa nil 99% 99% Aggregates < 300 kDa nil 90-99% 90-99% IgG fragments nil 0-50% 50-95% MVM nil 4 logs 5 logs MuLV nil 5 logs 9 logs Final turbidity 10-50 NTU 2 NTU 2 NTU IgG recovery 90-95% 98-99% 90-95% Ethacridine and Caprylate refer to hybrid methods that include solid phase adsorbents to enhance performance. © 2014, Bioprocessing Technology Institute, all rights reserved. 19 Managing chromatin Analytical SEC after different clarification methods © 2014, Bioprocessing Technology Institute, all rights reserved. 20 Benefits of managing chromatin Protein A, traditional clarification vs advance chromatin removal The contaminants in IgG eluted from protein A are not bound to IgG, and they do not co-elute with IgG. They dissociate from more strongly bound chromatin heteroaggregates at the same time IgG is dissociated from its interaction with protein A. Advance chromatin removal suspends this contamination pathway. © 2014, Bioprocessing Technology Institute, all rights reserved. 21 Benefits of managing chromatin Protein A, traditional clarification vs pre-elution wash Pre-elution washes pre-leach contaminant subsets from strongly retained chromatin heteroaggregates. This reduces the mass of contaminants that can leach during elution of IgG, but leachable contaminants remain. © 2014, Bioprocessing Technology Institute, all rights reserved. 22 Benefits of managing chromatin Protein A, purification summary, washes vs advance removal Param/Met. Traditional Pre-E wash AdvChromX Capacity 25 mg/mL 25 mg/mL 31 mg/mL DNA 13 ppm 6 ppm 2 ppm HCP 2,006 ppm 708 ppm < 1 ppm Turbidity 108 NTU 26 NTU 6 NTU The ability of pre-elution washes to improve purification performance is fractional compared to advance chromatin removal, they provide no improvement in dynamic binding capacity, and they do not reduce cumulative fouling of the adsorbent. © 2014, Bioprocessing Technology Institute, all rights reserved. 23 Benefits of managing chromatin Eshmuno™ HCX, traditional clarification vs advance chromatin removal Retained chromatin heteroaggregates obstruct pores and reduce IgG capacity more than 3-fold. They also interfere with elution. More than 95% of the contaminants in the IgG fraction are leached from chromatin heteroaggregates that remain bound under elution conditions. © 2014, Bioprocessing Technology Institute, all rights reserved. 24 Benefits of managing chromatin Purification summary: Clar. > Eshmuno HCX > Capto™ adhere Step/Clar. Traditional AdvChromX OM DNA His Nhis 4,722 ppm 28,359 ppm 377,976 ppm 4 ppm LOQ 27,302 ppm > 1,000x > 28,000x 14x HCX Capacity DNA His Nhis 29 mg/mL 65 ppm 1,474 ppm 125,507 ppm 94 mg/mL 4 ppm LOQ 3,704 ppm 3x 16x >1,400x 34x CA Capacity DNA His Nhis Aggr 9 mg/mL 60 ppb LOQ 2,682 ppm 0.05% 11 mg/mL 3 ppb LOQ 77 ppm 0.05% 20% 20x -----35x ------ Net recov. 71% 86% 17% His: histone host proteins. Nhis: non-histone host proteins. Aggr: aggregates © 2014, Bioprocessing Technology Institute, all rights reserved. Improvement 25 Benefits of managing chromatin Chromatin-directed clarification enables many capture options and a reduction of processing steps. Method HCP aggregates Protein A > Capto adhere < 1 ppm < 0.05% Cation exchange > Capto adhere < 1 ppm < 0.05% Cation exchange > hydroxyapatite < 1 ppm < 0.05% Multimodal (HCX) > VEAX < 1 ppm < 0.05% VEAX > Capto adhere < 1 ppm < 0.05% SXC > Capto adhere < 1 ppm < 0.05% VEAX: anion exchange chromatography in void exclusion mode. Nian et al, J. Chromatogr. A. 1282 (2013) 127-132. SXC: steric exclusion chromatography with magnetic nanoparticles. Gagnon et al, J. Chromatography A 1324 (2014) 171. All HCP values with Cygnus Generation III HCP ELISA. © 2014, Bioprocessing Technology Institute, all rights reserved. 26 Conclusions Chromatin is a keystone variable and defining element of the interface between upstream and downstream processing. It plays a role in aggregate formation and it influences the behavior of hundreds of host contaminant species. Its composition and chromatographic behavior are strongly influenced by the local chemical environment. It depresses capacity and fractionation performance of all purification methods. Its extreme physicochemical features make it a challenge for analytical methods. © 2014, Bioprocessing Technology Institute, all rights reserved. 27 Conclusions Awareness of chromatin’s bioprocessing impact provides a starting point for managing it. Integrating chromatin characterization into upstream operations could lead to cell culture processes that enhance performance and reproducibility of DSP. Characterizing chromatin during DSP development leads to more effective and better understood purification processes. These include many alternatives to the traditional protein A platform – many with only two chromatography steps. © 2014, Bioprocessing Technology Institute, all rights reserved. 28 Cautions Any recombinant protein produced in cells and harvested from preparations that contain dead cells will contain chromatin or its analogs. Even yeasts and bacteria require DNA-compaction proteins to organize their genetic material, and those proteins are all referred to as histones or histone-like. The issue extends to viruses, since their production typically kills the cells that host them. Whether recognized or not, chromatin is an important process variable for all biological product classes. © 2014, Bioprocessing Technology Institute, all rights reserved. 29 EndNotes If you know the enemy and know yourself, you need not fear the result of a hundred battles. –Sun Tzu Thanks to Sarah Maria Abdul Latiff, Jeremy Lee, Chiew Ling Lim, Cindy Chuah, Dawn Goh, Phyllicia Toh, Xinhui Wang, Qiaohui Yang, Aina Hoi, Denise Leong, Amrita Rai, Edmund Lim, Xuezhi Bi, Cheong Nge, and Jimmy Chao for performing experiments and analyses; Yuan Sheng Yang, Han Ping Loh, Su Jun Low, and Jake Chng for providing cell culture media. Thanks also to Validated Biosystems, to the Biomedical Research Council of the Singapore Agency for Science, Technology, and Research, and to Exploit Technologies Pte. Ltd. for supporting this work. Information/reprints: [email protected] © 2014, Bioprocessing Technology Institute, all rights reserved. 30
© Copyright 2025 Paperzz