The complement system consists of about 31 proteins, acting within a cascade-like reaction sequence, serving as control proteins or as cellular receptors. Some of the proteins are enzymes (C1r, C1s, C2, factor B, factor D), some cofactors, some inhibitors, and other are composed of membrane-integrated proteins.

Complement proteins are made mainly by the liver and circulate in the blood and extracellular fluid. A majority of the protein componetns of the system are syntehsized by liver hepatocytes; however, blood monocytes, tissue macrophages and the epithelial cells lining the gastrointestinal and genitourinary tracts can also synthesize some complement proteins. 

Both in vivo and in vitro studies have confirmed that cytokine mediators of the acute phase response, for example, the interleukins (particularly interlukin y), TNF and dexamethasone can increase the hepatic synthesis of complement proteins 2-5 fold in cultured hepatocytes. (Figueroa, Clinical Microbiology Reviews, July 1991, p. 359-395)

Most complement proteins are inactive until they are triggered by an infection. Most the circulating proteins are innnocuous and as such are regarded as “pro-enzymes”. However, upon initiation there occurs a sequential proteolysis and ensuing activation of these proteins. Activation of a complement protein changes its structure, revealing an “active” site then catalyzes, and usually cleaves, a portion of the next protein in the sequence, ultimately producing a multiple subunit protein complex that lyses the invading cell. 

They were originally identified by their ability to “complement” the action of antibodies but some components of complement are also pattern recognition receptors that can be activated directly by pathogen associated immunostimulants. Complement can be activated by any of three pathways, either the antibody-dependent classical pathway, the alternative pathway or the lectin pathway. On activation, these pathways result in the formation of unstable protease complexes, the C3-convertases. The classical pathway C3-convertase, C4b2a, and the AP C3-convertase, C3bBb, are both able to cleave the alpha chain of C3 generating C3b. C3b has the potential to bind covalently to biological surfaces which leads to opsonization for phagocytosis by polymorphonuclear cells and macrophages. When additional C3b is available, the C3-convertases can function as C5-convertases, cleaving C5 and initiating the assembly of the TCC, or the membrane attack complex (MAC), which mediates cellular lysis by insertion of pore-forming protein complexes into targeted cell membranes. (Kaleko, WO2008/106644).

Companies: Editas  Excision Bio Therapeutics Prime Medicine. Pairwise.  Tome Biosciences. EditCo

See also chimeric antigen receptors (CARs) under Cancer treatment

Programmable nucleases such as CRISPR-Cas9 make double strand DNA breaks (DSBs) that can disrupt genes by inducing mixtures of insertions and deletions (indels) at target sites. DSBs, however, are associated with undersired outcomes including complex mixtures of products, translocations and p53 activaiton. Moreover, the vast majority of pathogenic alleles arise form specific insertions, deletions or base substitutions tha require more precie editing technologies to correct. Homology-directed repair (HDR) stimulateed by DSBs has been used to insall precise DNA changes. HDR, however, relies on exogenous donor DNA repair templates, typically generates an excess of indels form end-joining repair of DSBs, and is inefficient in most therapeutically relevant cell types (T cells and some types of stem cells being important exceptions). (liu, Nature 2019, 576(7785): 149-157).

CRISPR-Cas componetns readily access the gehnome of bacteria as prokaryotes lack a nucleus. However, unmodified CRISPR-Cas components do not readily enter the nucleus of eukaryotic cells, wehre genomic DNA is located, which greatly limits the efficiency of DNA editing. A single nuclear localization signal (NLS) is typically sufficient to facilitate efficient nuclear entry of most prtoeins. However, multiple NLSs are necessary to drive Cas variants to the nucleus in eukaryotic cells, including Sterptococcus pyogenes (Sp) Cas9 and Neisseria meningitidis (Nm) Cas9, as well as base editors, prime editors, Cas12a, and nuclease dead Cas9. This may be because Cas9 is sequenstered in the cytoplams of mammalian cells, inn part, via interaction with the ribosome. Increasing the number of NLSs on Cas9 and/or increasing the amount of cytoplasmic guide RNA has the potential to outcompete ribosomal RNA binding and promote efficient nuclear localization of CRISPR-Cas9 variants. (Zylka, “Exploring the cytoplasmic retention of CRISPR-Cas9 in eukaryotic cells: the role of nuclear localizaiton signals and ribosomal interactions” CRISPR Journal, volume 00, number 00, 2025.)

Classes of CRISPR Nucleases:

In brief, CRISPR based gene editing reagents are generally used as two-component systems: a nuclease protein is complexed with a single-guid RNA (sgRNA) designed to target a specific site in the genome. Much work has gone into engineering both nucleases and guides to optimize function and stabilitiy across an array of cell types. The CRISPR associated nucleases most commonly used for double stranded DNA targeting belong to class II and can be put into two groups, type II and type V, that differ in the position of their protospacer-adjacent motif (PAM) vis-a-vis the spacer region (3′ vs. 5′) and the DNA ends that result from the clevage reaction (blunt vs. overhnag). (Lamothe, “Novel CRISPR-Associated gene-editing systems discovered in metagenomic samples enable efficient and specific genome engeering”, CRISPR Journal, 6(3), 2023)

Type II Nucleases:

The type II CRISPR-Cas system includes three components: (1) a crRNA molecule, which is called a “guide sequence” and “targeter-FNA”, (2) a tracr RNA, also known as an activtor-RNA and (3) a protein called Cas9. To alter a DNA molecule, the ssytem must acheive three intereactions: (1) crRNA binding by specific base pairing to a specific sequence in the dNA of interest (target DNA), (2) crFNA bing by specific base pairing at naother sequence to a tracer RNA, and (3) tracr RNA interacting with a Cas9 protein, which then cuts the target DNA at the specific site.

A CRISPR-Cas9 system is a combination of protein and ribonucleic acid (RNA) that can alter the genetic sequence of an organisms. In their natural environment, CRISPR-Cas systems protect bacterai agaisnt infection by viruses. Te system is now being developed as a powerful tool to modify specific deoxyribonucleic acid (DNA) in the genomes of other organisms, from plants to animals. With CRISP, scientists can create mouse models of human diseases much quicker, study indivdual genes much faster and easily change multiple genes in cells at once to study their interactions.

Although Streptococcus pyrogenes Cas9 is a highly active gene editing enzyme, its use is complicated by a low editing specificity. Furthermore, Cas9 is derived from a Streptococcus bacterium, a very commonly pathogenic genus. As a result, between one third and half of people have a preexisting immune response to the Cas9 enzyme and thus are less than optimal candidates for a Cas9 based gene editing therapy. (Lamothe, “Novel CRISPR-Associated gene-editing systems discovered in metagenomic samples enable efficient and specific genome engeering”, CRISPR Journal, 6(3), 2023)

Base Editors:

Base editors generate targeted base conversions without requiring DSBs. Cystosine base editors and adenine base editors for example comine deaminases with CRISPR systems to produce C:G-to T:A and A:T to G:C base transcritions, respectively. (Liu, “The CRISPR-Cas toolbox and gene editing technologies” Molecualr Cell 82, 2022)

Prime Editors (Prime editing):

Prime editing relies on Cas9 and a reverse transcriptase.

Prime editors are powerful tools for installing base substitutions and precise DNA insertions and deletions. They are composed of two components: an engineered Cas9 nickase (H840A) reverse transcriptase (RT) fusion protein and a prime editing guide RNA (pegRNA). The pegRNA contains an RT template (RTT) encoding the desired edits and a primer binding site (PBS) for hybridization of the 3′ end of the nicked DNA strand to initiate reverse transcription. After reverse transcription, the RT template is reverse transcribed, forming a 3′ DNA flap followed by a 5′ DNA flap, and this enables the desired edit to be integrated into the target site. (Liu, “The CRISPR-Cas toolbox and gene editing technologies” Molecualr Cell 82, 2022)

liu, (Nature 2019, 576(7785): 149-157) discloses a search-and-replace genome editing technology called “prime editing” that mediates targeted insertions, deletions, all 12 possible base to base conversions and combinations thereof in human cells without requiring DSBs or donor DNA templates. Prime editiors (PEs) use a reverse transcriptase (RT) fused to an RNA programmable nickage and a prime editing guide RNA (pegRNA) to direclty copy genetic informaiton from an extension on the pegRNA into the target genomic locus. The DNA nicked at the target site to expose a 3′-hydroxyl group can be used to prime the reverse transcription of an edit-encoding extension of the engineered guide RNA (prime editing guid RNA, or “pegRNA) directly into the target site. These initial steps result in a branched intermedaite with two redundant single-stranded DNA falps: a 5′ flap that contains the uneditind DNA sequence and a 3′ flap that contains the edited sequence opied form the pegRNA. %’ flaps are hte preferred substrate for structure-specific endonucleases such as FEN1 which excises 5′ flaps engenerated during lagging-strand DNA syntehsis and long-patch base excision repair. Alternativley, the reduncant uneidted DNA may be removed by 5′ exonucleases such as EXO1. 5′ flap excision and 3′ flap ligation can drive the incorproation of the edited DNA strand, creating heteroduplex DNA containing one edited strand and one unedited strand. DNA repair to resolved the hteroduplex by copying the informaiton in the edited strand to the complementary strand will permanently install the edit.

DNA-Dependent Polymerase (DDP) Editing (DPE):

DNA-dependent polymerase (DDP) editing presents adnvatages over prime editing by allowing researchers to cale materials up for clinical use. DPE benefits fro the highly processive and accurate synthesis abilities of DNA polyemrases. The approach avoids a range of problems associated with prime editing: fewer erros, less reliance on a cell cycle due to higher dNTP affinity, and easier template synthesis.

–Click Editing:

An even newer form of genome eidint that relies on a DDP, a nickase Cas9 (nCas9) and a histidine-hyrophogic histidine (HUH) endonuclease to alter the geneome allows for mass screening of DNA templates and futher refines DDNA polymerase editing (DPE). Click editing adds HUH edonucleases to DPE. HUH endonucleases are smalle (10-40 kDa) proteins common across all domains of life and which are often found playing a role in replication. HUH enzymes are guided by 8-40 nucleotide ssDNA regoniction sequences. Click editors eploy HUH endonculeases for teplate recruitment to teh target site, which covalently and sequence specifically bind ssDNA. Click editing invovles 5 components: nCas9, a single guid RNA (sgRNA), an HUH endonuclease, a DDP and a click DNA (clkDNA). clkDNA guides the DDP and contains the edit of itnerest, which an sgRNA directs nCas9. In this way, clkDNA mimics the role of DPETs. Click editing follows a methodical set of steps to alter the geome. Frist, the click editor binds and nicks the genome at the target site. The, HUH tethers or “clicks” -clkDNA to the target site. The primer binding sequence of the clkDNA subsequently binds to the flap created by the nickase, allowing clkDNA to serve as a tplate for polymerization. After the primer has synthesized the new sequence, the click editor dissociates and leaes the edited flap behind. Finally the new sequence is integrated into the gebome. Using a DNA based template has a range of benefits over prime editing, which uses a long pegRNA to direct and initate its reverse trasncriptase appraoch. Click editing extends these benefits by removing RNA form teh template to facilitate easier synthesis. (Seren hough, “Polymerase editing ‘clicks’ together  in trailbalzing study’ Tides Global, Oct 25, 2024.)

Applications:

See FDA Approved Cellular Therapies

Gene editing has been applied to cell therapy with many types of primary immune cells –especially T cells –via electroporation of Cas9 ribonucleoprotein particles (RNPs). Such engineering has been used to knockout the T cell receptor (TCR), checkpoint inhibitors, and ot knock in chimeric antigen receptors (CARs), among many examples. Gene editing systems (type II and type V) have also been used to edit B cells, NK cells, indicued pluripotent stem cells (iPSCs), and hematopoietic stem cells (HSCs). Lamothe, “Novel CRISPR-Associated gene-editing systems discovered in metagenomic samples enable efficient and specific genome engeering”, CRISPR Journal, 6(3), 2023)

HIV Gene Editing:

Manucuso et al. (“CRISPR based gene editing of SIV proviral DNA in ART treated non-human primates; Nature Communications 2020) discloses creating an adeno-associated virus mediated plasmid DNA vector that allows for simultaneous expression of Cas9 endonuclease and multiple guide RNA. The Cas9 endonucleas and gRNAs specifically recognize LTR and Gag region of the HIV-like simian immunodeficiency virus genome. Excision of large segments of the integrated proviral DNA spanning from cleavage site mitgates the chance of the replication -competent virus’s emergence.

Based on these promising results, Excision Bio Therapeutics has started clinical trials to evaluate the construct, termed EBT-101 as a potential cure of HIV infection.

Colesterol lowering/PCSK9:

Musunuro (“In vivo CRISPR base editing of PCSK9 durably lowers cholesterol in primates”  Nature, 2021) demonstrates durable editing in target organs of nonhuman primates is a key step before in vivo administration of gene editors to patients in clinical trials. Here we demonstrate that CRISPR base editors that are delivered in vivo using lipid nanoparticles can efficiently and precisely modify disease-related genes in living cynomolgus monkeys (Macaca fascicularis). We observed a near-complete knockdown of PCSK9 in the liver after a single infusion of lipid nanoparticles, with concomitant reductions in blood levels of PCSK9 and low-density lipoprotein cholesterol of approximately 90% and about 60%, respectively; all of these changes remained stable for at least 8 months after a single-dose treatment.

Deafness (Hearing Loss):

–Autosomal dominant deafness-50 (DFNA50) is a form of progressive hearing loss which starts with mild symptoms before getting significantly worse later in life. The condition is caused by a variant in the microRNA gene MIR96. A new CRISPR-Cas9 based treatment for FFNA50 has been desmontrated in an artile published in Science Translational Medicin (Zheng-Yi). The study showed that hearing loss was restored in mice after targeting and disrupting the DFNA50 causing version of MIR96. Chen’s team used an adeno-associated virsu (AAV) system to deliver Cas9 and a single guid RNA (sgRNA) specifically to teh cochlea in the inner ear, the region responsbiel for hearing. AAV has become a popualr choice for delivery becasue it is less likely to interate into the target cell. Moreover, different serotypes of AAV can be used to target specific parts of the body. On the other hand, the small cargo size of AAV prohibits the delivery of a large gene like traditional Cas9. To get around this limitation, the team used a specific kind of Cas9 derived form Staphylococcus aureus bacteria. This ortholog is smaller than the typical streptococcus pyogenes Cas9-making it an idea match for AAV delivery.

Sickle Cell Disease and beta-thalassemia:

–Casgevy (CRISPR Therapeutics & Vertex Pharmaceuticals): is a one time treatment for people age 12 and over for sickle-cell diesease as well as beta-thalassemia. Vertex recieved the FDA first approval for a CRIP based gene edited therapy, Casgevy for sickle cell disease. The following month, the FDA approved Casgevy to treat tranfusion dependent beta-thalassemia.

Beam Therapeutics also expects to conduct Phase I/II trails to asess its lead candicate BEAM-101 in severe sickle cell diseas.

Duchenne Mascular Dystrophy:

–ELEVIDYS (Sarepta Therapeutics): is a prescription gene therapy used to treat ambulatory children aged 4 through 5 years old with Duchenne muscular dystrophy (DMD) who have a confirmed mutation in the dystrophin gene. ELEVIDYS was approved under accelerated approval.

Animal Genetics:

Genus, a British animal genetics company with research facilites in Wisconsin and Tennessee, has developed a new generation of CISPR edited pigds that are resistant to procine reproductive and respiratory syndrome (PRRS) virus, a disease that has had a widespread impact on porcine populations around the world for decades. Several genes are invovled in viral infection, including CD163, which encoes the entry receptor for teh virus. In pigs, this particular protein is expressed on teh surface of macrophage and monocytes and mediates inflammation among other functions. A single modified CD163 allele was introduced into four geentically diverse, elete porcine lines. Genus scientifits injected CRISPR-Cas9 editing reagents into the genomes of pig zygotes. Their goal was to make a preice deletion in CD163 that removed a single expon encoding the domain that directly interacts with the virus. Importantly, the edit did not impact CD163’s function in the new population. The work produced healthy pigs that resisted PRRS virus infections. The company is now seeking regulatory approval in the US which is the only country which regulated intentional genetic alterations including single base pair deletions as an animal drug and reqires a new animal drug approval for commercialization. This regulatory path is very hard for small companies. Until the pigs are approved, they are considered unsalable.

Gene Editing in Plants:

Classical genome editing in plants involves a sgRnA guided Cas dsDNA nuclease with subsequent induction of the non-homologous end-joing (NHEJ) DNA repair pathway, frequently resulting in loss of function (LOF) mutaitons. Modulation or gain of function may be obtained through the use of sgRNA wity subsequent induction of the base excision repair pathway or by prime editor RNA (pegRNA) guided prime editors with subsequent induction of the NHEJ or the homology directed repair pathway, depending on cell type. Petersen “Strategies and Protocols for optimized genome editing in potato” CRISPR Journal, volume 00, number 00, 2024).

Diagnostics:

–Zika Detection:

Collins (“Rapid, low-cost detection of Zika virus using programmable biomolecular components” Cell 165, 1255-1266, 2016) demonstrate the rapid development of a diagnostic workflow for sequence-specific detection of Zika virus that can be employed in low-resource settings. Discriminatio of different viral strains at single-base resolution was achieved using a CRISPR-based tool. By simply boiling (95C) virsu samples for 2 min, sufficient quantities of RNA for amplication and detection were generated. NASBA is an extremely sensitive and has a proven track record in the field-based diagnostic applications. The amplificaiton process betgins with reverse transcription of a target RNA that is mediated by a sequence-specific reverse primer to create an RNA/DNA duplex. RNase H then degrades the RNA template, allowing a forward primer containing the T7 promoter to bind and initiate elongation of the complementary. strand, generating a double-stranded DNA product. T6-mediated transcription of the DNA template then creates copies of the target RNA sequence. Importantly, each new target RNA can be detected by a towhold switch sensors and also serve as starting material for futher amplication cycles. NASBA requires an initial heating step (65C), followed by isothermal amplication at 41 C. NASBA was performed on trigger RNA corresponding to Zika genomic regions for sensors 27B and 32B. Toehold switch sensors are programmable syntehtic riboregulators that control the translation of a gene via the binding of a trans-acting trigger RNA. The switches contain a hairpin structure that blocks gene translation in cis by sequestration of the ribosome binding stie (RBS) and start condon. Upon a switch binding to a complementary trigger RNA, sequenstraiton of the RBS and start condon is relieved, activating gene translation. To allow for colrimetric detection of trigger RNA sequencs, the sensors can be desigend to regulate translation of the enzyme LacA, which mediates a color change by converting a yellow substrate to a purpose product. Toehold switch sensors for sequence based detectin of Zika virus were generated using a modified version of a developed in silico design algorithm. The modifed algorithm screened the genome of the Zika strain prevalent in the American (Genbank: KU312312) for regions compatible with RNA amplication and toehold switch activation. The slected Zika genome regions were then computationally filtered to eliminate potential homology to the human transcriptome and to a panel of related virsues, incuding Dengue and Chikungunya. A total of 24 unique regions of the Zika genome compatible with downstream sensing efforts were identified. The presence of a train-sepcific PAm leads to the production of either truncated or full lenght trigger RNA, which differentially activate a towhold switch. The probability that a non-biased single nucleotide polymorphism (SNP) between two strains can be discriminated by CRISPR/Cas9 is 48%.

A species identified as a mAb monomer with an additional light chain covalently associated through a disulfide bond formed between a L chain cystein and an engineered cystein in either the H or L chains is known. Genetech researchers also characterized mAb species containing variants with one and two additional light chains present at 0.2% when produced in CHO cells. They determined that the relative quantity of this species was related to the redox environment of the cell culture, which implicates disulfide bonding as the mode of binding for tehe additional light chains. In addition, a report characterizaing a mAb size variant using SE-HPLC coupled with multi-angle light scattering (MALS), matrix-assisted laser desportion/ionization-time of light (MALDI-TOF) mass spectrometry, capillary gel elctrophoresis and N-terminal sequences showed a mAb with incorporation of a third light chain; however the extra L chain was associated through non-covalent interactions. A cell based potency assay showed the reduced aiblity of the extra L chain species to neutralzie the target antigen. Wollacot (mAbs, 5(6): 925-935 (2013)

Jensen (US 2016/0347833) discloses an antibody pre-monomer impurity that includes two heavy chains (HC) and three light chains (LC) of which one is non-covalently attached (LC2HC2: LC). Based on the biophysical, spectroscopic and function characterists of LC2HC2:LC, the molecular structure is an antibody where an addition light chain has taken up the position normally occupied by a HC. The additional LC is bound via non-covalent itneractions to another LC, which in turn is covalently bound to HC. The C-terminal cysteine in the non-covalently attached LC is capped by forming a disulfide bond with either glutathione or cysteine. 

Challenges Posed in removing Extra Light chains 

Due to the similarities to the monomer species, the separation of H2L3 antibodeis generates a challenge during downstream purificaiton of monoclonal H2L2 cysteine-modified antibodies. (Liu US 2019/0112359). 

Purification Strategies for Intermediate HMW species:

CEX:

Liu (US 2019/0112359) discloses a purificaiton strategy to seperate triple light chain (H2L3) antibodies from double light chain (H2L2) antibodies using CEX. using optimzied POROS SC strong CEX, the amount of H2L3 antibody can be reduced from 11% to less than 1%. In one embodiment, an antibody composition is applied to the CEX so that H2L3 antibodies and H2L2 antibodies bind and eluting with a pH of about 3.8 to about 5 and collecting an H2L2 composition eluted form the resin. In some embodiments, no more than 0.5 of the antibodies in the H2L2 composition are H2L3 antibodies. 

HIC

Jensen (US 2016/0347833) disclosing a method for removing an antibody pre-monomer that includes two H chains and 3 L chains (LC2HC2:LC) which includes the step of purifying the monomeric antibodies using hydrophobic interaction chromatography (HIC). Antibody products after the method include anti-IL21 comprising at most 1% antibody pre-monomer aggregates. Elution from the HIC was done by decreasing the ammonium sulphage concetnration, such as by using alinear gradient of ammonium sulphage to 0 mol/kg. In one embodiment, the monomeric antibody componets elutes as the first antibody componentIn such examples, the antibody solution of interest may be obtained by collecting the fractions eluting prior to the fractions including the majority of pre-monomers and antibody dimers. Pre-monomer mayb be excluded form the antibody composition by deselecting subsequent eluate fractions. 

Wollacot (mAbs, 5(6): 925-935 (2013) disclsoes a robust purificaiton method to reduce an intermedaite HMW species using hydrophobic interaction chromatography (HIC) from about 3% to less than 0.5% with good step recovery. 

SEC:

Size-exclusion chromatogrpahy (SEC) is the most commonly used method to separate and quantify mAbs size variants. SEC analysis of MAb-A resolved a peak, named Peak 1, which elutes between monomer and dimer peaks. Elctron spray ionization -time of light mass spectrometry (ESI-TOF MS) microfluidics capillary electrophoresis and sodium dodecyl sulfate-PAGE (SDS PAGE) results demonstrated that SEC Peak 1 contains two structural variants: MAb-A with one extra light chain (2H3L) and mAb-A with two extral light chains (2H4L). Teh C-terminal Cys of the extra light chain in Peak 1 variants is either a free thiol, capped by glutathione, cystein or another light chain. the Peak 1 fraction also shows two major peaks with calculated molecular weights of 175 kDa and 191 kDa which are the approximate sizes of 2H3L and 2H4L. (Liu, “Chracterization of monoclonal antibody size variants containing extra light chain” mAbs 5:1, 102-111, 2013). 

Companies:  ALV.  Brookhaven Instruments   Malvern. Wyatt  

Light scattering is one of the few absolute methods available for the determination of molecular mass and structure and is applicable over a broader range of Mw than any other methods. When size-exclusion chromtography (SEC) is used with on-line multi-angle laser light scattering (MALLS) detection, the weight, number and z-average values for obth mass and size may be obtained for most samples. Included in such measurements is the ability to caclulate both differential and accumulative distributions of the Mw and mean square radii. (Oliva, Applications of multi-angle laser light-scattering detection in the analysis of peptides and proteins” Current drug discovery technologies, 1(3), pp. 229-242 (2004). 

Liquid chromatography and in particular high performance size exclusive chromatography, HPSEC, is a useful tool for the characterization of polymers. Typically, samples are prepared and injected into a chromatgoraphy where they are pumped thorugh columns that seaprate the molecules based on their hydrodynamic size, smaller molecuels tend to remain longer in the interstices of the columns and thus elute at later times than large molecules. Historically, the chromatograph with its separating columns and concentraiton sensitive detector was claibrated by using nearly monodisperse polymeric standards spanning a broad range of molecular weight, MW. The MWs present in the unkown sample were thus derived form a measurement of the time required for each separated fraction of sample to pass through the chromatograph relative to the corresponding times for the narrow calibration standards. With the advent of in-line light scattering detectors, the need to calibrate was no longer required, since a light scattering detector combined with a concentraiton detector permitted the determiantion of MWs and sizes, and their distributions, on an absoltue basis. (Shortt US Patent No: 5,528366). 

Dynamic Light scattering (DLS):

products: 

Wyatt Technology DyanPro Plate Readr: can perform high-throughput screening with dyanmic light scattering (HTS-DLS), It contains 96, 384 or 1536 well plates and performs temeprature scans of all samples simultaneously for a temperature range of 4-85C. A biomolecule’s staility is not an entirely intrinsci property, as it is influenced by the concetnraiton at which the protein is formulated and buffer composition. Protein stability must be measrued as a function of specific ion type, ionic strengh, pH and excipient profile for an optimal and successful formulation. (Wyatt Technology “The diffusion interaction parameter (Kd) as an indicator of colloidal and thermal stability” 

High throughput light scattering applications are routinely applied in the pharmaceutical industry including dynamic light scattering (DLS) and multi-angle light scattering (MALS). DLS is based on the measurement of intensity fluctuations of scatterid light due to Brownian motion. Autocorrelation curves can then be calculated form the intesity flucutations and fitted to dervie the translational diffusion coefficient D. In the prsence of interactions the moelcuels diffuse according to an effective diffusion coefficient D whcih deviates form the diffusion coefficient of the moelcuels D0. This effective diffusion coefficient D exhibits a linear relationship as a function of mAb concetnration, D=D1(1+KDc), with the slope KD equal to the diffusion itneraction parameter. A positive slope indicates repulsive intermoelcuels interactions, whereas a negative slope indicates attractive intermoelcular interaction.  (Lorenzen Chapter 14, “Mesauring self-assocaition of antibody lead candidates with dynamic light scattering” in “Therapeutic Antibodies Methods and Protocols, Methods in Molecular Biology, springer Protocols, pp. 1-347, 2022)

Various studies suggest that undesriable solution behaviors such as elevated viscosity are caused by short live transient “clusters” that result form attractive prtoein-protein interactions (PPI), The protein interaction parameter (Kd) calculated form low-protein-concetnraiton dyynamic light scattering (DLS) mreasurements is often used to relate PPI to solution viscosity at high protein concetnration. For example, the potein interaction parameter (Kd) calculated from low protein concenration DLS measruements were related to solution viscosity at high protein concentration. These empirical correlations were used to conclude that negative KD values, and therefore attractive PPI, at least qualitatively, predict the large increases in viscosity observed at high mAb concetnration. (Woldeyes, “Molecular-scale understanding of protein interactions and solution viscosities” dissertation, 2018). 

DLS is based on the fluctuations of the scatterd light intensity. It is robust and amendable to high throughput methodeoloy (about 100 samples/day per instrucement). Colloidal stability can be measrued by measuring the concetnration depednence of the prtoein collective diffusion coefficeint via DLS with protein-protein interactions being parameterized via the interaction parameter KD and comparing diffusion coefficient values measrued usying Taylor disperson analysis. (Sikka, sirat “Studying Protein-Protein interactions using dynamic light scatteringa nd Taylor Disperson Analysis” Dissertation. 

A negative KD value (protein-protien interation parameter obtainable from DLS) can be used to udnerstand the effects of NaCL and ArgHCL on protein-protein interactions. A negative KD value indicates an attractive prtein-protein interaction, while a positive KD value indicates a repulsive protein-protein interaction. (Alsabih “Towards an improved predictor for the colloidal stability of unfolded prtoeins through probing aggregation behaviour in solutions containing chemical denaturants” Thesis. 

With UV absorbance (multi-angle light scattering (MALS) +UV):

Ion Exchange:

Bailey (US Patent Application 16/579,220, published as US 2020/0018771 and US 13/850,664, published as US 10481164; see also US 17/495,963 published as US 2022/0026442) disclose a method for determing a stop collection point during IEX which includes monitoring the eluate with a laser light scattering detector and a UV absorbance detector and calculating a fraction LS/UV ratio for each fraction until he peak max LS/UV is determined, calculating a normalized LS/UV ratio for all subsequent fractions, wherein an increase in the normalized LS/UV ratio indicates an increase in the amount of impurities in the eluate pool and terminating the eltuion when the normalized LS/UV ratio reaches a predetermined value. In one embodiment, the normalized LS/UV ratio is calculated by dividing the fraction LS/UV ratio by the peak max LS/UV ratio. When a purificaiton process is operated in bind and elute or gradeint elution mode, the protein product is eluted first and aggregated species are present in the tail of the elution peak. Real time monitoring of the change in the ratio allows for a real time determiantion of the stop collection point based on the properties of the desired product at the time of elution. A monomeric protein peak, for instance, will have a constant ratio of LS/UV. Any deviation in that ratio signifies the product stream is contaminated with non-monomer impurities. The ration of the light scattering (LS) to absorbance concentration (for example, UV) is determiend real time. In one embodiment, eluate from a protein A column was diverted using a slipstream port to a AKTA UV-900 by pump and then passed to a Heleous II (Wyatt Technology) light scattering detector. The signals were processed in real time using a software program that cacluated the LS/UV ration of the elution preaks, “fraction LS/UV ratio” using the signals form the light scattering detector and UV absorbance. The purest fraction was the peak max. The stop collection point for the elution was set at a predetermiend normalized LS/UV ratio in the elution pool. An increase in high molecular eight cotaminants in the elution was reflected in an increase in the normalized LS/UV ratio. 

With Size-exclusion (LS +SEC):

The SEC-MALLS technique does not rely on relative Mw standrds for column calibration and yields absolute Mw estimates direclty from the angular dependence of scatterd light intesity as a funciton of concentration, as formulated by light scattering theory. Oliva, Applications of multi-angle laser light-scattering detection in the analysis of peptides and proteins” Current drug discovery technologies, 1(3), pp. 229-242 (2004).

Harman “Charcterization and analysis of thermal denaturation of antibodies by size exlusion high-performance liquid chromatography with quadruple detection” (Analytical Biochemistry 325 (2004) 227-239) discloses that SEX coupled with online light scattering and UV visible spectroscopy provides a very powerful tool for studying protein size, shape and aggregation. 

Kalonia (US 2007/0178013) discloses appatuses and methods for simltaneous measurment of protein concentraiton and scatered light intensity. 

Mass spectrometry (MS) has gained importance in the anlytics of biopharmaceuticals, taking different complementary approaches ranging from peptide-based sequencing to direct analysis fo intact proteins and protein assmeblies. (Rosati, Nature Protocols, 9(4), 20140)p

A variety of “hyhenated chromatography-mass spectrometry” methods have demonstrated the capability of detecting low-abundance impurities in mAb products and providing highly detailed analyses that cannot be acheived by either SDS-PAGE or CE-SDS methods.  (Wang, US 16/223,463, published as US 2019/0194298).

Hydrophilic interaction chromatography (HILIC) + MS:

for characterization of glycosylation:

Glycosylation is one of the most common postranslational modifications of proteins. Protein heterogeneity introduced by glycosylation includes differences in the nature and structure of the attached oligosacharides, and the number and position of the glycans. Common methods to study glycosylation involve glycan release or protein digestion, followed by analysis of the reuslting glycans or clycopeptides. These methods offer detailed information about the sites of clycosylation and/or the carbohydrate composition. Electrospray ionization mass spectrometry (ESI-MS) enalbes the precise mass determination and characterization of intact (glyco) proteins. However, seperation prior to MS detection often is essential to acheive reliable assignment of intact glycoproteins and detection of low abundant glycoforms. HILIC-MS has shown highly useful for the analysis of a wide range of compounds including metabolites and pepties. (Somsen, “high-resolution glycofrom profiling of intact therapeutic proteins by hydrophilic interaction chromatography-mass spectrometry” Talanta 184 (2018) 375-381). 

Glycosylation is a post-translational modificaiton of proteins that occurs in all eukaryotic cells. The sugar chains on glucoproteins can mediate biologial activity, play a role in receptror-mediated recognition, increase solubility, regulate half-life and exert a stabilizing influence upon conformation. (Gillece-castro, “N-linked glycans of glycoproteins: a new column for improved resolution” Water Corporation (2009) .ueous mobile phase used in HILIC, which overcomes the drawbacks of the poor solubility often encounted in NP-LC. Expensive ion pair reagents are not required in HILIC, and it can be conveniently coupled to mass spectromtry (MS). (Busewski, “Hydrophilic interaction liquid chromatogrpahy (HILIC) – a powerful spearation technique, Anal Bioanal Chem (2012) 402: 231-247). 

HILIC has many specific advantages over conventional NP-LC and RP-LC. For example, it is suitable for anlyzing compounds in complex systems that always elute near the void in reserved-phase chromatography. Polar samples always show good solubility in the aq

D’Atri (Hydrophilic Interaction Chromatography hyphenated with mass spectrometry: a powerful analytical tool for the comparison of originator and biosimilar therapeutic monocloanl antibodies at the middle-up level of analysis” Anal. Chem 2017, 89, 2086-2092) discloses that using a sationary phase in combination with a highly organic mobile phase, HILIC is fully compatible with electrospray ionization mass spectrometry (ESL-MS) and offers a complementary retntion mechaism compared to RPLC, since analytes are eluted based on their hydrophilicity. 

Lauber (US 2015/0316515) discloses characterization of large biological molecules modified with polar groups such as antibody glycorms by hydrophilic interaction chromatography (HILIC) which further includes the step of identifying the glycopeptie with mass spectrometry. In one embodiment the sample is first prepared by reductyion, enzymatic digeston, denaturation, fragmentation, ehmical cleavage or a combination thereof. In another emobdiment the method for analyzing a glycosylated protenaceous sample, the chromatographic material is a porous material which includes at least one hydrophilic monomer and a poly-amide bonded pahse. In certain embodimetns, the method includes the step of denaturing the glycosylated proteinaceous sample at a temperature 80C or greater and deglycosylating the denatured sample. In certain embodiments, the method includes a mobile phase eluent such as acetonitrile, isopropanol or ethanol. 

Wang (J Pharmaceutical and Biomedical Analysis, 154: 468-476 (2018) disclsoes that hydrophilic interaction chromatography (HILIC) coupled with mass spectrometry analysis can characterize LMW impurites present within a purified mAb drug product sample. After mreoval of N-linked glycans, the HILIC method separates mAb related LMW impurites with asize based elution order. Free ligh chains, half-antibody, H2L species (antibody possessing a single light chain) and protein backbone truncated species can all be confidently identifed and elucidatesd in great detal, including the truncation sites and associated post translational modificaitons. 

Wang, (US 16/223,463, published as US 2019/0194298) discloses methods for charaterizing low molecular weight (LMW) protein drug product impurities which uses hydrophilic interaction chromatography (HILIC) coupled to mass spectrometry analysis. After removal of the N-linked glycans from teh protein drug product, for example an antibody drug product, the elution of LMW impurities from the HILIC column is determend by the size of the molecular weight species. 

IEX + Mass Spectrometry: 

Cation exchange (CEX) + MS:

Wang (Anal Cehm 87: 914-921 (2015) disclsoes drug product related impurities of an anti-Clostridium difficile IgG1 mAb were profiled by CEX liquid chromatogrpahy followed by teh CEX peaks being fraction collected for MS characterization. A reversed-phase liquid chromatography/mass spectrometry (LC/MS) methology was developed on a Thermo Q-Exactive orbitrap mass spectrometer for mass measurements of the mAb, its CEX fractioanted impurties and their respective H and L chains. 

Wang (US 16/259095, published as US 2019/0234959) discloses using IEX, preferably strong CEX  with an aqueous mobile phase coupled with native mass spectrometry analysis to detect and characterize size variant protein drug product imprities. In one embodiment the protein is first deglycosylated such as by treating the sample with IdeS.

SEC + Mass Spectrometry:

Kukrer (Pharm Res, 27: 2197-2204 (2016) discloses mass spectrometric analysis (MS of mAg aggregates fractionated by size-exclusion chromatography (SEC). IgG aggregates (dimers, trimers, tetramers and HMW oligomers) were created by subjecting an IgG formulation to several pH jumps. Protein oligomer fractions were isolated by high performance size exclusion chromatogrpahy (HP-SEC) dialyzed agaisnt ammonium acetate pH 6.0 and analyzed by native electrospray ionization time-of-flight mass spectrometry (ESI-TOF-MS). 

Wang (US 16/259095, published as US 2019/0234959) discloses using SEC with an aqueous mobile phase coupled with native mass spectrometry analysis to detect and characterize size variant protein durg product imprities. In one embodiment the protein is first deglycosylated such as by treating the sample with IdeS. 

Wollacot (mAbs, 5(6): 925-935 (2013) discloses using seize exclusion high-performance liquid chromatogrpahy (SE-HPLC) analysis of a hmAb to identify a mAb species that eluted between the monomeric and dimeric species.