Enumeration of Neural Stem Cells Using Clonal Assays

Neural stem cells (NSCs) have the ability to self-renew and generate the three major neural lineages &#8212; astrocytes, neurons and oligodendrocytes. ...

Agency for Science, Technology and Research (A*STAR)

Understanding how autocrine/paracrine factors regulate neural stem cell (NSC) survival and growth is fundamental to the utilization of these cells for therapeutic applications and as cellular models for the brain. In vitro, NSCs can be propagated along with neural progenitors (NPs) as neurospheres (nsphs). The nsph conditioned medium (nsph-CM) contains cell-secreted factors that can regulate NSC behavior. However, the identity and exact function of these factors within the nsph-CM has remained elusive. We analyzed the nsph-CM by mass spectrometry and identified DSD-1-proteoglycan, a chondroitin sulfate proteoglycan (CSPG), apolipoprotein E (ApoE) and cystatin C as components of the nsph-CM. Using clonal assays we show that CSPG and ApoE are responsible for the ability of the nsph-CM to stimulate nsph formation whereas cystatin C is not involved. Clonal nsphs generated in the presence of CSPG show more than four-fold increase in NSCs. Thus CSPG specifically enhances the survival of NSCs. CSPG also stimulates the survival of embryonic stem cell (ESC)-derived NSCs, and thus may be involved in the developmental transition of ESCs to NSCs. In addition to its role in NSC survival, CSPG maintains the three dimensional structure of nsphs. Lastly, CSPG's effects on NSC survival may be mediated by enhanced signaling via EGFR, JAK/STAT3 and PI3K/Akt pathways.

CSPG is a secreted factor that stimulates neural stem cell survival possibly by enhanced EGFR signaling.

The central nervous system (CNS) is a large network of interconnecting and intercommunicating cells that form functional circuits. Disease and injury of the CNS are prominent features of the healthcare landscape. There is an urgent unmet need to generate therapeutic solutions for CNS disease/injury. To increase our understanding of the CNS we need to generate cellular models that are experimentally tractable. Neural stem cells (NSCs), cells that generate the CNS during embryonic development, have been identified and propagated in vitro. To develop NSCs as a cellular model for the CNS we need to understand more about their genetics and cell biology. In particular, we need to define the mechanisms of self-renewal, proliferation and differentiation--i.e. NSC behavior. The analysis of pluripotency of embryonic stem cells through mapping regulatory networks of transcription factors has proven to be a powerful approach to understanding embryonic development. Here, we discuss the role of transcription factors in NSC behavior.

Transcription factors and neural stem cell self-renewal, growth and differentiation.

Neural stem cells (NSCs) are self-renewing multipotent cells that undergo symmetric and asymmetric cell division during development of the nervous system. The behavior of NSCs is tightly regulated by intrinsic processes such as transcriptional and post-transcriptional control, as well as the stem cell niche factors that activate ligand-receptor-mediated signaling pathways. However, the role of these niche factors that regulate NSC behavior is not clearly understood. We identified chondroitin sulfate proteoglycan, apolipoprotein E (ApoE) and cystatin C as factors derived from the mouse neurosphere conditioned medium. Here, we show that ApoE is an autocrine/paracrine factor that regulates NSC survival. Stimulation of NSC survival is mediated by ApoE receptor interaction and the downstream extracellular signal-regulated kinase/mitogen-activated protein kinase signaling pathway. In addition, ApoE also enhanced neurosphere formation of mouse embryonic stem cell-derived NSCs. Finally, in vitro differentiation studies with ApoE knock-out NSCs suggest a role for ApoE in oligodendrogenesis.

Identification of ApoE as an autocrine/paracrine factor that stimulates neural stem cell survival via MAPK/ERK signaling pathway.

Oct4 and Sox2 are two essential transcription factors that co-regulate target genes for the maintenance of pluripotency. However, it is unclear whether they interact prior to DNA binding or how the target sites are accessed in the nucleus. By generating fluorescent protein fusions of Oct4 and Sox2 that are functionally capable of producing iPSCs (induced pluripotent stem cells), we show that their interaction is dependent on the presence of cognate DNA-binding elements, based on diffusion time, complex formation and lifetime measurements. Through fluorescence correlation spectroscopy, the levels of Oct4 and Sox2 in the iPSCs were quantified in live cells and two diffusion coefficients, corresponding to free and loosely bound forms of the protein, were distinguished. Notably, the fraction of slow-diffusing molecules in the iPSCs was found to be elevated, similar to the profile in embryonic stem cells, probably due to a change in the nuclear milieu during reprogramming. Taken together, these findings have defined quantitatively the amount of proteins pertinent to the pluripotent state and revealed increased accessibility to the underlying DNA as a mechanism for Oct4 and Sox2 to find their target binding sites and interact, without prior formation of heterodimer complexes.

DNA-dependent Oct4-Sox2 interaction and diffusion properties characteristic of the pluripotent cell state revealed by fluorescence spectroscopy.

We introduce a chromatography method for purification of large proteins and viruses that works by capturing them at a non-reactive hydrophilic surface by their mutual steric exclusion of polyethylene glycol (PEG). No direct chemical interaction between the surface and the target species is required. We refer to the technique as steric exclusion chromatography. Hydroxyl-substituted polymethacrylate monoliths provide a hydrophilic surface and support convective mass transport that is unaffected by the viscosity of the PEG. Elution is achieved by reducing PEG concentration. Selectivity correlates with molecular size, with larger species retained more strongly than smaller species. Retention increases with PEG size and concentration. Salts weaken retention in proportion to their concentration and Hofmeister ranking. Retention is enhanced near the isoelectric point of the target species. Virus binding capacity was measured at 9.9×10(12) plaque forming units per mL of monolith. 99.8% of host cell proteins and 93% of DNA were eliminated. Mass recovery exceeded 90%. IgM capacity was greater than 60 mg/mL. 95% of host cell proteins were eliminated from IgM produced in protein-free media, and mass recovery was up to 90%. Bioactivity was fully conserved by both viruses and antibodies. Process time ranged from less than 30 min to 2 h depending on the product concentration in the feed stream.

Principles and applications of steric exclusion chromatography.

Tolerance of low pH is crucial for recombinant proteins to survive conditions that might be experienced during manufacturing such as virus inactivation and elution from bioaffinity columns. In this study, we exposed three different purified immunoglobulins M (IgMs) to pH 3.5 for 60 min at room temperature. Treated samples showed no significant aggregation or fragmentation and retained full immunoreactivity, an intact glycosylation profile, and unchanged thermal stability. Because IgMs are serious candidates for next-generation therapeutics, it is essential to know that some of them are stable at low pH.

Immunoglobulins M survive low-pH conditions used for virus inactivation and for elution from bioaffinity columns.

&#x6211;&#x4EEC;&#x63CF;&#x8FF0;&#x9634;&#x79BB;&#x5B50;&#x4EA4;&#x6362;&#x8272;&#x8C31;&#x6CD5;&#x5217;&#x6324;&#x6EE1;&#x4E86;&#x4F53;&#x73B0;&#x4F4E;&#x5BC6;&#x5EA6;&#x5E26;&#x7535;&#x7684;&#x805A;&#x5408;&#x7269;&#x533A;&#x652F;&#x6301;&#x7684;&#x66F4;&#x9AD8;&#x7684;&#x5BC6;&#x5EA6;&#x805A;&#x5408;&#x7269;&#x9AA8;&#x67B6;&#x7684;&#x591A;&#x5B54;&#x9897;&#x7C92;&#x4E2D;&#x7684;&#x65B0;&#x7684;&#x53D8;&#x91CF;&#x3002;IgG &#x53CD;&#x6297;&#x7684;&#x9634;&#x79BB;&#x5B50;&#x4EA4;&#x6362;&#x89C4;&#x8303;&#x548C;&#x88AB;&#x6392;&#x9664;&#x5728; pH 3-10 &#x548C; 0-4 &#x7684;&#x65E0;&#x6548;&#x5377; M &#x6C2F;&#x5316;&#x94A0;&#x3002;Fab&#x3001; F(ab&#39;)(2) &#x548C; IgM &#x4E5F;&#x51FA;&#x73B0;&#x65E0;&#x6548;&#x7684;&#x6392;&#x65A5;&#x3002;&#x5BBF;&#x4E3B;&#x7EC6;&#x80DE;&#x86CB;&#x767D;&#x6C61;&#x67D3;&#x7269;&#x5927;&#x591A;&#x662F;&#x6309;&#x7167;&#x60EF;&#x5E38;&#x89C4;&#x8303;&#x9634;&#x79BB;&#x5B50;&#x4EA4;&#x6362;&#x548C;&#x66F4;&#x5F3A;&#x70C8;&#x7684; ph &#x503C;&#x7684;&#x589E;&#x52A0;&#x548C;&#x51CF;&#x5C11;&#x7535;&#x5BFC;&#x7387;&#x7684;&#x7ED1;&#x5B9A;&#x3002;&#x793A;&#x4F8B;&#x7F13;&#x51B2;&#x533A;&#x7EC4;&#x6210;&#x4E86;&#x4E0D;&#x4F1A;&#x5F71;&#x54CD;&#x5206;&#x533A;&#xFF0C;&#x53EA;&#x8981;&#x5E94;&#x7528;&#x6837;&#x672C;&#x91CF;&#x4E0D;&#x8D85;&#x8FC7;&#x5217;&#x7684;&#x7C92;&#x5B50;&#x95F4;&#x7684;&#x7A7A;&#x9699;&#x5377;&#x3002;Void &#x6392;&#x9664;&#x6297;&#x4F53;&#x6D17;&#x8131;&#x5747;&#x8861;&#x7F13;&#x51B2;&#x533A;&#x4E2D;&#x3002;&#x8FD9;&#x4E2A;&#x770B;&#x4F3C;&#x77DB;&#x76FE;&#x7684;&#x884C;&#x4E3A;&#x96C6;&#x5408;&#x662F;&#x7531;&#x4F4E;&#x5BC6;&#x5EA6;&#x805A;&#x5408;&#x7269;&#x533A;&#x3001; &#x7535;&#x8377;&#x7279;&#x6027;&#x7684;&#x6297;&#x4F53;&#x7269;&#x79CD;&#x548C; ph &#x503C;&#x548C;&#x7535;&#x5BFC;&#x7387;&#x7684;&#x5747;&#x8861;&#x7F13;&#x51B2;&#x533A;&#x901A;&#x8FC7;&#x4ECB;&#x5BFC;&#x7684;&#x53EF;&#x53D8;&#x5927;&#x5C0F;&#x6392;&#x65A5;&#x51FD;&#x6570;&#x8C03;&#x548C;&#x7684;&#x3002;&#x5F53;&#x524D;&#x4EE3;&#x591A;&#x5B54;&#x9897;&#x7C92;&#x578B;&#x9634;&#x79BB;&#x5B50;&#xFF0C;&#x91C7;&#x7528;&#x5AC1;&#x63A5;&#x6280;&#x672F;&#x5B9E;&#x73B0;&#x9AD8;&#x7535;&#x8377;&#x5BC6;&#x5EA6;&#x8C03;&#x89E3;&#x65E0;&#x6548;&#x88AB;&#x6392;&#x65A5;&#x5230;&#x6700;&#x9002;&#x5408;&#x7684;&#x5B9E;&#x73B0;&#x5B8C;&#x5168;&#x6392;&#x9664;&#xFF0C;&#x4E0E;&#x5176;&#x4ED6;&#x4EBA;&#x4E0D;&#x540C;&#x7A0B;&#x5EA6;&#x5C3D;&#x53EF;&#x80FD;&#x5C11;&#x8FBE; 65%&#x3002;Perfusive &#x548C;&#x975E;&#x5AC1;&#x63A5;&#x7684;&#x57FA;&#x4E8E;&#x7C92;&#x5B50;&#x7684;&#x6362;&#x5668;&#x53EA;&#x8981; 50%&#x6392;&#x65A5;&#x8FDB;&#x884C;&#x8C03;&#x89E3;&#x3002;&#x6574;&#x4F53;&#x67F1;&#x8C03;&#x89E3;&#x4E0D;&#x6392;&#x65A5;&#xFF0C;&#x56E0;&#x4E3A;&#x4ED6;&#x4EEC;&#x7F3A;&#x4E4F;&#x7C92;&#x5B50;&#x95F4;&#x7684;&#x7A7A;&#x9699;&#x4F53;&#x79EF;&#x3002;&#x5408;&#x683C;&#x6362;&#x5668;&#x3001; &#x6280;&#x672F;&#x652F;&#x6301;&#x5927;&#x4E8E; 99%&#x51CF;&#x5C11;&#x5BBF;&#x4E3B;&#x86CB;&#x767D;&#x8D28;&#x3001; DNA&#x3001; &#x548C;&#x5185;&#x6BD2;&#x7D20;&#x3002;&#x75C5;&#x6BD2;&#x51CF;&#x5C11;&#x4E86; 99.9%&#x4EE5;&#x4E0A;&#xFF0C;&#x805A;&#x5408;&#x51CF;&#x81F3;&#x5C0F;&#x4E8E; 0.05%&#x3002;&#x8BE5;&#x65B9;&#x6CD5;&#x652F;&#x6301;&#x66F4;&#x597D;&#x7684;&#x8FDB;&#x7A0B;&#x63A7;&#x5236;&#x6BD4;&#x5176;&#x4ED6;&#x9634;&#x79BB;&#x5B50;&#x4EA4;&#x6362;&#x683C;&#x5F0F;&#x56E0;&#x4E3A; ph &#x503C;&#x8FDC;&#x8DB3;&#x4E00;&#x8D77;&#x4E0E;&#x76D0;&#x6D53;&#x5EA6;&#x7684;&#x53D8;&#x5316;&#x4E0D;&#x4F1A;&#x53D1;&#x751F;&#x76F4;&#x5230;&#x540E;&#x6297;&#x4F53;&#x5DF2;&#x6D17;&#x8131;&#x4ECE;&#x5217;&#x3002;

&#x4F5C;&#x5E9F;&#x6297;&#x4F53;&#x5AC1;&#x63A5;&#x914D;&#x4F53;&#x591A;&#x5B54;&#x9897;&#x7C92;&#x578B;&#x9634;&#x79BB;&#x5B50;&#x7684;&#x6392;&#x65A5;&#x3002;

We describe a new variant of anion exchange chromatography in columns packed with porous particles that embody charged low-density polymer zones supported by a higher density polymer skeleton. IgG defies the norms of anion exchange and is excluded to the void volume at pH 3-10 and 0-4M NaCl. Void exclusion also occurs with Fab, F(ab')(2), and IgM. Host cell protein contaminants mostly follow the usual norms of anion exchange and bind more strongly with increasing pH and decreasing conductivity. Sample buffer composition has no impact on partitioning so long as applied sample volume does not exceed the interparticle void volume of the column. Void-excluded antibody elutes in equilibration buffer. This seemingly conflicted collection of behaviors is reconciled by a variable size exclusion function mediated through the low-density polymer zones, the charge properties of the antibody species, and the pH and conductivity of the equilibration buffer. Current-generation porous particle anion exchangers that employ grafting techniques to achieve high charge density mediate void exclusion to varying degrees, with the best-suited achieving complete exclusion, and others as little as 65%. Perfusive and non-grafted particle-based exchangers mediate as little as 50% exclusion. Monoliths mediate no exclusion, due to their lack of an interparticle void volume. On qualified exchangers, the technique supports greater than 99% reduction of host proteins, DNA, and endotoxin. Virus is reduced more than 99.9%, and aggregates are reduced to less than 0.05%. The method supports better process control than other anion exchange formats because pH excursions in conjunction with changes in salt concentration do not occur until after the antibody has eluted from the column.

Void exclusion of antibodies by grafted-ligand porous particle anion exchangers.

Les auteurs d&eacute;crivent une nouvelle variante de chromatographie &eacute;changeuse d&#39;anion dans les colonnes emball&eacute;es avec des particules poreuses qui incarnent les zones charg&eacute;es polym&egrave;re de faible densit&eacute;, soutenus par un squelette de polym&egrave;re de densit&eacute; plus &eacute;lev&eacute;. IgG d&eacute;fie les normes d&#39;&eacute;change anionique et est exclu pour le volume de vide &agrave; pH 3-10 et 0-4 M de NaCl. Sub exclusion se produit &eacute;galement avec Fab, F(ab&#39;)(2) et IgM. Contaminants de prot&eacute;ine cellulaire h&ocirc;te suivent plus souvent les normes habituelles d&#39;&eacute;change d&#39;anions et de lier plus fortement avec l&#39;augmentation de pH et conductivit&eacute;. Composition du tampon &eacute;chantillon n&#39;a aucune incidence sur la r&eacute;partition tant qu&#39;appliqu&eacute;e volume d&#39;&eacute;chantillon ne d&eacute;passe pas le volume de vide entre les particules de la colonne. Vide-exclu anticorps &eacute;lu&eacute; dans le tampon de l&#39;&eacute;quilibration. Cette collection apparemment en conflit de comportements est rapproch&eacute;e par une fonction d&#39;exclusion de taille variable m&eacute;di&eacute;e par les zones de faible densit&eacute; de polym&egrave;re, les propri&eacute;t&eacute;s de la charge de l&#39;esp&egrave;ce d&#39;anticorps et le pH et la conductivit&eacute; de la m&eacute;moire tampon de l&#39;&eacute;quilibration. &Eacute;changeur d&#39;anions courants de particules poreuses qui emploient des techniques de greffage pour atteindre la haute densit&eacute; de charge nulle exclusion &agrave; des degr&eacute;s divers, &agrave; l&#39;exclusion compl&egrave;te r&eacute;alisation mieux adapt&eacute;e et d&#39;autres aussi peu de la m&eacute;diation de 65 %. Perfusive et non greff&eacute;es &eacute;changeurs ax&eacute;e sur les particules v&eacute;hiculent aussi peu que l&#39;exclusion de 50 %. Monolithes ne m&eacute;dient aucune exclusion, en raison de leur manque d&#39;un volume de vide entre les particules. Sur les &eacute;changeurs qualifi&eacute;s, la technique prend en charge plus de r&eacute;duire de 99 % des prot&eacute;ines de l&#39;h&ocirc;te, l&#39;ADN et endotoxines. Virus est r&eacute;duite de plus de 99,9 %, et les agr&eacute;gats sont r&eacute;duits &agrave; moins de 0,05 %. La m&eacute;thode prend en charge le meilleur contr&ocirc;le du processus qu&#39;autre &eacute;changeuse d&#39;anions formats car excursions de pH en conjonction avec les changements dans la concentration de sel ne se produisent pas jusqu&#39;apr&egrave;s &eacute;lution de l&#39;anticorps de la colonne.

L&#39;annulation de l&#39;exclusion des anticorps par l&#39;&eacute;changeur d&#39;anions greff&eacute;s-ligand particules poreuses.

Wir beschreiben eine neue Variante des Anion-Austausch-Chromatographie in Spalten verpackt mit por&ouml;sen Partikeln, die aufgeladene niedriger Dichte Polymer-Zonen, unterst&uuml;tzt durch eine h&ouml;here Dichte Polymer Skelett zu verk&ouml;rpern. IgG trotzt die Normen der AAT und ist ausgeschlossen, das nichtig Volumen bei pH 3-10 und 0-4 M NaCl. Privatevoid Ausschluss tritt auch bei Fab, F(ab&#39;)(2) und IgM. Host Zelle Protein Kontaminanten folgen meist die &uuml;blichen Normen der AAT und Bind st&auml;rker mit zunehmenden pH-Wert und Leitf&auml;higkeit verringern. Beispiel Puffer Zusammensetzung hat keine Auswirkungen auf die Partitionierung so lange angewendet Probenvolumen interparticle nichtig Notenband der Spalte nicht &uuml;berschreitet. Leere ausgeschlossenen Antik&ouml;rper elutes in Gleichgewichtherstellung Puffer. Diese scheinbar widerspr&uuml;chliche Sammlung von Verhaltensweisen wird durch eine Variable Gr&ouml;&szlig;e Ausgrenzung Funktion vermittelt durch die niedriger Dichte Polymer-Zonen, die kostenlos-Eigenschaften der Antik&ouml;rper-Arten, und die pH und Leitf&auml;higkeit des Puffers Gleichgewichtherstellung vers&ouml;hnt. Aktuellen Generation por&ouml;se Partikel-Anion-W&auml;rmetauscher, die hohe Ladungsdichte erreichen Unterlagsreben Techniken vermitteln nichtig Ausgrenzung in unterschiedlichem Umfang, mit der am besten erreichen v&ouml;llige Ausschluss und andere so wenig wie 65 %. Perfusive und nicht veredelt Partikel-basierte W&auml;rmetauscher vermitteln weniger als 50 % ige Ausschluss. Monolithen vermitteln kein Ausschluss durch ihren Mangel an einem interparticle nichtig-Volume. Auf qualifizierte Exchangers unterst&uuml;tzt die Technik gr&ouml;&szlig;er als 99 % Reduzierung der Host Proteine, DNA und Endotoxin. Virus ist mehr als 99,9 % reduziert, und Aggregate sind auf weniger als 0,05 % reduziert. Die Methode unterst&uuml;tzt bessere Prozesskontrolle als andere AAT Formate, da pH-Wert Ausfl&uuml;ge in Verbindung mit Ver&auml;nderungen in der Salzkonzentration nicht bis erfolgen, nachdem der Antik&ouml;rper aus der Spalte eluiert wurde.

Ausschluss von Antik&ouml;rpern durch por&ouml;se Partikel veredelt-Ligand Anion Austauscher ung&uuml;ltig.

Descriviamo una nuova variante di cromatografia a scambio anionico in colonne imballate con particelle porose che incarnano zone carica polimeri a bassa densit&agrave;, supportati da uno scheletro di polimero di densit&agrave; superiore. IgG sfida le norme di scambio anionico e viene escluso il volume Sub a pH 3-10 e 0-4 M di NaCl. Sub esclusione si verifica anche con Fab, F(ab&#39;)(2) e IgM. Host cell protein contaminanti principalmente seguono le usuali norme di scambio anionico e legano pi&ugrave; fortemente con l&#39;aumentare il pH e diminuendo la conducibilit&agrave;. Composizione di Sample buffer non ha alcun impatto sul partizionamento purch&eacute; applicata volume del campione non superi il volume Sub interparticella della colonna. Vuoto-escluso anticorpo eluisce nel buffer di equilibrazione. Questa collezione apparentemente contrastante di comportamenti &egrave; riconciliata da una funzione di esclusione di dimensione variabile mediata attraverso le zone di polimeri a bassa densit&agrave;, le propriet&agrave; di carica della specie dell&#39;anticorpo e il pH e la conducibilit&agrave; del buffer equilibrazione. Scambiatori di anioni particella porosa attuale generazione che impiegano tecniche di innesti per raggiungere elevate densit&agrave; di carica mediano esclusione Sub a vari gradi, con l&#39;esclusione di completa raggiungimento pi&ugrave; adatto e altri come poco come il 65%. Scambiatori basati su particelle perfusive e non innestate mediano poco come esclusione del 50%. Monoliti non mediano esclusione, a causa della loro mancanza di un volume Sub interparticella. Su scambiatori qualificati, la tecnica supporta superiore al 99% di riduzione di host proteine, DNA ed endotossine. Virus &egrave; ridotto pi&ugrave; del 99,9%, e gli aggregati sono ridotti a meno di 0,05%. Il metodo supporta il migliore controllo del processo di scambio altri anioni formati perch&eacute; escursioni di pH in concomitanza con i cambiamenti nella concentrazione salina non si verificano fino a dopo l&#39;anticorpo &egrave; eluito dalla colonna.

Sub esclusione degli anticorpi mediante scambiatori di anioni particella porosa innestate-ligando.

&#x6211;&#x3005; &#x306F;&#x3088;&#x308A;&#x9AD8;&#x3044;&#x5BC6;&#x5EA6;&#x9AD8;&#x5206;&#x5B50;&#x9AA8;&#x683C;&#x306B;&#x3088;&#x3063;&#x3066;&#x30B5;&#x30DD;&#x30FC;&#x30C8;&#x3055;&#x308C;&#x3066;&#x3044;&#x308B;&#x8377;&#x96FB;&#x5BC6;&#x5EA6;&#x30DD;&#x30EA;&#x30DE;&#x30FC; &#x30BE;&#x30FC;&#x30F3;&#x3092;&#x5177;&#x73FE;&#x5316;&#x3059;&#x308B;&#x591A;&#x5B54;&#x8CEA;&#x7C92;&#x5B50;&#x3092;&#x8A70;&#x3081;&#x3089;&#x308C;&#x305F;&#x30B3;&#x30E9;&#x30E0;&#x306E;&#x9670;&#x30A4;&#x30AA;&#x30F3;&#x4EA4;&#x63DB;&#x30AF;&#x30ED;&#x30DE;&#x30C8;&#x30B0;&#x30E9;&#x30D5;&#x30A3;&#x30FC;&#x306E;&#x65B0;&#x3057;&#x3044;&#x4E9C;&#x7A2E;&#x306B;&#x3064;&#x3044;&#x3066;&#x8AAC;&#x660E;&#x3057;&#x307E;&#x3059;&#x3002;IgG &#x306E;&#x9670;&#x30A4;&#x30AA;&#x30F3;&#x4EA4;&#x63DB;&#x306E;&#x898F;&#x7BC4;&#x3092;&#x6311;&#x767A;&#x3057;&#x3001;pH 3 10 &#x3068; 0 4 &#x30DC;&#x30A4;&#x30C9;&#x3092;&#x9664;&#x5916; M &#x306E; NaCl&#x3002;Void &#x306E;&#x9664;&#x5916;&#x3082; igm &#x6297;&#x4F53; Fab &#x3068; F(ab&#39;)(2)&#x3001;&#x767A;&#x751F;&#x3057;&#x307E;&#x3059;&#x3002;&#x30DB;&#x30B9;&#x30C8; &#x30BB;&#x30EB;&#x306E;&#x86CB;&#x767D;&#x8CEA;&#x306E;&#x6C5A;&#x67D3;&#x7269;&#x306F;&#x3001;&#x307B;&#x3068;&#x3093;&#x3069;&#x306E;&#x9670;&#x30A4;&#x30AA;&#x30F3;&#x4EA4;&#x63DB;&#x304A;&#x3088;&#x3073; pH &#x306E;&#x5897;&#x52A0;&#x3068;&#x96FB;&#x6C17;&#x4F1D;&#x5C0E;&#x5EA6;&#x6E1B;&#x5C11;&#x3092;&#x3088;&#x308A;&#x5F37;&#x304F;&#x30D0;&#x30A4;&#x30F3;&#x30C9;&#x306E;&#x901A;&#x5E38;&#x306E;&#x898F;&#x7BC4;&#x306B;&#x5F93;&#x3063;&#x3066;&#x304F;&#x3060;&#x3055;&#x3044;&#x3002;&#x9069;&#x7528;&#x3055;&#x308C;&#x308B;&#x9650;&#x308A;&#x3001;&#x30D1;&#x30FC;&#x30C6;&#x30A3;&#x30B7;&#x30E7;&#x30F3;&#x5206;&#x5272;&#x306E;&#x5F71;&#x97FF;&#x30B5;&#x30F3;&#x30D7;&#x30EB; &#x30D0;&#x30C3;&#x30D5;&#x30A1;&#x30FC;&#x69CB;&#x6210;&#x3092;&#x6301;&#x305F;&#x306A;&#x3044;&#x30B5;&#x30F3;&#x30D7;&#x30EB; &#x30DC;&#x30EA;&#x30E5;&#x30FC;&#x30E0;&#x5217;&#x306E;&#x7C92;&#x5B50;&#x9593;&#x306E;&#x7A7A;&#x9699;&#x3092;&#x8D85;&#x3048;&#x3066;&#x3044;&#x307E;&#x305B;&#x3093;&#x3002;Void &#x9664;&#x5916;&#x6297;&#x4F53;&#x306E;&#x5E73;&#x8861;&#x30D0;&#x30C3;&#x30D5;&#x30A1;&#x30FC;&#x3067;&#x6EB6;&#x51FA;&#x3057;&#x307E;&#x3059;&#x3002;&#x3053;&#x306E;&#x52D5;&#x4F5C;&#x306E;&#x4E00;&#x898B;&#x76F8;&#x53CD;&#x3059;&#x308B;&#x30B3;&#x30EC;&#x30AF;&#x30B7;&#x30E7;&#x30F3;&#x306F;&#x4F4E;&#x5BC6;&#x5EA6;&#x306E;&#x9AD8;&#x5206;&#x5B50;&#x5316;&#x5408;&#x7269;&#x306E;&#x30BE;&#x30FC;&#x30F3;&#x3092;&#x4ECB;&#x3057;&#x3066;&#x3001;&#x6297;&#x4F53;&#x7A2E;&#x3068; pH &#x306E;&#x96FB;&#x8377;&#x7279;&#x6027;&#x3068;&#x5C0E;&#x96FB;&#x7387;&#x306E;&#x5E73;&#x8861;&#x30D0;&#x30C3;&#x30D5;&#x30A1;&#x30FC;&#x3092;&#x4ECB;&#x3057;&#x305F;&#x53EF;&#x5909;&#x30B5;&#x30A4;&#x30BA;&#x6392;&#x9664;&#x6A5F;&#x80FD;&#x306B;&#x3088;&#x3063;&#x3066;&#x548C;&#x89E3;&#x3067;&#x3059;&#x3002;&#x9AD8;&#x96FB;&#x8377;&#x5BC6;&#x5EA6;&#x3092;&#x9054;&#x6210;&#x3059;&#x308B;&#x305F;&#x3081;&#x306B;&#x79FB;&#x690D;&#x306E;&#x6280;&#x8853;&#x3092;&#x63A1;&#x7528;&#x73FE;&#x5728;&#x4E16;&#x4EE3;&#x591A;&#x5B54;&#x8CEA;&#x7C92;&#x5B50;&#x306E;&#x9670;&#x30A4;&#x30AA;&#x30F3;&#x4EA4;&#x63DB; void &#x306E;&#x9664;&#x5916;&#x307B;&#x3068;&#x3093;&#x3069;&#x3068;&#x3057;&#x3066;&#x3001;&#x6700;&#x9069;&#x306A;&#x3092;&#x9054;&#x6210;&#x3059;&#x308B;&#x5B8C;&#x5168;&#x306A;&#x6392;&#x9664;&#x3068;&#x3001;&#x7A0B;&#x5EA6;&#x306E;&#x5DEE;&#x306F; 65% &#x3092;&#x4EF2;&#x4ECB;&#x3057;&#x307E;&#x3059;&#x3002;50% &#x9664;&#x5916;&#x3068;&#x3057;&#x3066;&#x5C11;&#x3057; perfusive &#x3068;&#x975E;&#x79FB;&#x690D;&#x306E;&#x7C92;&#x5B50;&#x30D9;&#x30FC;&#x30B9;&#x306E;&#x4EA4;&#x63DB;&#x3092;&#x4EF2;&#x4ECB;&#x3057;&#x307E;&#x3059;&#x3002;&#x30E2;&#x30CE;&#x30EA;&#x30B9;&#x3092;&#x9664;&#x5916;&#x3001;&#x7C92;&#x5B50;&#x9593;&#x306E;&#x7A7A;&#x9699;&#x7387;&#x306E;&#x4E0D;&#x8DB3;&#x306F;&#x3001;&#x4EF2;&#x4ECB;&#x3059;&#x308B;&#x306A;&#x3044;&#x3067;&#x3059;&#x3002;&#x4FEE;&#x98FE;&#x306E;&#x4EA4;&#x63DB;&#x306E;&#x6280;&#x8853;&#x306F; 99% &#x524A;&#x6E1B;&#x30DB;&#x30B9;&#x30C8;&#x86CB;&#x767D;&#x8CEA;&#x3001;DNA &#x304A;&#x3088;&#x3073;&#x30A8;&#x30F3;&#x30C9;&#x30C8;&#x30AD;&#x30B7;&#x30F3;&#x306E;&#x3088;&#x308A;&#x5927;&#x304D;&#x3044;&#x3092;&#x30B5;&#x30DD;&#x30FC;&#x30C8;&#x3057;&#x307E;&#x3059;&#x3002;&#x30A6;&#x30A4;&#x30EB;&#x30B9;&#x3092; 99.9% &#x4EE5;&#x4E0A;&#x524A;&#x6E1B;&#x3001;&#x96C6;&#x8A08; 0.05% &#x672A;&#x6E80;&#x306B;&#x7E2E;&#x5C0F;&#x3055;&#x308C;&#x307E;&#x3059;&#x3002;&#x30E1;&#x30BD;&#x30C3;&#x30C9;&#x6297;&#x4F53;&#x30AB;&#x30E9;&#x30E0;&#x304B;&#x3089;&#x6EB6;&#x51FA;&#x304C;&#x5F8C;&#x5869;&#x6FC3;&#x5EA6;&#x306E;&#x5909;&#x5316;&#x3068;&#x7D44;&#x307F;&#x5408;&#x308F;&#x305B;&#x3066; pH &#x30C4;&#x30A2;&#x30FC;&#x307E;&#x3067;&#x306F;&#x767A;&#x751F;&#x3057;&#x307E;&#x305B;&#x3093;&#x306E;&#x3067;&#x4ED6;&#x306E;&#x9670;&#x30A4;&#x30AA;&#x30F3;&#x4EA4;&#x63DB;&#x5F62;&#x5F0F;&#x3088;&#x308A;&#x3082;&#x3088;&#x308A;&#x3088;&#x3044;&#x30D7;&#x30ED;&#x30BB;&#x30B9;&#x5236;&#x5FA1;&#x3092;&#x30B5;&#x30DD;&#x30FC;&#x30C8;&#x3057;&#x3066;&#x3044;&#x307E;&#x3059;&#x3002;

&#x9670;&#x30A4;&#x30AA;&#x30F3;&#x4EA4;&#x63DB;&#x6A39;&#x8102;&#x591A;&#x5B54;&#x8CEA;&#x63A5;&#x304E;&#x6728;&#x914D;&#x4F4D;&#x5B50;&#x306B;&#x3088;&#x308B;&#x6297;&#x4F53;&#x306E;&#x9664;&#x5916;&#x3092;&#x7121;&#x52B9;&#x306B;&#x3002;

&#xC6B0;&#xB9AC;&#xAC00; &#xC124;&#xBA85; &#xC5F4; &#xB2E4;&#xACF5;&#xC131; &#xC785;&#xC790; &#xCDA9;&#xC804;&#xB41C; &#xC800;&#xBC00;&#xB3C4; &#xD3F4;&#xB9AC;&#xBA38; &#xC601;&#xC5ED; &#xB192;&#xC740; &#xBC00;&#xB3C4; &#xACE0;&#xBD84;&#xC790; &#xACE8;&#xACA9;&#xC5D0; &#xC758;&#xD574; &#xC9C0;&#xC6D0; &#xB41C; &#xD3EC;&#xC7A5;&#xC5D0; &#xC74C;&#xC774;&#xC628; &#xAD50;&#xD658; &#xD06C;&#xB85C;&#xB9C8;&#xD1A0;&#xADF8;&#xB798;&#xD53C;&#xC758; &#xC0C8;&#xB85C;&#xC6B4; &#xBCC0;&#xC885;. IgG &#xC74C;&#xC774;&#xC628; &#xAD50;&#xD658;&#xC758; &#xADDC;&#xBC94;&#xC744; &#xBB34;&#xC2DC; &#xD558; &#xACE0; pH 3-10, 0-4&#xC5D0;&#xC11C; &#xBB34;&#xD6A8; &#xBCFC;&#xB968; &#xC81C;&#xC678; M NaCl. &#xD339;, F(ab&#39;)(2), &#xBC0F; IgM &#xBB34;&#xD6A8; &#xC81C;&#xC678;&#xB3C4; &#xBC1C;&#xC0DD;&#xD569;&#xB2C8;&#xB2E4;. &#xD638;&#xC2A4;&#xD2B8; &#xC138;&#xD3EC; &#xB2E8;&#xBC31;&#xC9C8; &#xC624;&#xC5FC; &#xBB3C;&#xC9C8;&#xC740; &#xC8FC;&#xB85C; &#xC74C;&#xC774;&#xC628; &#xAD50;&#xD658; &#xBC0F; &#xC804;&#xB3C4;&#xC131;&#xC744; &#xAC10;&#xC18C; &#xD558; &#xACE0; pH &#xC99D;&#xAC00; &#xD568;&#xAED8; &#xB354; &#xAC15;&#xB825; &#xD558; &#xAC8C; bind&#xC758; &#xC77C;&#xBC18;&#xC801;&#xC778; &#xADDC;&#xBC94;&#xC744; &#xB530;&#xB985;&#xB2C8;&#xB2E4;. &#xC0D8;&#xD50C; &#xBC84;&#xD37C; &#xAD6C;&#xC131; &#xBD84;&#xD560;&#xB85C; &#xC801;&#xC6A9;&#xC5D0; &#xC544;&#xBB34;&#xB7F0; &#xC601;&#xD5A5;&#xC774; &#xC0D8;&#xD50C; &#xBCFC;&#xB968; &#xC5F4;&#xC758; interparticle &#xBB34;&#xD6A8; &#xBCFC;&#xB968;&#xC744; &#xCD08;&#xACFC; &#xD558;&#xC9C0; &#xC54A;&#xC2B5;&#xB2C8;&#xB2E4;. &#xBB34;&#xD6A8; &#xC81C;&#xC678; &#xD56D; &#xCCB4; &#xD3C9;&#xD615; &#xBC84;&#xD37C;&#xC5D0;&#xC11C; elutes. &#xC774; &#xAC89;&#xBCF4;&#xAE30; &#xAC08;&#xB4F1;&#xB41C; &#xB3D9;&#xC791; &#xCEEC;&#xB809;&#xC158; &#xAC00;&#xBCC0; &#xD06C;&#xAE30; &#xC81C;&#xC678; &#xAE30;&#xB2A5; &#xD56D; &#xCCB4; &#xC885; &#xBC0F; &#xC0B0;&#xB3C4;&#xC758; &#xCDA9;&#xC804; &#xC18D;&#xC131;&#xACFC; &#xD3C9;&#xD615; &#xBC84;&#xD37C;&#xC758; &#xC804;&#xB3C4;&#xC131; &#xD3F4;&#xB9AC;&#xBA38; &#xC800;&#xBC00;&#xB3C4; &#xC601;&#xC5ED;&#xC744; &#xD1B5;&#xD574; &#xC911;&#xC7AC;&#xC5D0; &#xC758;&#xD574; &#xD654;&#xD574; &#xC774;&#xB2E4;. &#xB192;&#xC740; &#xC804; &#xD558; &#xBC00;&#xB3C4; &#xB2EC;&#xC131; &#xD558;&#xAE30; &#xC704;&#xD574; &#xC774;&#xC2DD; &#xAE30;&#xBC95;&#xC744; &#xC0AC;&#xC6A9; &#xD558;&#xB294; &#xD604;&#xC7AC; &#xC138;&#xB300; &#xB2E4;&#xACF5;&#xC131; &#xC785;&#xC790; &#xC74C;&#xC774;&#xC628; &#xAD50;&#xD658;&#xAE30; &#xC911;&#xC7AC; &#xBB34;&#xD6A8; &#xC81C;&#xC678; &#xC870;&#xAE08; &#xB113;&#xC73C;&#xBA70; &#xB2EC;&#xC131; &#xC644;&#xB8CC; &#xC81C;&#xC678; &#xBC0F; &#xB2E4;&#xB978; &#xB2E4;&#xC591; &#xD55C; &#xC218;&#xC900;&#xC73C;&#xB85C; 65%. Perfusive &#xBC0F; &#xBE44; &#xD22C;&#xC785; &#xC785;&#xC790; &#xAE30;&#xBC18; &#xAD50;&#xD658;&#xAE30; &#xC911;&#xC7AC; &#xAC70;&#xC758; 50% &#xC81C;&#xC678;. Monoliths &#xC911;&#xC7AC; interparticle &#xBB34;&#xD6A8; &#xBCFC;&#xB968;&#xC758; &#xADF8;&#xB4E4;&#xC758; &#xBD80;&#xC871;&#xC73C;&#xB85C; &#xC778;&#xD574; &#xC544;&#xBB34; &#xC81C;&#xC678;. &#xC790;&#xACA9; &#xAC16;&#xCD98;&#xB41C; &#xAD50;&#xD658;&#xAE30;&#xC5D0; &#xAE30;&#xC220;&#xC744; &#xD638;&#xC2A4;&#xD2B8; &#xB2E8;&#xBC31;&#xC9C8;, DNA, &#xC120;&#xCC98;&#xB9AC;&#xC758; 99% &#xAC10;&#xC18C; &#xBCF4;&#xB2E4; &#xD070; &#xC9C0;&#xC6D0;&#xD569;&#xB2C8;&#xB2E4;. &#xBC14;&#xC774;&#xB7EC;&#xC2A4;&#xB97C; 99.9% &#xC774;&#xC0C1; &#xAC10;&#xC18C; &#xD558; &#xACE0; &#xC9D1;&#xACC4;&#xB294; 0.05% &#xBBF8;&#xB9CC;&#xC73C;&#xB85C; &#xAC10;&#xC18C;. &#xBA54;&#xC11C;&#xB4DC;&#xB294; &#xB2E4;&#xB978; &#xC74C;&#xC774;&#xC628; &#xAD50;&#xD658; &#xD615;&#xC2DD; &#xC5F4;&#xC5D0;&#xC11C; &#xD56D; &#xCCB4;&#xAC00; eluted &#xD6C4; &#xC18C;&#xAE08; &#xB18D;&#xB3C4;&#xC758; &#xBCC0;&#xD654;&#xC5D0; &#xB530;&#xB77C; &#xD568;&#xAED8;&#xC5D0;&#xC11C; &#xC0B0;&#xB3C4; &#xC5EC;&#xD589;&#xAE4C;&#xC9C0; &#xBC1C;&#xC0DD; &#xD558;&#xC9C0; &#xC54A;&#xAE30; &#xB54C;&#xBB38;&#xC5D0; &#xBCF4;&#xB2E4; &#xB354; &#xACF5;&#xC815; &#xC81C;&#xC5B4;&#xB97C; &#xC9C0;&#xC6D0; &#xD569;&#xB2C8;&#xB2E4;.

&#xB2E4;&#xACF5;&#xC131; &#xC785;&#xC790; &#xD22C;&#xC785; &#xB9AC;&#xAC04;&#xB4DC; &#xC74C;&#xC774;&#xC628; &#xAD50;&#xD658;&#xAE30;&#xC5D0; &#xC758;&#xD574; &#xD56D; &#xCCB4;&#xC758; &#xBB34;&#xD6A8; &#xC81C;&#xC678;.

Describimos una nueva variante de la cromatograf&iacute;a de intercambio ani&oacute;nico en columnas repleto de part&iacute;culas porosas que representan zonas de pol&iacute;meros de baja densidad cargada apoyadas por un esqueleto de pol&iacute;mero de densidad m&aacute;s alto. IgG desaf&iacute;a las normas de intercambio de aniones y queda excluida en el volumen vac&iacute;o a pH 3-10 y 0-4 M de NaCl. Exclusi&oacute;n de vac&iacute;o tambi&eacute;n se produce con Fab, F(ab&#39;)(2) e IgM. Anfitri&oacute;n celular prote&iacute;nas contaminantes sobre todo seguir las normas habituales de intercambio de aniones y atar m&aacute;s fuertemente con el aumento de pH y disminuyendo la conductividad. Composici&oacute;n del tamp&oacute;n de muestra no tiene ning&uacute;n impacto particionar mientras aplica el volumen de muestra no exceda el volumen vac&iacute;o entre part&iacute;culas de la columna. Anticuerpo excluidos vac&iacute;o elutes en buffer de equilibrado. Esta colecci&oacute;n aparentemente contradictorio de comportamientos es reconciliada por una funci&oacute;n de exclusi&oacute;n de tama&ntilde;o variable mediada a trav&eacute;s de las zonas de baja densidad del pol&iacute;mero, las propiedades de la carga de la especie de anticuerpos y el pH y conductividad del b&uacute;fer equilibrado. Intercambiadores de aniones part&iacute;culas porosas de la generaci&oacute;n actual que emplean t&eacute;cnicas de injertos para alcanzar la alta densidad de la carga median exclusi&oacute;n vac&iacute;o en diferentes grados, con la exclusi&oacute;n completa de lograr m&aacute;s adecuada y otros tan poco como el 65%. Intercambiadores basadas en part&iacute;culas perfusive y no-injertado median tan poco como la exclusi&oacute;n del 50%. Monolitos no median ninguna exclusi&oacute;n, debido a su falta de un volumen vac&iacute;o entre part&iacute;culas. En los intercambiadores calificados, la t&eacute;cnica admite mayor que 99% de reducci&oacute;n de anfitri&oacute;n prote&iacute;nas, ADN y endotoxinas. Virus se reduce m&aacute;s del 99,9%, y agregados se reducen a menos de 0.05%. El m&eacute;todo admite mejor control del proceso de otro intercambio de aniones formatos porque excursiones pH junto con cambios en la concentraci&oacute;n de sal no se producir&aacute; hasta despu&eacute;s de que el anticuerpo se eluyen de la columna.

Anular la exclusi&oacute;n de anticuerpos por intercambiadores de aniones part&iacute;culas porosas injertado-ligando.

We observed that IgM monoclonal antibodies and aggregates in mammalian cell culture supernatants were associated nonspecifically with nucleosomes, DNA, and histone proteins derived from nuclei of host cells that died during antibody production. A series of multimodal sample treatments were evaluated for their ability to selectively remove these contaminants without significant antibody loss. The first consisted of adding 2,5-dioxo-4-imidazolidinyl urea (allantoin) and the DNA intercalating agent 7-ethoxyacridine-3,9-diamine (ethacridine), then flowing the supernatant through a column of mixed porous particles bearing metal affinity, anion exchange, and cation exchange functionalities. A one-step variant of the method was to mix chromatography particles with the allantoin-ethacridine-treated supernatant. An alternative one-step treatment consisted of passing untreated cell supernatant through a chelating monolith in tandem with an anion exchange monolith. All methods eliminated high molecular weight aggregates, and reduced smaller aggregates to 2-4%. They also achieved 98% DNA reduction, 99% reduction of nucleosomes and histones, 30-70% reduction of general host proteins, and 98% IgM recovery. Size exclusion chromatography analysis indicated that IgG monoclonal antibodies benefit similarly from treatment. Subsequent IgM purification reduced DNA levels beneath the level of detectability by fluorescent dye intercalation, histones to less than 10 parts per million by ELISA, and aggregates to less than 0.05% by size exclusion chromatography. The results point to chromatin catabolites as promoters of antibody aggregate formation.

Characterization and removal of aggregates formed by nonspecific interaction of IgM monoclonal antibodies with chromatin catabolites during cell culture production.

Chromatin released from dead host cells during in vitro production of IgG monoclonal antibodies exists mostly in complex hetero-aggregates consisting of nucleosomal arrays (DNA+histone proteins), non-histone proteins, and aberrant forms of IgG. They bind immobilized protein A more aggressively than IgG, through their nucleosomal histone components, and hinder access of IgG to Fc-specific binding sites, thereby reducing dynamic binding capacity. The majority of host cell contaminants in eluted IgG are leachates from chromatin hetero-aggregates that remain bound to protein A. Formation of turbidity in eluted IgG during pH titration is caused by neutral-pH insolubility of chromatin hetero-aggregates. NaOH is required at 500 mM to remove accumulated chromatin. A chromatin-directed clarification method removed 99% of histones, 90% of non-histone proteins, achieved a 6 log reduction of DNA, 4 log reduction of lipid-enveloped virus, and 5 log reduction of non-enveloped retrovirus, while conserving 98% of the native IgG. This suspended most of performance compromises imposed on protein A. IgG binding capacity increased ~20%. Host protein contamination was reduced about 100-fold compared to protein A loaded with harvest clarified by centrifugation and microfiltration. Aggregates were reduced to less than 0.05%. Turbidity of eluted IgG upon pH neutralization was nearly eliminated. Column cleaning was facilitated by minimizing the accumulation of chromatin.

Nonspecific interactions of chromatin with immunoglobulin G and protein A, and their impact on purification performance.

Chromatin heteroaggregates comprising nucleosomal arrays, DNA, histone and non-histone host cell proteins contributed 28% of the host contaminant mass in an IgG cell culture harvest. A subset comprising soluble particles of 50-400nm was retained through its histone component by an electronegative multimodal chromatography adsorbent. Accumulated heteroaggregates hindered pore access and depressed IgG capacity by 69% compared with capacity determined with purified IgG. Heteroaggregate retention persisted in 2M NaCl but some associations within them were dissociated. This manifested during IgG elution as contaminant leaching from the retained heteroaggregate mass, which contributed more than 95% of the contaminants in the IgG. Retained heteroaggregates also interfered with pore egress of eluted IgG, increasing peak width ∼3-fold. IgG recovery was 80%. Remaining heteroaggregates were removed with 1M NaOH. Advance reduction of heteroaggregates with a hybrid fatty acid-solid phase clarification method reduced DNA more than 3 logs, histones beneath the level of detectability, and non-histone proteins by 90% while conserving 90% of the IgG. Non-lipid-enveloped virus was reduced by 5 logs and lipid-enveloped virus by 9 logs. IgG capacity on the multimodal adsorbent was 94mg/mL, compared with 95mg/mL when loaded with protein A-purified IgG. Non-histone host protein reduction increased to 98%. IgG eluted in a sharp concentrated peak and step recovery was >95%. Residual fatty acids did not bind the adsorbent and were mostly eliminated during sample loading. A single polishing step with an electropositive multimodal adsorbent reduced non-histone host proteins to <100ppm, DNA to <10ppb, and aggregates to <0.05%. IgG recovery across the entire process was >80%. These results qualify electronegative multimodal adsorbents as an IgG capture option, but more importantly reveal chromatin heteroaggregate content as a keystone process variable at all stages of purification process development.

Chromatin-mediated depression of fractionation performance on electronegative multimodal chromatography media, its prevention, and ramifications for purification of immunoglobulin G.

Practical effects of advance chromatin removal on performance of protein A affinity chromatography were evaluated using a caprylic acid-allantoin-based extraction method. Lacking this treatment, the practice of increasing loading residence time to increase capacity was shown to increase host protein contamination of the eluted IgG. Advance chromatin extraction suspended that compromise. Protein A ligand leakage from columns loaded with chromatin-extracted harvest was half the level observed on protein A columns loaded with non-extracted harvest. Columns loaded with chromatin-extracted harvest were cleaned more effectively by 50-100mM NaOH than columns loaded with non-extracted harvest that were cleaned with 250-500mM NaOH. Two protein A media with IgG capacities in excess of 50g/L were loaded with chromatin-extracted harvest, washed with 2.0M NaCl before elution, and the eluted IgG fraction titrated to pH 5.5 before microfiltration. Host protein contamination in the filtrate was reduced to <1ppm, DNA to <1ppb, protein A leakage to 0.5ppm, and aggregates to 1.0%. Caprylic acid and allantoin were both reduced below 5ppm. Step recovery of IgG was 99.4%. Addition of a single polishing step reduced residual protein A beneath the level of detection and aggregates to <0.1%. Overall process recovery including chromatin extraction was 90%.

Hui Theng Gan

Neural Stem Cell Laboratory,
Institute of Medical Biology,
Neural Stem Cell Laboratory, Institute of Medical Biology

Agency for Science, Technology and Research (A*STAR)

Enumeration of Neural Stem Cells Using Clonal Assays

Hui Theng Gan

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Neural Stem Cell Laboratory,Institute of Medical Biology,Neural Stem Cell Laboratory, Institute of Medical Biology

Agency for Science, Technology and Research (A*STAR)

Enumeration of Neural Stem Cells Using Clonal Assays

Neural Stem Cell Laboratory,
Institute of Medical Biology,
Neural Stem Cell Laboratory, Institute of Medical Biology