The Copyright Office has published a report on copyright and artifical intelligen (AI) which can be found at chrome-https://www.copyright.gov/ai/Copyright-and-Artificial-Intelligence-Part-1-Digital-Replicas-Report.pdf.

The Report uses the term “digital replica” to refer to a video, image, or audio recording that has been digitally created or manipulated to realistically but fasely depict and individual. 

The Report notes many deficiencies with respect to existing laws as they apply to digital replicas. For example, digital replicas that are produced by ingesting copies of preexisting copyright works, or by altering them, such as superimposing someone’s face onto an audiovisual work or simulating their voice singing they lyrics of a musical work, may implicate copryight exclusive rights, including the rights to reproduce a work and to prepare derivative works. Copyright does not, however, protect an individual’s identiy in itself, even when incorproated into a work of authorship. 

Under the Lanham Act, claims such as false endorsement involving a digital replica are limited to unauthorized commercial uses, and most federal courts also require a showing of consumer awareness of the depicted individual in order to establish a likelihood of confusion, limiting the Lanham Act’s protection to well-known figures and commercial circumstances. It may be difficult for many individuals, including less famous artists and performers, to prove that the challnged conduct is likely to confuse consumers regarding the plaintiff’s association with, or approval of, the defendant’s commerical activiteis. And issues like AI-gnerated “revenge porn” would likely fall beyond its reach. 

The report reccomends a new federal law which covers all individuals, at least for the individual’s lifetime that targets digital replicas, whether generated by AI or otherwise, taht are so realistic taht they are difficult to distinguish from authentic depictions. Liability should arise from teh distribution or making available of an unauthorized digital replica, but not the act of creation alone. It should nott be limtied to commercial uses,a s the harms caused are often personal in nature. It should require actual knowledge both tthat hte representation was a digital replica of a particular indivdual and that it was unauthorized. The statute should include a safe harbor mechanism that incentivizes online servie providers to remove unauthorized digital replicas after receiving effective notice or otherwise obtaining knowledge that they are unauthorized. Individaul should be albe to licesne and monetize their digital replica rights, subject to gaurdrails, but not to assign them outright. Effective remedies should be provided, both injunctive releif and monetary damages as well as statutory damages and/or prevailing party attorney’s fees. 

Introduction; Definitions:

The storage of electrical energy has become a key technology today. There is an urgent need for better storage options for renewable energies (water, wind, and solar) in order to be able to further reduce CO2 emissions and thus secure the environment. Batteries offer higher storage capacities adn are more suitalbe for long term storage of large amounts of energy. Supercapcitors can be charged and dischared very quickly and deliver very high power densities. These supercapacitors servers for example as storage buffers in wind turbines, and for rapid recuperation of braking energy in electric vehicles. 

In most batteries electrical energy is stored chemically and released again in a controlled manner when needed. A classic embodiment consists of a cathode and an anode, each in an electroyte solution, separated by a separation membrane. (Meerholz, US 2022/0190339)

Annode: is the electrode at which oxidation occurs. (Oxidation involves the loss of an electron by a molecule, atom or ion. (ie., loss of H or gain or O). Oxidation is sometimes referred to as gaining a positive charge. ) Electrons flow away from the annode and the conventional current towards it. 

Cathode: At the cathode, reduction takes place with the electrons arriving from the wire connected to the cathode and are absorbed by the oxidizing agent. (Reduction involves the uptake of an electron by a molecule, atom or ion (i.e., gain of H or loss of O).)

Lithium-ion Batteres and Supercapacitors:

Lithium-ion technologies are currently the sector in the energy stroage field with the alrgest investments in the world. Compared to conventional batteries, they are much more powerful and eficient. Their main applicactiones includes light and starter batteries for vehicles, consumer electronics (e.g., cell phones, PCs, tablets) as well as in the industrial sector. In the automative industry, the use of lithium-ion technology is steadily increasing. (Meerholz, US 2022/0190339)

The cost of raw materiasl accounts for 40-70% of the cost of manufacturing Li-ion batteries with cathod csots about 27%. Cathode materiasl include LiCoO2, LiMn2O4, LiMPO$. The main price drivers for these etched cathode materials are the raw material prices for cobalt and nickel. (Meerholz, US 2022/0190339)

Organic Redox-Active polymers (ORP) based Electrode materials:

ORP baed electrode materials consist mainly of carbon, nitrogen and oxgyen (Cn, N, O) from reseource saving raw amterails. Other limtied raw materails are not needed, and ORPs casue fewer problems in recovery, disposal and recycling. It is currently not foreseable that metal-free (polyer) electrodes reach the energy desnsity of Li-ion batteries since the molar mass of a redox unit in the established Li-ion batteries cathod materials (e..g, LicoO2=98 g/ml) is lower than in typical redox systems of active organic compounds (e.g., tripheny amine-245 g/ml). Nevertheless, due to ther advantageous properties, organic metal-free electrodes could in the future offer interesting environmentally friendly alternative. Starting from that technical background, polytriphenylamine compounds promise a vareity of advantages. The cathode consists of a highly conductive poly-p-phenylene backbone which ihas a high rate stroage and a high electronic conductive potential. (Meerholz, US 2022/0190339)

Non-conjugated polymer backbones:

(Meerholz, US 2022/0190339) discloses a non-conjugated system within the organic compound to guarantee independent charging and discharging. The approach is a non-conjugated polymer backbone, to which the redox-units are attached in a way that they are not electorically coupled to each other. In such a system, the redox protential of each uit will be marginally, if at all, influenced by the charging state of neighbouring redox-units. Non-conjugated triphenylamine is combined with a porous matrix system. The metal-free polymeric electrode material can be adjsuted in its porosity by a special processing method, whereby the pore size and thus the contact surface to the electrolyte solution is adapted. In a first aspect, an electrode includes an organic compound prepared by polymerization of a triarylamine haivng at least one reactive polymerizable group, characterized in that in the organic compound at least a part of the aryl moieites of the triaryl amine are non-conjugatedly connected to each other. This means that the aryl moeities of the organic compound are connected to each other over a backbone which is not electronically conjugated which means that the aryl moeiteis of the organic ompound are connected to each other over a backbone whcih does not include conjugated double bonds. This structure of the organic compound in which the aryl moieties are decoupled from each other allows to realize a more constant voltage curve over the entire duration of the discharing and charging of the electrode. In anther aspect, the invention is characterized in that the organic compound has at least a bimodal pore size distribution. Based on the at least modal pore size distribution, channels and/or pores are available in the resulting electrode material in which the electrolyte solution (solvent and salt can penetrate). The large surface of the elctrode allows faster charge exchagne and thus faster charging and discharing. The polymerized triaryl amine compound used in the electrode is usually applied on the electrode substrate with a binder. Anything that binds the conductive carbon component and the redox-active component together and imrproves adhesion to the electrode substrate/current collector is used as the binder. Examples include polyvinylidene fluoride. The organic compound used in the electrode can be provided in geometrical form of partciels, platelets or fibers. A polymeric network and not jsut a polymeric chain is preferred. The density of the network is determined by the degree of cross-linking, i.e., by the number of cross-linkable groups. The electrode may additionally include at least one additive which is preferably selected from the group consisting of non-redox active materials such as polystyrene, PVK, polyrethane. The triaryl amine having at elast one reactive polymerizable group and being polymerized allows a so-called balanced ion movement. Due to the addition of a salt (in case I) preferably a polymeric salt, to the triaryl amine having at least one reactive polymerizable group and being polymerized, or due to the covalent attachment of ionic groups (case II) to the triaryl amine having at least one reactive polymerizable group and polymerized, the charge balance during charging or discharging by the transport of both ion types (positive and negative) at the same time (balanced ion movement, BIM) can be achieved more easily. There occurs an ion movement in both directions (into and out of the electrode material) during each charge and discharge process of a batttery in which the electrode is used. During charging, a cathod material is oxidized. For charge equalization anions migrate form the electrolyte (sovlent plus salt) into the cathode material and cations of the polyelectrolyte added to the redox-active material from the composition material into the electrolyte. The migration of (solvated) ions into and out of the cathode material cahnges its volume during charging and discharging. Simultatneou migration of ions into the cathode and migration of ions out of the cathode occurs. As a result, the volume change of the cathode is reduced and thus it is reduced or avoided that hte electrode is subjected to mechanical stress and cracking of the material, loss of adhesion to the current colector or spalling of parts of the redox materail. By optimizing mixing ratio or depending on the tyep of ion, a volume change may be reduced or completely avoided. In case I (the addition of a salt-polymeric anion), the cathod material is admixed with a sale consisting of a polyanion and LMW cations. An example couple be the lithium or soidum sale of polystyrene sulfonic acid. Curing charing, the cathode material is oxidized. For cahrge equalization anions migrate from the electrolyte (solvent plus salt) into the material and cations of the polyelectrolyte added tot he redox-active material migrate from the composition material into the electrolyte. In case II (coavlent attachmetn of annionic groups), the cathod material is chemically modified (covalent attachmetn) and thus beomces a salt itself, consisting of a directly attached anion and LMW cations. The anionic groups covaelntly bonded to the redox system coule be sulfonate, phosphate, acetate groups of the like. Suitable cationic counterions are for example Na+ or Li2+. As in Case 1, the cathod material is oxidized. For charge equalizaiton, anions migrate form the electrolyte (solvent plus salt) into the material and cations of the caovelntly bonded ionic groups into the cationic redox system migrate from the materail into the electrolye. 

Cell Culutring, Harvesting & Preservation:

Kinoshita (US 2023/0380417) discloses a storage method for HCECs or their precursor cells by culturing the cells in a medium that contains a ROCK inhibitor and in which the content of epidermal growth factor (EGF) is less than a concentraiton that will cause a transformation are harvested at a timing when any one or plurality of conditions (a)-(d) have been met and then placing the cells in a suspension state and then preserved. (a) morphology: immediately after the HCECs has shifted from a spindle fibroblast-like shape having irregular elongated projections to a tessellated shape whose major axis-minor axis ration incluidng the projections is close to 1, until immediately before boundaries between tehe cells become indistinct. (b) during a period when the expression of CD44 becomes equalto or less than half a maximum value observed aft the most recent subculturing until this expression level reaches a plateau. (c) cell density: when the cell density of the HCECs is not less than 900 cells/mm2 and not more than 2500 cells/mm2 and (d): when the number of culturing days since the most recent subculturing is not less than 4 days and not more than 14 days. The inventors discovered that if the cells are harvested immediately after a timing when the destiny of the cell population to be differentated as funcitonal human corneal endotheial cells had been decided which was also a timing when the proliferation viability was still high and then placing theis populaiton in a suspension state, then it was possible to preserve functional HCECs while maintaining a hihg survival rate. 

Macrophages are specialist cells that have developed an innate capacity to recognize subtle differences in the structure of cell surface epressed identification tages, so called molecular patters. They arrive later to a site of infection as compared to neutrophils. Unlike neutrophils (PMNs), macrophages are also longer lived and they are present in tissues as well as in blood. Macrophages patrol the body’s tissues, searching for signs of infection. When they detect a foreign protein, they set off the inflammatory response. In particular, they engulf and destroy the invader bearing that protein and secrete a suite of cytokines, some of which raise an alarm that recruits other cells to the site of infection and puts the immune system on alert. 

What Do Macrophages Do?

Anti-Bacteria Activity: Macrophages are in a resting state but can be activated by bacterial products like LPS as well as cytokines secreted by activated . like IFN-y which is secreted by TH1 cells. Activated macrophages have increased phagocytic and microbicidal activity (such as oxygen dependent mechanisms that catalyzes the reduction of oxygen to superoxide anion (O2-) which generates other oxidizing agents harmful to bacterial like hydrogen peroxide).  A group of antimicrobial and cytotoxic peptides known as defensins are also present in activated macrophages.

Macrophages play important role in host defense and inflammation. First, macrophages can act as effector cells in killing phagocytosed and extracellular microbes and tumor cells by producing reactive oxygen intermediates, nitric oxide (NO), and lysosomal enzymes.

Mqs endocytose particles or soluble glycoconjugates taht are bound by the mannose receptor, a C-type lectin with broad carbohydrate specificty, Mqs also have a receptor for LPS.

Cytokines secreted by Macrophages: Production of cytokines by macrophages is considered one of the important immunoregulatory function sof macrophages. Macrophages regulate the immune response of T cells through producing important cytokines such as IL-12 and IL-10. These two cytokines can enhance Th1 or Th2 cell differentiation, respectively, result in enhancing or suppressing of cell-mediated immunity. Macrophages are also progenitors of inflammatory cytokines, such as TNF-alpha, IL-1 and Il-6.

Tissue macrophages respond to the perception of microorganisms with phagocytosis of the pathogens and the production of cytokines. Among these TNFa, IL-12 p70 and IL18 induce the secretion of IFNy from NK cells. IFNy then converts macrophages harboring pathogens to a a microbicidal state, in which bactericidal agents such as NO and reactive oxygen intermediates are porduced to kill the bacteria.

Phagocytosed antigen is digested within the endocytic processing pathway into peptides that associate with class II MHC molecules which then move to the macrophage membrane. Activation of macrophages induces increased expression of both  class II MHC molecules and the co-stimulatory B7 family of membrane molecules which allows macrophages to function more effectively as antigen presenting cells. Macrophages also secrete a number of proteins central to development of immune responses such as interleukin 1 which acts on  and provides a costimulatory signal required for activation following antigen recognition. Thus, macrophages and  facilitate each other’s activation during the immune response.

The macrophage membrane has receptors for certain classes of antibody and thus will bind better to antigen which is complexed with antibody. Thus antibody functions as an opsonin, a molecule that binds to both antigen and macrophage and enhances phagocytosis. Opsonization is the process by which particular antigens are rendered more susceptible to phagocytosis. 

Macophages express substantial levels of the F4/880 antigen, and for the most part express little or no NHC class II.

Specialized Types of Macrophages in the Body:

Kupffer cells (KCs): reside within the lumen of the liver sinusoids and form the largest population of macropahges in the body. Although KCs have markers in common with other tissue resident macrophages, they perform specialized functions geared towards efficient clearance of gut dervied bacterial, microbial deris, bacterial endotoxins, immune complexes and dead cells present in portal vein blood draining from the microvascular system of the digestive tract. Efficient binding of pathogens to the KC surface is a crucial step in the first line immune defense against pathogens. A central role for KCs in the rapid clearance of pathogens from the circulation is illustrated by the significanlty increased mortality in mice depleted of KCs.

Companies: BioLife Solutions

For thawing cells, conventional practice is to warm the cells quickly in a warm water bath (e.g., 37 C) to just about the point to which the last bit of ice is about to melt and then to dilute the cells slowly into growth media. If the sample is allowed to get to warm the cells may start to metabolize, and be poisoned by the DMSO (dimethyl sulfoxide) that is used in the feezing prcoess. (Schryver, US Patent Application 15,602,711 published as US 2017/0257908 and US 10,917,941, which is a continuation of US Application 14.712,120 published as US 10,555,374)

Thawing Devices and Methods:

Pluristem LTD (CA 2883826) discloses a system for heating a biological material which includes a processor configured to receive an input associated with a target temperature and transmit a signal to controllably move a heating deice relative to the base for a time period, wherein the time period is detemriend based on the target emperature and content volumne. A method for thawing the frozen biological material includes the steps of controllably moving the heating device for a specific time period, wherein the time period is determiend based on the target emperature, the vial content material and the content volumne. Initially, a thawing cycle may active only after heating device has reached an initial temperature. This may reduce the affect of room and/or device temperature on the thawing procedure.

Thawing Protocols for Specific types of Cells: 

Thawing Melanoma cells:

Preparing Media (Dulbeo’s Modification Eagles Medium “DMEM”  ): 

(1) spray under hood with ETOH

(2) Add 50 ml of FBS to our bottel of DMEM. (We have a 500 ml volume and need to use 10% FBS so we will use 50 ml FBS.)

(3) Add 5ml of antibiotic solution (P/S/A “penicillin, streptomycin “antimicotic ) and 5 ml of L-glutamine (since we want a 1:100 dilution)

(4) invert the jugs a couple of times and store at 4c

(5) label “DMEM 10 + P/S/A JL date”

Thawing:

(1) obtain large conical tube (yellow cap) and transfer 30 ml of the media above. Use sterile conditions under the hood.

(2) get cells out from liquid N2 (ex. rack 10, Box 6, B-16; keep rack right on top of machine)

(3) thaw cells quickly in water bath and transfer with 2 ml pipette just as soon as cells have thawed.

(4) mix tube by inverting and then use 25 ml pipette to add the cells in the tube to sterile flask which has been labelled (ex. “B1678H1 F:2/27/94 T: 6/23/03 JLR”)

(5) observe cells under microscope in flask to access survivability  (“alive cells will adhere to bottom of flask and become dendritic. floating cells will be dead by tommorroo)

(6) grow these cells at 37.5 with falsk in horizontal position

US Patent Pub # 20040109851; Use of immature dendritic cells to selence antigen specific cd8cell function 

Immature DCs: Another way to generate suppressor T cells in vitro involves the stimulation of naive T cells with iDCs. Repetive stimulation of naive cord-blood T cells with allogeneic iDCs has reportedly generated a population of poorly growing T cells that primarily produce IL-10. Although these cells produced IL-10, their suppressor phenotype resembled that of CD25+ T cells, as it was contact dependent, antigen non-specific and APC independent. Furthermore, suppression could be overcome partially by the addition of IL-2. These cells differ from  in that IL-10 is not required for their generation because iDCs do not produce IL-10. The precursors of these suppressor cells in cord blood do not express CD25 so it is unlikley that they are derived form a CD25+ T cell population that has not fully differentiated.

Immature DCs are the ideal population to prime regulatory T cells as they are deficient in co-stimulatory moleucles, and priming with antigen-iDC complees might even be able to downregulated preexisting antigen specific immune responses.

Induction of Antigen specific CD8T-IL-10 producing cells: Injection of iDCs into human subjects reportedly results in inhibition of MP-specific CD8+ T cell effector function in freshly isolated T cells. Before immunization, MP-specific IFNy producing T cells were detectable in both human subjects as expected because most adults have been exposed to the influenza virus. However, after DC immunization, there was a decline in MP specific IFNy producing cells. Moreover, this decline was associated with IL10 but not IL4 producing T cells. These postimmunization IL-10 producers were CD8+CD4- cells.

Reversal of Anergic state with Antigen-processing mature DCs: 

CD25+CD4+ T cells can be expanded both in vitro and in vivo with antigen-loaded BM DCs from Balb/c mice.

CD4+CD25+ T cells proliferation vigorously to stimulation with anti-CD3 in the presence of mature bone marrow-derived DC (BMDC).

A number of studies have shown that CD4+CD25+ T cells from TCR transgenic mice will proliferate following transfer in vivo when stimulated with their cognate Ag presented by DC.

CD25-mediated suppression of T cell proliferation is also abrogated when BMDC are used as APC.

Treg anergy is dependent on TLR activaiton of BMDCs and involves the potentiation of Treg responsiveness to IL-2 by cooperative effects of IL-6 and IL-1, both of which are produced by TLR activated mature DCs.

Reversal of suppressive function with Mature DCs:

The suppressive function of Tregs is readily reversed by the maturation of DCs induced by GM-CSF and does not require TLR activation of either DCs or Tregs. Thus BMDCs derived by short-term culture (day 5-6) were permissive for suppression by CD25+CD4+ T regs, whereas BMDCs derived by long term culture (day 10-11) were not. Activation of day 10-11 BMDCs by a TLR ligand (LPS), however, does further enhance T cell proliferation.

Using splenic DCs, Il-6 was required to block the suppressive activity of Tregs. However, IL-6 was not required when using BMDCs.

Therapies Using iDCs

One possible therapy application relates to the use of iDCs to generate antigen specific regulatory CD8+T cells in vitro, which may then be used for adoptive immunotherapy in vivo. In this system, T cells are co-cultured with immature DCs in vitro at a DC to T cell ratio of about 1:10-100 and the resulting T cells are then injected for the purpose of suppressing an active immune responde.

Model of Tolerance

In one proposed model, the maintenance of self tolerance is dependent on the ratio of T4 cells to potentially pathogenic autoreactive T cells that respond to a given periopheral antigen. In a given lymph node this ratio fluctuates depending on the infectious status of the local tissue. In the steady-state, immature DCs may traffic through peripherotic debris arising form normal cell turnover in the tissue without becoming activated. Evein in the absence of inflammation, a few of these iDCs will migrate to the draining lymph nodes where they will present a panel of self peptides to both TR cells and Tpath cells. However, autoimmunity will not occur, perhaps because the autoreactive T cells are insufficiently activated by iDCs or, alternatively, because the iDC preferentially stimulate TR cells. Consistent with the latter hypothesis, TR cells can respond to much lower concnetrations of cognate peptide ligands than conventional naive CD4+T cells and iDCs express relatively low amounts of MHC class II and costimulatory molecules. Check effects of EGCG treated DCs In contrast, the presence of an infectious agent will induce DC activation and migration so that high numbers of mature DCs will arrive in the lymph node and present peptides derived from the pathogen and from self antigen. This stimulus may be potent enough to transiently override TR cell activity, permitting an expansion of anti-pathogen T cells but also allowing expansion of T path cells. This transient loss of TR cell activity will also be associated with proliferation of the TR cells themselves, analygous to the in vitro hyperstimulation of CD4+CD25+TR cells with anti-CD3 + IL-2 of anti-CD28, which results in proliferation of the TR ceels accompanied by a transient loss of suppressive activity.

About the Author: 

James Rogers is a patent attorney and former patent examiner with the United States Patent and Trademark Office. Previous to his work as a patent examiner, James was in house counsel for a diagnostic company and worked for several law firms in the intellectual property area. James received his law degree from Suffolk University Law School. He received an MS degree in biology with an emphasis on DNA recombinant technology from New York University and a PhD in molecular medicine from the University of South Florida College of Medicine with an emphasis on immunology. He is a member of the New York and Massachusetts bars.

Ypatent has additional information and links which supplements The Complete Patent Kit, Fourth Edition.

Companies:

Biotechne Simple Western (after preparing reagents they are loaded into a Simple Western plate which is placed with a cpaillary cartridhe into a Simple Western instrument. The Simple Western does protein analysis thorugh separation and immunoprobing in a capillary system.) 

Capillary electrophoresis-sodium dodecyl sulfate (CE-SDS): (see also capillary electrophoresis under Diagnostic Techniques)

Capillary electrophoresis-sodium dodecyl sulfate (CD-SDS) has emerged as a modern equilvalent of SDS-PAGE, offering superior reproductibility, sensitivity and throughput. (Wang WO 2019/152303)

Capillary electrophoresis-sodium dodecyl sulfate (CE-SDS) is a modern equivalent of SDS-PAGE discussed below and offers superior reproducibility, sensitivty and throughput. During CD-SDS analysis of mAb products, minor peaks with shorter migraton times (LMW forms) than the intact antibody can be routinely observed. Unliked SDS-PAGE analysis, these LMW impurites cannot be extracted or subjected to further analyses. As a result, the idenitites of LMW impurites obersrved in CE-SDS methods are often proposed solely based on empirical knowledge. (Wang WL 2019/152303)

CE-SDS has emerged as a valuable alternative to conventional sodium dodecyl sulfate polycarylamide gele electrophoresis (SDS-PAGE) for the characterizaiton of automatic quantitation of biomolecules and particularly monoclonal antibodies. High separation efficiency, ease of operation and automation and reduced operating costs are the main reasons for this success. As a result, CE-SDS has found wide acceptance within the biopharmaceutical industry for the development, manufacturing and release of therapeutic mAbs. CE-SDS methods are in routine use for in process monitoring, purity assessment, detecing low level impurites and measuring size variant hterogeneity. (Schneider, “Tracking of antibody reduction fragment by capillary gel electrophoresis during the coupling to microparticles surface” J. Pharma. and Biomed. Analysis 53 (2010). 

CE-SDS is widely used for purity analysis of mAb therapeutics for release and stability to demonstrate product consistency and shelf life during the manurfacturing and life cycle of the product. The method separates molecule specific fragments and other impurities based on their electrophoretic mobility under denaturing conditions. Recently, US Pharmacopeia (USP) suggested that a universal mAb system suitability reference standard be adopted for CE-SDS and size exclusion chromatography. During sample prepration, antibodies are denatured in the presence of an ionic detergent which masks the native charge of the protein providing a net negative charge enabling electrophoretic mobility speration based almost exclusively on the hydrodynamic radius of the protein molecule(s). The disulfide bonds which are responsible for the tetrameric structure of mAbs are disrupted (reducing) or protected (non-reducing) during heat denaturation by reducing or alkylating reagents respectively. (Esterman “implementation of USP antibody standard for system suitability in capillary electrophoresis sodium dodecyl sulfate (CE-SDS) for rlease and stability methods” J. Pharmaceutical and Biomedical Analysis 128 (2016))

CE-SDS was used for seperation of denatured prtoein size variants under non-reduced or reduced conditions For non-reduced CE-SDS (nrCE-SDS) samples were denatured with sodium dodecyl suflate at 70C for 5 min in the present of iodoacetamide. for reduced CE-SDC (rCE-SDS), 2-mercaptoethanol (Bio-Rad) was added to the protein denaturation step to reduce the disulfide bonds. After denaturation, both non-reduced and reduced samples were injected int a bar fused silica capilary (Beckman Coulter) and separated based on hydrodynamic size resulting form an applied electric field in which migraiton of smaller sized proteins is inversiy related to overal size. (Katterle “How sable are new biologicals” Pharm, 80(11) 1557-1563 (2018). 

For Activatable Antibodies:

Carman (US 2019/0117789) discloses activatable antibodies that bind CD166. The activatable antibody (AA) includes an antibody that binds to CD166 including a heavy chain and light change, a masking moeity (MM) coupled to the Ab wherein the MM inhibits the binding of the AB to the CD166 when the AA is in an uncleaved state and a cleavable moeity (CM) coupled to the Ab, wehrein the CM is a substrate that functions for a protease. In some embdoiments an agent such as maytansinoid is conjugated to the AA. The anti-CD166 conjugated activatable antibody was activated with matriptase or MMP14 for 2 hours at 37C and mixed with intact conjugated activatable antibody. The mixture was then analysed by the The WES Capillary electrophoresis system using anti-human IgG (American Qualex Catalog #A110UK). Carman showed the ability to separate matriptase-activated or MMP14 activated conjugated activatable antibodies form intact conjugated activatable antibodies. 

Desnoyers (US Patent Application No: 16/632265) discloses a method of quantitating a level of activation of an activatable antibody which includes the steps of 1) loading at least one capillary with a stacking matrix and a separation matrix. the “stacking matrix” refers to a highly porous (relative to the separation matrix) material that functions to concentrate proteins present in the sample and “stack” them at the interface with the separation matrix so that the proteins start migration under electrophoresis conditions form the same physical starting point. Suitable stacking matrices include those used to prepare stacking gels for Western blotting such as acrylamide, SDS. Capillaries pre-loaded with stacking matrix and separation matrix are available commercially as Wes Separation Module. Electrophoresis causes the compounds in the sample to migrate through the separation gel at differential rates according to molecular size/weight. 2) the HMW and LMW compounds are immobilzied within each capilalry as by using UV light. 3) Each capilarry is immunoprobed with a first reagent such as an idiotypic antibody that is specific for the activatable antibody. 3) Detection of the first reagent can be accomplised for example with a second reagent that binds to the first reagent. Exemplary detectably labeled second r agents include HRP-conjugate anti-mouse secondary antibody. 

Vasiljeva (US Patent Application 2020/0377602) discloses polypeptides that include at least a first cleavable moiety (CM1) that is a substrate for at oast one matrix metalloprotease (MMP) and at leaset a second clevable moeity (CM2) that is a substrate for at least one serine protease to activatable antibodies which include these polypeptides. Vasiljeva disclosing measuring cleavage by capillary electrophoresis and analyzation with the Wes Western Blot protocol using goat anti-human IgG antibodies and anti-goat secondary anitobodies. The fraction of the cleaved activatable antibody was determined by quantifying the mobility of the higher mobility polypeptide corresponding to the cleaved antivatable antibody. 

Capillary electrophoresis-mass spectrometry (CE-MS):

CE-MS is a pwerful orthogonal technique capable of filling in gaps int he dientification, quantitation and isomeric resoluion of many small hydrophilic and charged metabolites. The metabolome is a large complex mixture of molecuels for which not one technique can optimally identify and measure it in it’s entirety. LC-MS, GC-MS and NMR have been widely used for metabolomics for teh past 20 years for a wide range of applications. CE-MS captures a unique metabolic chemical space beyond these standard methods pvoiding another window into metabolomics profiling. CE-MS is the most suited technique for analyzing posphorylated metabolites, amino acids or metabolites form teh TCA cycle and glycolytic pathways. A fused silica capillary contains surface charges of sianol groups present on the inner walls. The silanol groups on the capillary inner wall are ionized presenting an overall negative surface. Opposing ions in the electrolyte solution are attracted to the inner wall surface to achieve a balance of electric charges, resulting in the formation of a double layer with ionized silanol groups. Under these conditions, a potential diffference is created very close to the inner wall. The applicaiton of a voltage to both ends of the capillary attracts the positively charged ions of the diffuse double alyer to an anode. In contrast, the silanol groups cannot move due to the fixation on the wall surface and the entire electrolyte solution in the capillary is directed toward the anode with the migration of the positively charged ions, thereby generating a flow. The degree of mobility of any copound relative to tothers is due to variation in their ionic radius and size and charge of the electrolyte filing the capillary. A compound or metabilite with a larger ionic radius and smaller charge would ahve limited mobility compared to small, more polar species. Compounds with a small ionic radius and higher charge would have high obility. Hence controlling the electrical gradient across the capillary and pH of teh electrolyte solution are two of the most important parameters in controlling metabolite separation into the mass spectrometer. (Buko “Capillary electrophoresis mass spectrometry based metabolomics” J Applied Bioanalysis, 2017). 

Imaged capillary isoelectric focusing (iCIEF):

Charge profiels/isoelectric points (pI) of analytes can be determeind by imaged capillary isoelectric focusing (icIEF) analysis using an iCE3 system (ProteinSimple) equipped with a 5 cmx100 uM ID flurocarbon coated capillary Sampels are spearated according to theri pI and detected by using a whole column UV adsorption detector (280 nm) that avoids distrubing focused protein zones. Imaged capillary isoelectric focusing fo tthe analysis of therapeutic antibodies and antibody drug conjugates provides a fast separation and resolution of acidic, neutral and basis variants of an antibody. Katterle “How sable are new biologicals” Pharm, 80(11) 1557-1563 (2018).

Companies which assist in clinical trial development: Amarex

Center for Biologics Evaluation and Research (CBER)  (the center within the FDA which regulates biological products for human use under applicable federal laws).

Medicine and Healthcare Products Regulatory Agency (MHRA) (drug regulation in UK)

Chemical vs. Biological Products:

RNA:

While mRNA vaccines are considered as biological products, the classificaiton of chemical or biological products and the definition of gene therapy medicinal products may require updating or alignment to accommodate advancements in mRNA technology. Currently, plasmid DNA serves as the starting material for manufacturing mRNA va in vitro transcription method. Essentially, this involves using the plasmid. With advancements in cell free production technology, there may be a future where mRNA will be entirey synthetically produced. This raises the challenge of defining what constitutes a biological product.

Currently smaller RNAs such as siRNAs are typically considered chemical products due to their purely syntehtic nature. Barker, “Evolution of mRNA-based vaccines: a regulator’s view” Vaccine Insights 2024; 3(3), 97-105)

However, as mRNA becomes more syntehtically produced, utilizing cell free systems without biological components like plasmid, questions arise about whether they should still be classified as biological products. According to regulatory definitions, biological products typically involve substances produced or extracted form a biological source, necessitating specific testing and product controls. While mRNA has some similarities to shorter oligonucleotides like siRNAs, its great complexity and size warrant advanced characterization tools to ensure structural integrity, morphology and interactions with drug delivery systems. These are then more similar to biological products than to a well defined chemical product. Given that mRNA vacines fall under the category of immunogloical medicinal products, it may be appropriate for them to be classified as biological products. However, a mRNA production transitions away from plasmids toward cell free systems, updates to the definition may be necessary to accurately reflect the nature of these products. (Barker, “Evolution of mRNA-based vaccines: a regulator’s view” Vaccine Insights 2024; 3(3), 97-105)

Upstream manufacturing process of mRNA is maturing. cGMP quality plasmid, polymerases, and enzymes needed for in vitro synthesis of mRNA are available but can be costly. Poly(A) tails can be created by inclusion in the teplate or by use of enzyme. Capping options include high-efficiency, co-synthesis reagetns such as TriLink Biotechnology’s CleanCap and enzyme treatment with high efficiency. (Cytiva, “mRNA vaccines and therapeutics: current trends and perspectives” Fe 17, 2025.)

Accelerated Approval:

Accelerated Approval.   (FDA may approve drugs for serious or life-threatening diseases or conditions where there is an unmet medical need and the drug is shown to have an effect on a surrogate endpoint that is reasonably likely to predict a clinical benefit to patients (improving how patients feel or function, or whether they survive longer).

In 2012, Congrewss passed the Food and Drug Adminsitration Safety Innovations ACT (FDASIA). Section 901 of FDASIA amends the Federal Food, Drug, and Cosmetic Act to allow the FDA to base accelerated approval for drugs for serious conditions that fill an unmet medical need on whether the drug has an effect on a surrogate or an intermediate clinical endpoint. A surrogate endpoint used for accelerated approval is a marker -a laboratroy measurement, radiographic image, physical sign or other measure that is thought to predict clincial benefit, but is not itself a measure of clinical benefit. Likewise, an intermediate clinical endpoint is a measure of a therpaeutic effect that is considered reasonably likely to predict the clinical benefit of a drug, such as an effect on irreversible morbidity and mortality (IMM).

The FDA bases its decision on whether to accept the propsoed surrogate or intermedate clincial endpoint on the scientific support for that enpoint. Studies that demonstrate a drug’s effect on a surrogate or intermediate clincial endpoint msut be adequate and well control as required by the FD&*C Act.

Using surrogate or intermediate clincial endpoints can save valuable time in the drug approval process. Instead of haivng to wait to elarn if a drug actually extends survival for cnacer pateints, for exaple, the FDA may approve a drug based on evidence that the drug shrinks tumors, becasue tumor shrinkage is considered reasonably likely to predict a real clinical benefit.

Fast Track:

Fast track is a process designed to facilitate the development, and expedite the review of drugs to treat serious coditions and fill an unmet medical need. Determining wehther a condition is serious generally is based on wehther the drug will ahve an impact on such factors as survival, day-to-day functioning, or the likelihood that the codition, if left untreated, will progress form a less severe condition to a more serious one. AIDS, Alheimer’s, heart failrue and cancer are obvious examples of serious conditios.

Filling an unmet medical need is defined as providing a therapy where none exists or providing a therpay which may be potentially better than available therapy.

Fast track designation must be requrested by the drug company. A request for the designation may be made at the same time, or any time after, the applicaton submission for the investigation (study) of the drug under section 505(i) or section 351(a)(3) of the Public Health Service Act. FDA will review the request and make a deciion with 60 days based on whether the drug fills an unmet medical need to a serious condition. Once a drug recives Fast Track designation, early and frequent communication between the fDA and a drug company is encouraged throughout the entire drug development adn erview process.

Laws: 

FDA Development & Approval Process.

Priority Review.

Orphan Products Development

CFR Title 21:

21 CFR 211.65 (equipment construction)

21 CFR 600.11 (Physical establishment, equipment, animals and care)21 CFR 600.11 (Physical establishment, equipment, animals and care)

ICH Q7: Good Manufacturing Practice for Active Pharmaceutical Ingredients, US Fed. Reg. 66(186) 2000: 49-28-49029

FDA:  FDA blog

European Union good manufacturing practice

BIOSECURE act (proposed; US House of Representatives bill H.R. 7085; US Senate bill S. 3558) would restrict the US activity of 5 Chinese biotech companies on national security grounds. The act is expected to drive mroe business to CDMOs outside China).

Definitions:

ANDA: In order to being manufacturing a generic product, the manufacturer must submit an Abbreviated New Drug Application (ANDA) to the Orange Book before the patent on the branded product expires, certifying that the generic dose not infringe on the current patent. However, when submitting this ANDA, the eneric manufacturer must also certify for each unexpired patent in the ORange Book. Depending on the number of patents associated with a single product, this can create a substantial hurdle for generic entry, as it requires the gneeric manufacturer to go to the epxense of assessing each patent to determine infringement potential. (Campanelli, “Feeling evergreen: a case study of humira’s patent extension strategeis and retroactive assessment of seocnd-line patent validity”, 2022).

Biosimilars: Biosimilars cannot be approved by the FDA until the reference biologic has been on the market for at least 12 years.. This is different than in the small molecule drug space, where generics can enter a market after 5 years. As a result, biosimilars can only enter the market towards the end of the lifecycle of the reference biologic drug, which limits their overall potential earnings. (Campanelli, “Feeling evergreen: a case study of humira’s patent extension strategeis and retroactive assessment of seocnd-line patent validity”, 2022).

Section 351(K) of the Public Health Service Act (PHS) provides an abreviated licensure pathway for biological products shown to be biosimilar to, or interchangeable with, an FDA licensed reference product. As part of a demonstration of biosimilarity, a 351(k) applicaiton generally will contain data derived form a clinical study or studies sufficient to demonstrate safety, purity and potency in one or more appropriate conditions of use for which the reference product is licensed and for which the biosimilar or interchangeable biosimilar product applicant is seeking licensure. 351 (K)(2)(A)(i)(I)(cc) of the PHS Act. (Labeling for biosimilar and Interchangeable Biosimilar Products Guidance for Industry” September 2023).

FDA recommends that biosimilar and interchangeable biosimilar product labeling incorporate relevant data and information form the reference product labeling, with appropriate modificaitons.

Evergreening (“patent thickening”): a legal strategy employed by any company with a proprietary patent by which they intend to extend the life of the original patent either through taking out new patents or by buying out a frustrating competitor. “Patent thicketing” invovles many patents being conferred at the same time, oten on overlapping aspects of the innovation. Each component of the drug is protected separately, for example the cotaing, the delivery system, the formulation, and the composition would all be protected under separate patetns. Onre reprot revealed that hte top 12 products on the market are protected by an average of 71 patents per drug. (Campanelli, “Feeling evergreen: a case study of humira’s patent extension strategeis and retroactive assessment of seocnd-line patent validity”, 2022).

Orange Book: The FDA maintains a list of approved drug products with therapeutic equivalence evlautions, known as the “Orange Book”. This keeps tract of all branded drugs with generic coutnerparts, as well as the use codes or cases for the branded products.

Orange Book Preface

The Orange Book

US Orphan Drug Act:

The US Orphan Drug Act was passed in 1983 to encourage research and development of therapies for rare diseases thorugh tax inventives, market exclusivity, and user fee exemptions. The act results in a marked increase in rare diease research, from fewer than 10 drugs before its passage to more than 300 drugs for rare dieases over the past 25 years.

Biosimilar Markets and Regulation

The pipeline of follow-on (biosimilar) products in developmetn includes nearly 800 biosimilars. See Biosimilarspipeline

BPCI Act:  

Biosimilars are defined as “similar” or highly similar” to the reference medicinal products following the EMA (European Medicines Agency ) adn the US FDA regulatory guildielines. In 2012, the EMA issued guidelines on the development of biosimilar monoclonal antibodies. In 2012 the FDA released draft guidance for teh development and regulatory review of biosimilars. In India, the biosimilars are termed as “similar biologics” in accordance with the guidelines issued by the Central Drugs Standard Control Organization (CDSCO).  (Bandyopadhyay “physicochemical and functional characterization of a biosimilar adalimumab ZRC-3197, Biosimilars, 2015: 5, pp. 1-18).

The BPCA Act was enacted to establish an abbreviated pathway for FDA licensure of biological products that are demonstrated to be biosimilar to or interchangeable with an FDA licensed reference product.

Guidance for Industry: Clinical Pharmacology Data to Support a Demonstration of Biosimilarity to a Reference Product, May 2014

Quality Considerations in Demonstrating Biosimilarity of a Therapeutic Protein Product to a Reference Product: Guidance for Industry

Contract Testing Organization (CTOs) and Contract Research Organization (CROs)

CTOS are specialized laboratories offering epxertise with characterization and preclinical testing. Examples include Charles River Laboratoreis and SGS. Others include BioAgilytix, Creative Proteomics, Eurofins Scientific, Intertek, Myoderm and SSCI.

CROs focus on managing clinical trials. Quintiles IMS and Parexel are examples. Others include Altasciences, IDDI, INC Research, inVentiv Health and Pharmaceutical Product Devleopment (PPD).

Good Manufacturing practice (CGMP):

World Health Organization (WHO)

21 CFR 211.65 states that equipement shall be constructed so that surfaces that contact components, in process materials, or drug products shall not be reactive, additive, or adsorptive so as to alter the safety, identity, strengh, quality, or purity of the drug product beyond the official or otehr established requriements.

US FDA CGMP for biologicals, 21 CFR 600.11 provies that all surfaces that come in contact with products shall be clean and free of surface solids, leacable contaminants, and other materials that will hasten the deterioration of the product or otherwise rend it less suitable for the intended use.

ICH Q7 Good Manufacturing Practice for Active Pharmacetuical Ingredients guidelines tates that equipment hsould be constructed so that surfaces that contact raw materials, intermediates, or APIs do not alter the quality of the itnermediates and APIs beyond official or other established specifications.

FDA Process:

Orange Book

A drug compound must pass three phases of huamn clinical trials in order to obtain FDA approval.

IND:

A threshold step is to gain the FDA’s permission to test the coumound in humans in the first place. After a drug developer has conducted preclinical studes (i.e., tested the compound in vitro and in animals, it submits an Investigational New Drug (IND) application to the FDA. An IND submission includes an investigator’s brochere, which disclsoes informaiton such as animal safety and preclinical efficacy data, clinical trail proposlas, and toxicology data. If the FDA approves the IND, then Phase I studies can commence.

Phase I:

Phase I studies involve administering the compound to a small group of healthy volunteers or advanced cancer patients with a vareity of tumor types. Phase I studies are conducted primarily to evaluate safety, to determine a safe dosgin range, and to idnetify any side effects.

Phase II:

Clinical trails do not focus on efficacy until Phase II, which typically involved adminsitering the compound to a specific patient population. The goals of a Phase II study include evaluating efficacy in specific patient populaiotns, determining dose tolerance and optimal dosage and identifying possible adverse effects and safety risks.

Phase III:

Phase III studies are large scale and are undertaken to evaluate clinical efficacy and safety in an expanded patient population. After completing Phase III studies, a developer submits a New Drug Applicaiton to the FDA for approval.

Simulated Moving Bed (SMB) chromatography:

Introduction:

A specific way of operating continous chromatography is called simulated moving bed (SMB) chromatography. In SMB all the chromatography columns are periodically and simultaneously moved in the direction opposite the sample flow. The movement of the columns is realized by appropriate redirections of inlet and outlet stream to/from the columns which requires a sophisticaed setup. Rose (WO 2017/140081)

The simulated moving bed (SMB) technology as been patetned about 40 years ago. it is a multi-column continous chromatographic binary separator involving a coutner-current between the liquid (eluent) and the separating medium (stationary pahse) packed in the columns. (Nicoud “Recent aspects in simulated moving bed” Aanlusis Magazien, 1998, 26, N 7). 

For large scale separations in actual productions continuous processes are much more economic than batch processes. The advantages of a continous process is for example high yield, less solvent consumption, less costly fractionation and analyses, better flexibility for the auntities to be purified. One way to realize a continuous chromatographic process is the so called simulated moving bed process (SMB). (Aumann, WO 2006/116886). 

In continous chromatography (SMB chromatography), several identical columns are connected in an arrangement that allows columns to be operated in series and/or in parallel, depending on the method requirements. Thus, all columns can be run in principle simultaneously, but slightly shifted in method steps. Compared to “conventional” chromatography, where a single chromatography cycle is based on consecutive steps of loading, washing, elution and regeneration, in continous chromatography based on multiple identical columns all these steps occur simulataentously but on different columns each. Continous chromatography operation results in a better utilization of chromatography resin, reduced prcoessing time and reduced buffer requriements. Rose (WO 2017/140081)

Aumann (US2011/0042310; US 2009/0050567; US8,496,836; US7,837,881) teaches a process for continuous purification of a mixture using columns that are grouped into at least four sections. After a switch time, the last column from the first section is moved to the first position of the second section, the last column of the second section is moved to the first position of the third seciton, the last column of the third section is moved to the first position of the fourth section and the last column of the fourth seciton is moved to become the first column of the first section. The column can be run in at least one batch mode step in which the outlet of one column is used to collect the desired intermediate product as well as in continous mode where the outlet of at least one column is fluidly connected with the inlet of at least one other column.

Bryntesson (WO2008/153472A1 and US2010/0176058) discloses a SMB method where a feed compriing a target compound is passed across a 1st adsorbent, the outflow passes to a 2nd adsorbent, the feed is redicted to the 2nd adsorbent, a wash liquid is passed across the 1st adorbent, this wash liquid outlfow is directed to a 3rd adsorbent and the second adsorbent outflow is directed to the 3rd adsorbent, the 1st adsorbent is regenerated, the feed is redicted to the 3rd adsorbent, wash liquid is passed across the 2nd adorbent, directed the wash liquid outflow to the 1st adsorbent, subsequently directly the outflow from the 3rd adsorbent to the 1st adsorbent, regenerating the 2nd adsorbent, redicteing the feed to the 1st adsorbent, washing the 3rd adsorbent and directly its outlfow to the 2nd adsorbent, directing the otuflow from the `st adsorbent to the 2nd adsorbent, regenerating the 3rd adsorbent and repeating all the steps.

Jeon (US 13/643146, published as US. 2013/0046080) discloses a chromatographic method for purifying an antibody where a sample is loaded on a 1st column, loading the outflow to a 2nd column, before washing the 1st column, stopping the loading of the sample and loading the sample on the 2nd column, loading the outflow from the 2nd column onto a third column and before washing of the 2nd column, stopping the loading of the sample on the 2nd column and loading the sample on the 3rd column and repeating the steps from the 1st to 3rd and 3rd to 1st columns.

Muller-Spath (US2014/0299547) teaches reducing the number of column to twofor the isolation of a product like an antibody comprising a first batch step, where said column are disconnected and a first column is loaded with feed and its outlet directed to waste and from a second column desired product is recovered and subsequently the second column is regnerated, a first interconnected step where the outlet of the first column is connect to the inlet of the second column, the first column is laoded beyond its DBC with feed and the outlet of the second column is directed to waste, a second batch step analogus to the first batch step but with exchanged column and a second interconnected step analogous to the first interconnected step but with exchanged column.

Thommes (US2004/0241878) teaches separating an antibody using a simulated moving bed (SMB) system where at certain points liquid streams may be introduced and at other points effluent streams may be withdrawn. Thommes (US 2007/0215534 and US Patent No: 8608960) also discloses a SMB system which incorporates a plurality of zones  such as an association zone, a wash zone, an elution zone. Suitable solid phases may include Protein A or G but can also include CEX. Use of continous chromatography in a continous counter-current mode where the columns are loaded to equilibrum capacity requires smaller column valumes compared to batch chromatgoraphy wehre the column are loaded to there DBC and requires using columns of 2-3 times the volume. In one embodiment, the feed is applied to 2 columns in parallel and makes a second pass through the 2 parallel columns. A carousel or similar apparatus may be used to move the columns relative to stationary valves and inlet and outlet streams. The columns moving to the left from the adsorption zone enter the adsorption wash zone. The effluent of the adsorption wash zone is fed back into the adsorption zone to minimize product loss. The adsorption was zone consists of 2 columns in series thus reducing the amount of buffer reuqired by about 40%. The product is leuted in an elution zone consisting of 2 columns in series. The eltuion zone is followed by an elution wash zone. 

Commercial SMB columns:

SMB systems have been designed by Novaept (Pompey, France), Tarpon (Worcester, Ma), Semba (Madison, Wi), Contichrom (Knauer, Germany) and GE Healthcare. (Gdawat “Periodic counter-current chromatogrpahy- design and oeprational considerations for integrated and continous purificaiotn of proteins” Biotechology Journal7, 1496-1508, 2012)

Cature SMB:

In captureSMB process, two identical columns are used to recover the target compound form the feed mixture. After the columns have been equilibrated, they are interconnected by connecting the outlet of the first column to the inlet of the second column. For a certain time, the feed solution is loaded onto the first column, The fact that there is a second column behind the first one allows for longer feeding compared to the batch case, because the eventual breakthrough is cought by the second column instead of being lost. After this time, the first column enters the recovery and regeneration step, which is equivalent to the batch process. During this step, the now diconnected second column is further loaded with feed. As soon as the first column is re-equilibrated and ready to be loaded again, the columns swap roles and ocntinue with the next interconnected step.  (Baur, Daniel, “Design, modeling and optimization of multi-column chromatographic processes” Doctoral Thesis, 2017). 

Periodic Counter-Current Chromatography systems (PCCS or MPCC):

In multi-colunn periodic counter-current chromatography (MPCC) the breakthrough protein form the first column is captured by the second column. The first column stops laoding when a set protein breakthrough percentage is reached and the loading switches tothe second column. The first column is then eluted and regenerated and multiple columns are alternated to acheive continuous chromatogrpahic separation, thereby improving productivity, resin capacity utilization and reducing buffer consumption and equipemnt size. Thus for a three column MPCC, columns 1 and 2 are connected in series for laoding and column 3 is eluted and subjected to recovery and regenreation R-R). When column 3 is completely eluted and regeneration, column 1 also reaches the set protein brekathrough percetnage. Columns 1 and 3 a re connected and unadsorbed protein in column 1 is washed to column 3. At the same time, feed is swtiched to column 2. After column 1 is cwashed, it is disconencted form column 3 and column 2 is connected to column 3 for continous loading, while column 1 is subjected to R-R. When column 1 is completely eluted and regeenrated column 2 also reaches the set protein breakthrough percetnage . Column 2 and 1 are connected in series and the unadsorbed rptoein in column 2 is washed to column 1. At the same time, the feed is switched to column 3. After column 2 is completely washed, it is disconnected form column 1 and column 3 is connected to column 1 for continous laoded bhile column 2 is subjected to the R-R. When column 2 is completely eluted and regenreated, column 3 also reaches the set protein brekathrough percetnage. Column 3 and 2 are connected in series and the unadsorbed rptoein in column 3 is washed to column 2. At the same time, the feed is switched to column 1. Lin (US Patent Application No: 18/117,479, published as US 20230203092)

In PCC, multiple columns are used to perform the same steps in parallel such that continuous feed is acheived and all other process steps are discrete in time. For PCC, a cycle is defined when each one of the columns has completed equilibraiton, load, wash, elution and regeneration steps in sequence. To proceed from one step to another, an automated column switching algorithm is employed. There are essentailly two decision points required for every column cycle –one at the start of protein brekathrough and the other at column saturation. These switch points in PCC are detemriend by a UV based control strategy developed by GE Healthcare that is based on a difference between the feed and column outlet V. In principle, column switching can be determined by an process analytical technology tool capable of in-line measurement of product concentration. (Gdawat “Periodic counter-current chromatogrpahy- design and oeprational considerations for integrated and continous purificaiotn of proteins” Biotechology Journal7, 1496-1508, 2012)

Konstantinov (US 2014/0255994, issued as US 9,650,412) discloses an integrated continous biomanufacturing process for producing a therapeutic protein such as an antibody which includes feeding a liguid culture medium into a first multi-column chromatogrpahy system (MCCS1) to capture the protein and then continously feeding the eluate which contains the protein into a second multi-column chromatgraphy ystem (MCCS2) to purify and polish the protein. The process can include the use of two or more mutli-column chromatography systems (MCCSs). A MCCS can include two or more chromatogrpahy columns or membranes or a combination of columns and membranes. The first and/or second MCCS can be a periodic counter current chromatogrpahy system (PCCS) which includes towo or more columns that are switched in order to allow for teh continous elution of the protein from the two or mroe columns. In PCCSs, multipe columns are used to run the same steps discretely and continously in a cyclic fasion. Since the columns are operated in series, the flow through and wash form one column is capture by another column. This unique feature allows for loading of the resin close to its static binding capacity instead of the DBC and is typical during batch mode chromatography. Once all the steps in the cycle are competed, the cycle is re-started. The system can include column switching based on teh UV absorbance difference betweenthe feed inlet and column outlet. During column laoding, the PCCC control system dtermiend the impurity baseline when the absorbance stabilizees. As the recombinant therapeutic prtoein breaks through, there is an increase in the outlet UV signal above the impurity baseline. At the point when deltaUV ahs reached a rpe-detemriend threshold (such as 3% breakthrough of the reocmbinatn therapetuic protein), the flow-through from column 1 is directed onto column 2 ineasted of to the wate. When column 1 is nearly saturated with recobmiannt threptuic protein adn the delta UV has reached a pre-detemriend vlue, the feed is switched to column 2. An important adantage of this deltaUV based column switching streegy is that it allows for uniform loading of the columns irrespective of the feed recobminant therapetuic prdcut concetnraiton and the oclumn capacity. In PCCCS, the resdience time (RT) of the protein on the column can be decreased without increasing the column size becasue the breakthrough form the frist column in the system can be captured on the second column in the system. 

3-column periodic counter-current chromatography (PCC) (3-C PCC):

The 3-column PCC process uses three identical columns. The first part of a switch consists of an interconnected wash step where the first, fully loaded column is washed and the wash is recylced into the third column, while the second column is loaded in a disconnected mode. Since the feed is applied to a disconnected column, this phase is denoted as athe batch pahse, with the duration tB and the feed flow rate BB. When the interconnected wash step is finished, the first and the third column are disconnected and the first column continues in the recvoery and regeneration procedure, with further wash steps, the elution, the CIP and the equilibraiton step. In the meantime, the two other columns are loaded in an interconnected manner using a flow rate QIc; therefore this part of the switch is called the interconnected phase, which last for the time tIC. After the interconnected phase is complete, all columns are moved one position upstream relative to the liquid phase flow, which denotes the beginning of a new switch. When three swithes have passed and the columns are back in the initial configuation, one cycle has passed. (Baur, Daniel, “Design, modeling and optimization of multi-column chromatographic processes” Doctoral Thesis, 2017). 

Gadgil (US 16/340,822, published as US 2019/0263855) discloses a multi-column chromatography system such as a periodic counter current chromatography system (PCC) which includes carboxypeptidase B immobilized on sepharose.  The C-terminal lysine residues on H chain can be truncated by passing a harvest recovered form perfusion cell culture on a column which has CPB on sepharose. The CPB preferentially acts upon the basic amino acids, such as arginine and lysine and thus the resin can be used for removal of charged isoforms belowing to any class of antibodies. In one embodiment, a continuous process for reducing heterogeneity of an antibody includes a CPB-Sepharose column connect to a Protein A column. The flow through from the CPB-Sepharose column is directly loaded ontto the protein A column for capture step. The third and fourth columns can be selected from AEX, CEX, HIC and MM chromatography. In one embodiment, the multicolumns can be run either in series or in parallel. In one embodimentm the prcoess was carried out on AKTA pcc (three column periodic counter curent chromatography, 3C PCC) GE Healthcare, where one CPB-CNBR activated Sepharose 4B column (column 1) and two prtoein A columns (column 2) are connected parallely. The residence time maintined on column 1 is equivalent to 1.7 min and column 2 was about 4.4 min (as resin used is MabSelect SuRE LX). 

4-column periodic counter-current chromatography (PCC) (4-C PCC):

The 4-column PCC process uses four identical columns that are loaded and eluted sequentially.  IIn the first part of the switch, the column in the first position, which has been washed in the previous switch, is eluted. Meanwhile, the second column undergoes the first part of the wash step, during which product from the liquid phase of the first column is directed to the cleaned and regenerated column in the last position. During this time, the column in the third position is disconnceted and loaded with feed. When the intercconencted part of the wash step is completed, the second column is disconnected and the recovery and regeneration continues. Product elution from the first column finishes during this phase, and CIP and regeneration follow. Meanwile, the other two column are interconnected and feed is applied to the third column. After this, one switch has passed and the columns are moved one position upstream. The 4-column PCC prcoess differs form the other processes in that there is more than one column in the recvoery and regeneration phase at any one time. If there is no constraint that the switching times in all positions must be the same, the recvoery and regeneraiton can be scheduled mroe efficiently in the 4-column PCC process. (Baur, Daniel, “Design, modeling and optimization of multi-column chromatographic processes” Doctoral Thesis, 2017). 

Gdawat (“Periodic counter-current chromatogrpahy- design and oeprational considerations for integrated and continous purificaiotn of proteins” Biotechology Journal7, 1496-1508, 2012) discloses a 3 column PC cycle. At the begining of a cycle, the feed is laoded onto column 1 and the flow through goes to waste. (step 1) When the product start to breakthrough, the flow-through from column 1 is directed to column 2 to capture the unbound product. (step 2) Once column 1 is fully loaded, the feed is directly loaded onto column 2, while column 1 is washed onto column 3, eluted regenerated and requilibrated for the next cycle. (steps 3-4) Column 2 is now subjected to steps 3-6, which are identical to steps 1-4 for column 1. Finally, the same steps occur on column 3. 

–Commercial examples:

Gdawat (“Periodic counter-current chromatogrpahy- design and oeprational considerations for integrated and continous purificaiotn of proteins” Biotechology Journal7, 1496-1508, 2012) discloses a 4 column PCC system which is a custom modified AKTA system (GE Healthcare). The system was equipped with five UV minotors, three pmumps, multiple vales and a pH and conductivity meter and was pwoered by a Unicorn based custom control strategy. MabSelect SuRe, iminodiacetate adn hydrophobic interaction chromatgoraphy HIC) media were packed into three of four 1.6 cm x 6 cm, 16.6 cm x 10 cm, or 0.66 cm x 6 cm columns. 

Purification of Target Proteins using Continous Multi-column chromatography  each with the same ligand for each of the Target proteins

Bataille (US Patent application 15/125483, published as US 2017/0073396) disclsoes purification of three separate plasma proteins, immunoglobulin, albumin and fibrinogen, where muticolumn affinity chromatography comprising several chromatogrpahy columns having the same chromatography support are linked in series, the chromatography in series being used to purify the threee separate proteins. For example, a first mutlicolum affinity chromatography step where each of the columns includes an affinity ligand that binds to immunoglobulins is first conducted, the flow through fraction is then subjected to a second muticuloumn IEX or mixed mode or HIC multicolumn step where the fraction containing albumin is collected and the flow through is usbjected to a third multicolumn affinity chromatography series having an affinity ligand that binds to fibrinogen, at the conclusion of which the fration containing fibrinogen is collected. Each of the multicolumn chormatrpahy steps can for example be 3 columns in series and performed such that once loading and washing of the first column is completed, the first column is separated from the other columns and undergoes elution while the second column becomes the first column in the series and once the equilibration is completed, the first column is replaced in series in last position and the bycle of steps is repeated. 

Optimization of PCC:

Protein adsorption to a chromatogrpahy resin is dependent on the bidning conditions, size, biochemical proeprties of the target protein, the resin bead morphology (bead size, pore structure and phase ratio) and the functional ligand properties (type and density). Collectively, these factors influence the thermodynamic and mass transfer kinetics of protein-resin interations. The thermodynamics govern the equilibrium binding properties, such as static binding capacity and the binding isotherm, while mass transfer kinetics govern the rate of protein adsorption onto the resin. The quantitative aspect of mass trasnfer kinetics in protein binding is demonstrated by the shape and nature of the protein breakthrough curve. Protein breakthrough form a column occurs before all the protein accessible binding sites are unitized on the resin due to mass transfer resistance. Since columns are operated at a finite flow rate with a finite protein residence time, only a poriton of the static binding capacity can be utilzied. This capacity is the dynamic binding capacity at a particular residence time. Chromatographers have traditionally deisgned batch processes with relatively long target protein residence times to increase resin capacity utilization. However, this approach also resutls in low throughput and subsequent oversizing of the chromatogrpahic column to aceive the desired target throughput. In contrast to batch chromatogrpahy, target protein breakthrough is laoded onto a second column in PCC until the first column is almost fully saturated. This leads to full capacity utilization irrespective o most cmmonly used residence times. This can also be acheived in batch mode in convective systems (perfusion media with large proes and membrane chromatography) at short residence times wehre the mass transfer effects are negligible.  (Gdawat “Periodic counter-current chromatogrpahy- design and oeprational considerations for integrated and continous purificaiotn of proteins” Biotechology Journal7, 1496-1508, 2012)

(Gdawat “Periodic counter-current chromatogrpahy- design and oeprational considerations for integrated and continous purificaiotn of proteins” Biotechology Journal7, 1496-1508, 2012) isclsoes a mathematical framework for designing PCC for a given protein capture resin system. The scheduling required to perform continous captrue of a target protein using PCC is governed by the target prtoein breakthrough curve. For a given protien-capture resin system, the scheduling parameters can be detemriend from a mathematical transformation of the breakthrough curve. The optimal PCC operation can be defined using one of many potential bjective functions, including maximization of resin capacity, utlization, minimum recovery loss during was, resin lifetime usage or minimization of residence time. 

Countercurrent Tangential Chromatography:

Countercurrent trangential chromatography overcomes many of the limitations of conventional column chromatography by having the resin (in the form of a slurrry) flow through a series of static mixers and hollow fiber membrane modules. The microporous hollow fiber membranes retain the large resin particles while letting all disssolved species pass through the membrane and into the permeate. Thus, during the binding operation, the protein of interest stays bound to the resin while the impurites flow through the membrane and are removed as waste. The buffers used in the binding, washing, and elution steps flow countercurrent to the resin, enabling high resolution separation while reducing the amount of buffer needed for protein purification. For example, a concentrated slurry is pumped from slurry tank 1 into a first statis mixer (SM1) where it is mixed with recycled permeate form a second hollow fiber mrembrane module (HF2). The diluted slurry then passes into the frist hollow fiber module (HF1) where sufficient permeate is removed to return the slurry to its origianl concentration. This provides true countercurrent contacting, the slurry moves form stage 1 to stage 2 while the buffer (permeate) starts in stage 2 and moves to stage 1. For binding operations the concentrated slurry is then mixed with the protein mixture in the second statis mixer (SM2), pumped into HF2 and collected as a reconcentraed slurry in slurry tank 2. Permeate from the first module is sent to the permeate collection tank. For washing and elution, the concentrated slurry is mixed with an appropriate buffer in the second static mixer. (Zydner “Countercurrent tangential chromatography for large-scale protein purificaiton” Biotechnology and Bioengineering, 108(3), 2011), disclosing using a Macroprep 25Q strong anion exchange resin consisting of a methacryalte based modified with a quaternary amine functionality). 

The CCTX process utilizes the resin in the form of a slurry which flows thorugh a series of static mixers and hollow fiber membrane modules. The micro-porous hollow fiber membranes retain the large resin partciles while letting all dissolved species, including proteins and buffer components pass through the membrane an into the permeate. The buffers used in the binding, washing, elution, strippeing and equilbiraiton steps flow coutnercurrent to the resin slurry in a multi-stage confirguation, enabling hihg resolution separation while reducing the amoutn of ubffer need for protein purificaiton. (Shinkazh “Purificaiton of monoclonal antibodies form clarified cell culture fluid using Protein A capture continous coutnercurrent tangential chromatography” J Biotechnology. 213 (2015) 54-64). 

Shinkazh (US 2010/0193434; see also US 2017/0045483) discloses a module that consists of two or more interconnected tangential flow filters and static mixers. The chromatography resin flows thorugh this module in a single pass, while similar operations to a regular chromatographic process are performed on the resin (binding, washing, elution, regeneration and equilibration). The buffers for these operations are pumped into the module in a countercurrent direction to the flow of resin, and permeate solutiosn from latter stages are recyled back into previous stages. This creates concentration gradients in the permeate solution of the tangential flow filters in the countercurrent direction to resin flow, thus saving buffer volume and increasing process efficiency. The permeate solutions from binding, washing, equilibration and regeneraiton operations are put to waste. The permeate solution form the elution operation is the purified product stream which is collected in a separate product tank. In one embodiment, the module for CCTC includes a first input prot for receiving an input solutions, a first mixer for mixing the input solutions with a recyled solution from a second input port to produce a first mixer output, a stage 1 filter for concentrating the first mixer output to produce stage I retentate, wehrein stage I permeate exists the module from the stage 1 filter via a first output port, a second mixer for mixing the stage 1 retentate form teh stage I filter and an optional buffer solution from a second inpurt port and a stage 2 filter for concetnrating an output from the second mixer to produce stage 2 retnentate which exits the module form teh stage 2 filter via a second output port, wehrein stage 2 permeate exists the module form the stage 2 filter via a third output port. 

Shinkazh (US 2020/0030717) disclsoes replacing the hollow fiber membranes of CCTC systems above by spiral micro-fluidic partcile sorter devices. These dvices seaprate resin form the dissolved species using Dean Vortices at the curved outer wall of the device channel to selectively attract partciles to one wall of the device. The devices accordingly do not reuqire a physical barreir such as a membrane for separating resin partciles from dissolved speies.